CN101663395B

CN101663395B - Novel asparaginase and uses thereof

Info

Publication number: CN101663395B
Application number: CN2008800127838A
Authority: CN
Inventors: 简·米特斯卡·拉恩·范德; 马克·克里斯蒂安·斯托; 艾瑟·兰格·德; 利瑟特·默赫曼恩
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2007-04-20
Filing date: 2008-04-17
Publication date: 2012-09-05
Anticipated expiration: 2028-04-17
Also published as: CN101663396B; CN101663395A; CN101663396A

Abstract

The present invention relates to asparaginase having a breadth of pH activity range of at least 3.5. Furthermore, the present invention relates to newly identified nucleic acids according to SEQ ID NO: 2 or SEQ ID NO: 4 and variants thereof and polynucleotide sequences encoding such novel asparaginase variants. Furthermore, the present invention relates to the use of these novel asparaginase variants in industrial processes.

Description

Novel asparaginase and uses thereof

Technical field

The present invention relates to new asparaginase polypeptide variants that comes out and the polynucleotide sequence that comprises the gene of these novel asparaginases of encoding identified.The present invention relates to the aminoacid sequence of total length functional protein and the function equivalent of gene or aminoacid sequence.The invention still further relates to the method for in industrial technology, using these misfolded proteins.The present invention also comprise transformed through polynucleotide according to the present invention, be suitable for producing these proteic cells and wherein according to the present invention albumen carried out genetic modification to strengthen or to reduce its expression level and/or active cell.The invention still further relates to and in industrial technology, use these proteic methods.

< > Background of invention <>

Recently, acrylamide is disclosed at a lot of appearance (Tareke et al.Chem.Res.Toxicol.13 in the heat food product, 517-522(2000)).Be considered to maybe be carcinogenic to the animal and human in view of acrylamide, and this discovery causes having produced worldwide concern.Further research discloses, and through curing, can detect the acrylamide of suitable content in the common food of frying and baking box preparation, and shows that the appearance of acrylamide is the result of heating process in the food multiple.

Mottram et al.Nature 419:448(2002) result as Maillard reaction has been proposed from the approach of amino acid with reducing sugar formation acrylamide.According to this hypothesis, acrylamide can form between the reaction period at Maillard.Curing and toasting (roasting) during, color, aroma and flavor are responsible in the Maillard reaction.The reaction relevant with Maillard is amino acid whose Strecker degraded, proposed to form the approach of acrylamide.When temperature surpasses 120 ℃, can detect the formation of acrylamide, about 170 ℃, observed the highest formation speed.When having l-asparagine and glucose, can be observed the highest levels of acrylamide, and glutamine and aspartic acid only cause trace.

Official's migration restriction that the plastics that contact from food to acrylamide among the EU move the feed thing is set to every kg 10 micrograms of 10ppb().Though the acrylamide that forms during the culinary art still is not provided with official's restriction, a lot of products (especially cereals, bread product and based on the product of potato or corn) have caused people's attention above this fact of this value.

Known some material of vegetable origins contain the l-asparagine of higher level.In potato; L-asparagine is to account for main total free aminoacids (940mg/kg; 40%) of corresponding total amino acid content; In whole meal flour; L-asparagine exists with the level of about 167mg/kg; Corresponding to the 14%(Belitz and Grosch in Food Chemistry-Springer New York of total free amino acid, 999).Acrylamide mainly forms from l-asparagine (combination reducing sugar), the high level of acrylamide in the plant prod of this true soluble warp frying, baking box culinary art or baking.Therefore, with regard to the interest of public health aspect, need have much lower levels of acrylamide in a hurry, perhaps preferred foodstuff products completely without acrylamide.

The solution of multiple minimizing acrylamide content has been proposed; Perhaps through changing processing variable; For example, the temperature of heating steps or time length, perhaps prevent that through chemical mode or enzymatic mode acrylamide formation from perhaps realizing through the acrylamide of removing formation.

In some patent applications, disclose asparaginase and be used to the purposes that reduces the l-asparagine level and reduce the amount of the acrylamide that forms thus.Produced the suitable asparaginase that is used for this purpose from some kinds of originated from fungus, for example, from Aspergillus niger(WO2004/030468) and Aspergillus oryzae(WO04/032648 in).

Though all same chemical conversions of catalysis of all altheine enzymes, this does not also mean that they are suitable for same application.Multiple different application has proposed different requirement to the operated prerequisite of enzyme.Physics and the chemical parameters that possibly have influence on Enzymatic transformation speed is the concentration etc. of existence, substrate and product of activator or suppressor factor of structural integration, the enzyme of temperature (chemical reaction rate is had favourable influence, but possibly negative impact arranged to enzyme stability), moisture content, pH, salt concn, food matrix.

Therefore, people are just needing improved asparaginase, are used to have the multiple application that improves character.

< > Goal of the invention <>

An object of the present invention is to provide the polynucleotide of novel asparaginase variant polypeptide and this type of variant of coding.Another purpose provides the recombinant bacterial strain that produces this type of l-asparagine enzyme variants.Fusion polypeptide, manufacturing and use also are parts of the present invention according to the method for polynucleotide of the present invention and polypeptide.

Summary of the invention

The invention provides the polypeptide variants of novelty with asparaginase activity.Especially, the invention provides variant asparaginase with aminoacid sequence shown in any among SEQ ID NO:2 or the SEQ ID NO:4 and with they at least one its further variant with at least 85% homology.Typically, this type of further variant of the present invention will be the function equivalent of polypeptide any one among SEQ ID NO:2 or the SEQ ID NO:4.Term " function equivalent " is represented to require this type of function equivalent polypeptide must have asparaginase activity at least in this article.

The present invention also provides has at least 3.5, and preferably at least 4, the more preferably asparaginase of the wide pH field of activity width of at least 5 pH units.In context of the present invention, pH field of activity width is the width (calculating with pH unit) of enzyme 50 to 100% the pH scope that shows its maximum activity.The pH field of activity width of enzyme can calculate according to shown in the embodiment.Having the asparaginase of at least 3.5 pH field of activity width separable can be artificial asparaginase from natural origin or its.A kind of preferred embodiment in; Have at least 3.5; Preferably at least 4, more preferably the asparaginase of the wide pH field of activity width of at least 5 pH units be according to the variant asparaginase with aminoacid sequence shown in any among SEQ IDNO:2 or the SEQ ID NO:4 and with they at least one its further variant with at least 85% homology.

In one embodiment of the invention; The invention provides the separated polynucleotide that comprise following nucleotide sequence; Said nucleotide sequence codedly have the polypeptide of the aminoacid sequence shown in SEQ ID NO:2 or the SEQ ID NO:4 or any variant in them, for example, and function equivalent.

In another embodiment, the invention provides separated polynucleotide, its coding is according at least one functional domain of polypeptide any among SEQ ID NO:2 or the SEQ ID NO:4 or any variant in them, for example, and function equivalent.

In addition, the invention provides novel polynucleotide, its coding is according to the polypeptide variants of novelty of the present invention.

In addition, the invention provides the nucleotide sequence of the following asparaginase of coding, said asparaginase has at least 3.5, and preferably at least 4, more preferably at least 5 pH field of activity width that pH unit is wide.

The invention still further relates to and comprise according to the carrier of polynucleotide sequence of the present invention and can be used for increasing or detecting primer, probe and fragment according to polynucleotide of the present invention.

Another preferred embodiment in; Following carrier is provided; Wherein, according to polynucleotide sequence of the present invention and the functional connection of at least a regulating and controlling sequence that is suitable in proper host cell (for example filamentous fungus, for example Aspergillus), expressing the aminoacid sequence that is encoded.The present invention also provides the method for preparation according to polynucleotide of the present invention and carrier.

The invention still further relates to and contain with good grounds variant of the present invention (allos) host cell polynucleotide, recombinant production.

In another embodiment, the invention provides following recombinant host cell, wherein, significantly increase according to the expression of variant asparaginase of the present invention, perhaps, wherein, the activity of the asparaginase of generation increases.

In another embodiment; The invention provides the host cell of following recombinant production; Said cell contains with good grounds polynucleotide of the present invention; Therefore, wherein, said cell can produce according to functional variant asparaginase of the present invention; Preferably; The cell of ability overexpression according to variant asparaginase of the present invention is provided, for example, comprised Aspergillus niger bacterial strain according to polynucleotide of the present invention.

Aspect another, purified variant polypeptide is provided of the present invention.Polypeptide according to the present invention comprises the polypeptide according to polynucleotide encoding of the present invention.Especially preferred is according to variant polypeptide any among SEQ IDNO:2 or the SEQ ID NO:4 or any variant in them, for example, and function equivalent.

Comprise also within the scope of the invention according to the fusion rotein of polypeptide of the present invention.

The present invention also provides the method according to variant polypeptide of the present invention of making.

The invention still further relates to the purposes of variant asparaginase according to the present invention in any industrial technology as described herein.

< > Accompanying drawing is described <>

The comparison of the disclosed A.niger wild-type of the SEQ IDNO:3 asparaginase among figure 1SEQ ID NO:2 and SEQ ID NO:4 and the WO2004/030468.

< > Detailed Description Of The Invention <>

Specification sheets of the present invention and accompanying claims in full in, " comprising " and " comprising " with and various part of speech form all represent to be included.That is possibly comprise other element or the integer of specifically not mentioning when, these words are used to be illustrated in the situation permission.

The present invention relates to have variant l-asparagine enzyme sequence or its further variant of the sequence shown in any among SEQ ID NO:2 or the SEQ ID NO:4.

Be shown specifically some concrete peptide sequences below.

The sequence of SEQ ID NO:2 comprises following replacement :T25A and 377V than the WO 2004/030468 disclosed wild-type l-asparagine enzyme sequence that obtains from Aspergillus niger.

The WO 2004/030468 disclosed wild-type l-asparagine enzyme sequence that obtains from A.niger is disclosed as SEQ ID NO:3(aminoacid sequence therein).Asparaginase shown in the application's the SEQ ID NO:2 temporarily is called ASN001.

The variant of the sequence shown in the SEQ ID NO:2; For example; Function equivalent; Can comprise the 25th Ala; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; One or more among the 375th Val or the 377th 's the Val, said position defines with reference to SEQ ID NO:2.

That is, during sequence alignment shown in the variant of the sequence shown in the SEQ ID NO:2 and the SEQ ID NO:2, this variant can have following in a place or many places:

(in the variant) locational Ala of the 25th corresponding to SEQ ID NO:2;

(in the variant) locational Thr of the 28th corresponding to SEQ ID NO:2;

(in the variant) locational Tyr of the 30th corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 35th corresponding to SEQ ID NO:2;

(in the variant) locational Leu of the 53rd corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 63rd corresponding to SEQ ID NO:2;

(in the variant) locational Ala of the 64th corresponding to SEQ ID NO:2;

(in the variant) locational Asp of the 65th corresponding to SEQ ID NO:2;

(in the variant) locational Asn of the 66th corresponding to SEQ ID NO:2;

(in the variant) locational Ala of the 74th corresponding to SEQ ID NO:2;

(in the variant) locational Ile of the 77th corresponding to SEQ ID NO:2;

(in the variant) locational Ile of the 79th corresponding to SEQ ID NO:2;

(in the variant) locational Gln of the 80th corresponding to SEQ ID NO:2;

(in the variant) locational Thr of the 81st corresponding to SEQ ID NO:2;

(in the variant) locational Glu of the 88th corresponding to SEQ ID NO:2;

(in the variant) locational Pro of the 106th corresponding to SEQ ID NO:2;

(in the variant) locational Val of the 108th corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 111st corresponding to SEQ ID NO:2;

(in the variant) locational Leu of the 114th corresponding to SEQ ID NO:2;

(in the variant) locational Ala of the 117th corresponding to SEQ ID NO:2;

(in the variant) locational Thr of the 119th corresponding to SEQ ID NO:2;

(in the variant) locational Glu of the 122nd corresponding to SEQ ID NO:2;

(in the variant) locational Asp of the 126th corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 131st corresponding to SEQ ID NO:2;

(in the variant) locational Val of the 161st corresponding to SEQ ID NO:2;

(in the variant) locational Ala of the 168th corresponding to SEQ ID NO:2;

(in the variant) locational Gln of the 181st corresponding to SEQ ID NO:2;

(in the variant) locational Pro of the 189th corresponding to SEQ ID NO:2;

(in the variant) locational Ala of the 190th corresponding to SEQ ID NO:2;

(in the variant) locational Leu of the 197th corresponding to SEQ ID NO:2;

(in the variant) locational Val of the 205th corresponding to SEQ ID NO:2;

(in the variant) locational Phe of the 208th corresponding to SEQ ID NO:2;

(in the variant) locational Ala of the 210th corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 211st corresponding to SEQ ID NO:2;

(in the variant) locational Val of the 224th corresponding to SEQ ID NO:2;

(in the variant) locational Asn of the 228th corresponding to SEQ ID NO:2;

(in the variant) locational Ala of the 231st corresponding to SEQ ID NO:2;

(in the variant) locational Ile of the 232nd corresponding to SEQ ID NO:2;

(in the variant) locational Val of the 233rd corresponding to SEQ ID NO:2;

(in the variant) locational Lys of the 236th corresponding to SEQ ID NO:2;

(in the variant) locational Tyr of the 238th corresponding to SEQ ID NO:2;

(in the variant) locational Tyr of the 240th corresponding to SEQ ID NO:2;

(in the variant) locational Thr of the 250th corresponding to SEQ ID NO:2;

(in the variant) locational Thr of the 251st corresponding to SEQ ID NO:2;

(in the variant) locational Thr of the 252nd corresponding to SEQ ID NO:2;

(in the variant) locational Val of the 254th corresponding to SEQ ID NO:2;

(in the variant) locational Arg of the 255th corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 259th corresponding to SEQ ID NO:2;

(in the variant) locational Tyr of the 267th corresponding to SEQ ID NO:2;

(in the variant) locational Gln of the 270th corresponding to SEQ ID NO:2;

(in the variant) locational Gln of the 273rd corresponding to SEQ ID NO:2;

(in the variant) locational Asp of the 279th corresponding to SEQ ID NO:2;

(in the variant) locational Asp of the 282nd corresponding to SEQ ID NO:2;

(in the variant) locational Asn of the 283rd corresponding to SEQ ID NO:2;

(in the variant) locational Lys of the 286th corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 293rd corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 297th corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 299th corresponding to SEQ ID NO:2;

(in the variant) locational Gly of the 301st corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 303rd corresponding to SEQ ID NO:2;

(in the variant) locational Asp of the 304th corresponding to SEQ ID NO:2;

(in the variant) locational Asp of the 307th corresponding to SEQ ID NO:2;

(in the variant) locational Ile of the 309th corresponding to SEQ ID NO:2;

(in the variant) locational Ala of the 310th corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 311st corresponding to SEQ ID NO:2;

(in the variant) locational Thr of the 312nd corresponding to SEQ ID NO:2;

(in the variant) locational His of the 313rd corresponding to SEQ ID NO:2;

(in the variant) locational Ser of the 314th corresponding to SEQ ID NO:2;

(in the variant) locational Ile of the 317th corresponding to SEQ ID NO:2;

(in the variant) locational Leu of the 319th corresponding to SEQ ID NO:2;

(in the variant) locational Thr of the 321st corresponding to SEQ ID NO:2;

(in the variant) locational Gly of the 324th corresponding to SEQ ID NO:2;

(in the variant) locational Pro of the 330th corresponding to SEQ ID NO:2;

(in the variant) locational Phe of the 345th corresponding to SEQ ID NO:2;

(in the variant) locational Ala of the 351st corresponding to SEQ ID NO:2;

(in the variant) locational Ala of the 360th corresponding to SEQ ID NO:2;

(in the variant) locational Glu of the 361st corresponding to SEQ ID NO:2;

(in the variant) locational Gly of the 364th corresponding to SEQ ID NO:2;

(in the variant) locational Phe of the 365th corresponding to SEQ ID NO:2;

(in the variant) locational Lys of the 366th corresponding to SEQ ID NO:2;

(in the variant) locational Arg of the 369th corresponding to SEQ ID NO:2;

(in the variant) locational Glu of the 370th corresponding to SEQ ID NO:2;

(in the variant) locational Lys of the 374th corresponding to SEQ ID NO:2;

(in the variant) locational Val of the 375th corresponding to SEQ ID NO:2; Or

(in the variant) locational Val of the 377th corresponding to SEQ ID NO:2;

Variant l-asparagine enzyme polypeptide of the present invention can comprise two in the following amino acid; Three; Four; Five; For example; At least 10; At least 15; At least 20; For example at least 25; At least 30; At least 40; At least 50; At least 60; At least 70; At least 80 or whole: the 25th Ala; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; The 375th Val or the 377th 's Val, said position defines with reference to SEQ ID NO:2.

Therefore; Variant l-asparagine enzyme polypeptide of the present invention comprises the 25th Ala; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; Two or more any combination among the 375th Val or the 377th 's the Val, said position defines with reference to SEQ ID NO:2.

Preferably; Variant l-asparagine enzyme polypeptide of the present invention comprises the 25th Ala; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; Whole among the 375th Val or the 377th 's the Val, said position defines with reference to SEQ ID NO:2.

These positions corresponding to the 25th and 377 in the variant l-asparagine polypeptide of the present invention can be identified like this: compare through for example using the GAP with the homologous sequence of finding through GAP program (details about this program sees below), the sequence of variant polypeptide and the sequence of SEQ ID NO:2 are compared.The 25th and 377 bit positions corresponding to SEQ ID NO:2 in the variant can be come out by evaluation thus, and according to calling with reference to defined those positions of SEQ ID NO:2.

The sequence of SEQ ID NO:4 comprises following replacement :A35S and 377V than the WO 2004/030468 disclosed wild-type l-asparagine enzyme sequence that obtains from Aspergillus niger.

The sequence of ID NO:4 comprises aminoacid deletion everywhere than the disclosed wild-type l-asparagine enzyme sequence that obtains from Aspergillus niger of WO 2004/030468 in addition: promptly, the disclosed SEQ ID of this paper NO:4 does not comprise the 2004/030468 disclosed wild-type l-asparagine enzyme sequence (among WO 2004/030468, being shown SEQ ID NO:3(aminoacid sequence) that obtains from Aspergillus niger corresponding to WO) in the amino acid of D336, T337, A338 and T339.The asparaginase shown in the SEQ ID NO:4 temporarily is called as ASN002 among the application.

The variant of the sequence shown in the SEQ ID NO:4; For example; Function equivalent; Can comprise the 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; One or more among the 372nd Gly or the 373rd 's the Val, said position defines with reference to SEQ ID NO:4.

That is, during sequence alignment shown in the variant of the sequence shown in the SEQ ID NO:4 and the SEQ ID NO:4, variant can have following in a place or many places:

(in the variant) locational Ser of the 35th corresponding to SEQ ID NO:4;

(in the variant) locational Leu of the 53rd corresponding to SEQ ID NO:4;

(in the variant) locational Ser of the 63rd corresponding to SEQ ID NO:4;

(in the variant) locational Ala of the 64th corresponding to SEQ ID NO:4;

(in the variant) locational Asp of the 65th corresponding to SEQ ID NO:4;

(in the variant) locational Asn of the 66th corresponding to SEQ ID NO:4;

(in the variant) locational Ala of the 74th corresponding to SEQ ID NO:4;

(in the variant) locational Ile of the 77th corresponding to SEQ ID NO:4;

(in the variant) locational Ile of the 79th corresponding to SEQ ID NO:4;

(in the variant) locational Gln of the 80th corresponding to SEQ ID NO:4;

(in the variant) locational Thr of the 81st corresponding to SEQ ID NO:4;

(in the variant) locational Glu of the 88th corresponding to SEQ ID NO:4;

(in the variant) locational Pro of the 106th corresponding to SEQ ID NO:4;

(in the variant) locational Val of the 108th corresponding to SEQ ID NO:4;

(in the variant) locational Ser of the 111st corresponding to SEQ ID NO:4;

(in the variant) locational Leu of the 114th corresponding to SEQ ID NO:4;

(in the variant) locational Ala of the 117th corresponding to SEQ ID NO:4;

(in the variant) locational Thr of the 119th corresponding to SEQ ID NO:4;

(in the variant) locational Glu of the 122nd corresponding to SEQ ID NO:4;

(in the variant) locational Asp of the 126th corresponding to SEQ ID NO:4;

(in the variant) locational Val of the 161st corresponding to SEQ ID NO:4;

(in the variant) locational Ala of the 168th corresponding to SEQ ID NO:4;

(in the variant) locational Gln of the 181st corresponding to SEQ ID NO:4;

(in the variant) locational Pro of the 189th corresponding to SEQ ID NO:4;

(in the variant) locational Ala of the 190th corresponding to SEQ ID NO:4;

(in the variant) locational Leu of the 197th corresponding to SEQ ID NO:4;

(in the variant) locational Val of the 205th corresponding to SEQ ID NO:4;

(in the variant) locational Phe of the 208th corresponding to SEQ ID NO:4;

(in the variant) locational Ser of the 211st corresponding to SEQ ID NO:4;

(in the variant) locational Val of the 224th corresponding to SEQ ID NO:4;

(in the variant) locational Asn of the 228th corresponding to SEQ ID NO:4;

(in the variant) locational Ala of the 231st corresponding to SEQ ID NO:4;

(in the variant) locational Ile of the 232nd corresponding to SEQ ID NO:4;

(in the variant) locational Val of the 233rd corresponding to SEQ ID NO:4;

(in the variant) locational Lys of the 236th corresponding to SEQ ID NO:4;

(in the variant) locational Tyr of the 238th corresponding to SEQ ID NO:4;

(in the variant) locational Thr of the 250th corresponding to SEQ ID NO:4;

(in the variant) locational Thr of the 251st corresponding to SEQ ID NO:4;

(in the variant) locational Val of the 254th corresponding to SEQ ID NO:4;

(in the variant) locational Arg of the 255th corresponding to SEQ ID NO:4;

(in the variant) locational Ser of the 259th corresponding to SEQ ID NO:4;

(in the variant) locational Tyr of the 267th corresponding to SEQ ID NO:4;

(in the variant) locational Gln of the 270th corresponding to SEQ ID NO:4;

(in the variant) locational Asp of the 279th corresponding to SEQ ID NO:4;

(in the variant) locational Asp of the 282nd corresponding to SEQ ID NO:4;

(in the variant) locational Asn of the 283rd corresponding to SEQ ID NO:4;

(in the variant) locational Lys of the 286th corresponding to SEQ ID NO:4;

(in the variant) locational Ser of the 293rd corresponding to SEQ ID NO:4;

(in the variant) locational Ser of the 297th corresponding to SEQ ID NO:4;

(in the variant) locational Ser of the 299th corresponding to SEQ ID NO:4;

(in the variant) locational Ser of the 300th corresponding to SEQ ID NO:4;

(in the variant) locational Gly of the 301st corresponding to SEQ ID NO:4;

(in the variant) locational Ser of the 303rd corresponding to SEQ ID NO:4;

(in the variant) locational Asp of the 304th corresponding to SEQ ID NO:4;

(in the variant) locational Ile of the 309th corresponding to SEQ ID NO:4;

(in the variant) locational Ser of the 311st corresponding to SEQ ID NO:4;

(in the variant) locational Thr of the 312nd corresponding to SEQ ID NO:4;

(in the variant) locational His of the 313rd corresponding to SEQ ID NO:4;

(in the variant) locational Ser of the 314th corresponding to SEQ ID NO:4;

(in the variant) locational Ile of the 317th corresponding to SEQ ID NO:4;

(in the variant) locational Thr of the 321st corresponding to SEQ ID NO:4;

(in the variant) locational Gly of the 324th corresponding to SEQ ID NO:4;

(in the variant) locational Pro of the 330th corresponding to SEQ ID NO:4;

(in the variant) locational Glu of the 333rd corresponding to SEQ ID NO:4;

(in the variant) locational Gln of the 336th corresponding to SEQ ID NO:4;

(in the variant) locational Phe of the 341st corresponding to SEQ ID NO:4;

(in the variant) locational Ala of the 356th corresponding to SEQ ID NO:4;

(in the variant) locational Gly of the 360th corresponding to SEQ ID NO:4;

(in the variant) locational Glu of the 362nd corresponding to SEQ ID NO:4;

(in the variant) locational Arg of the 365th corresponding to SEQ ID NO:4;

(in the variant) locational Glu of the 366th corresponding to SEQ ID NO:4;

(in the variant) locational Lys of the 370th corresponding to SEQ ID NO:4;

(in the variant) locational Val of the 371st corresponding to SEQ ID NO:4;

(in the variant) locational Gly of the 372nd corresponding to SEQ ID NO:4; Or

(in the variant) locational Val of the 373rd corresponding to SEQ ID NO:4.

Variant l-asparagine enzyme polypeptide of the present invention can comprise two in the following amino acid; Three; Four; Five; For example; At least 10; At least 15; At least 20; For example at least 25; At least 30; At least 40; At least 50; At least 60; At least 70 or whole: the 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; One or more among the 372nd Gly or the 373rd 's the Val, said position defines with reference to SEQ ID NO:4.

Therefore; Variant l-asparagine enzyme polypeptide of the present invention comprises the 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; Two or more any combination among the 372nd Gly or the 373rd 's the Val, said position defines with reference to SEQ ID NO:4.

Preferably; Variant l-asparagine enzyme polypeptide of the present invention comprises the 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; Whole among the 372nd Gly or the 373rd 's the Val, said position defines with reference to SEQ ID NO:4.

These positions corresponding to the 35th and 373 in the variant l-asparagine polypeptide of the present invention can be identified like this: compare through for example using the GAP with the homologous sequence of finding through GAP program (details about this program sees below), the sequence of variant polypeptide and the sequence of SEQ ID NO:4 are compared.The 35th and 373 bit positions corresponding to SEQ ID NO:4 in the variant can be come out by evaluation thus, and according to calling with reference to defined those positions of SEQID NO:4.

In addition; Variant l-asparagine enzyme polypeptide of the present invention does not typically comprise one or more corresponding in the amino acid of disclosed Asp 336, Thr 337, Ala 338 and the Thr339 from the wild-type l-asparagine enzyme sequence that Aspergillus niger obtains of WO2004/030468; For example, two, three or whole.Promptly; Disclosed during from wild-type asparaginase sequence alignment that Aspergillus niger obtains as variant l-asparagine enzyme polypeptide of the present invention and WO2004/030468, it will typically not comprise one or more corresponding in the amino group of amino acids acid of the 336th, 337,338 and 339 discovery of wild-type A.niger sequence.

In another embodiment, the present invention also provides has at least 3.5, and preferably at least 4, the more preferably asparaginase of the wide pH field of activity width of at least 5 pH units.A kind of preferred embodiment in; Have at least 3.5; Preferably at least 4; More preferably the asparaginase of the wide pH field of activity width of at least 5 pH units is according to the asparaginase with variant asparaginase of the aminoacid sequence shown in any among SEQ ID NO:2 or the SEQ ID NO:4, and with they at least one its further variant with at least 85% homology.

< > Polynucleotide <>

Shown in above, the invention provides coding and have the polynucleotide of the variant asparaginase of the aminoacid sequence shown in any among SEQ ID NO:2 or the SEQ ID NO:4 and other variant thereof, for example, function equivalent.Therefore, the invention provides according to polynucleotide any among SEQ ID NO:1 or the SEQ ID NO:3 and other variant polynucleotide thereof.

For clarity sake, SEQ ID NO:1 is the polynucleotide sequence of coding according to the polypeptide of SEQ ID NO:2; SEQ ID NO:3 is the polynucleotide sequence of coding according to the polypeptide of SEQ ID NO:4.

The invention provides and comprise polynucleotide sequence and the encoding sequence thereof of coding according to the gene of asparaginase of the present invention.Therefore, the present invention relates to nucleotide sequence, it comprises:

A)DNA sequence, it is encoded according to any one asparaginase among SEQ ID NO:2 or the SEQ ID NO:4;

B)DNA sequence, its coding and the asparaginase that has at least 85% homology according to any one polypeptide among SEQ ID NO:2 or the SEQ ID NO:4;

C)DNA sequence, its with according to a) or b) dna sequence dna have at least 80% homology;

D)DNA sequence, its under the tight degree of height with according to a) or b) the complementary strand hybridization of dna sequence dna;

E) have at least 100 Nucleotide according to a), b), c) or the subsequence of d) dna sequence dna; Or

F) according to a), b), c), d) or the complementary strand of e) dna sequence dna.

The invention still further relates to the separated polynucleotide of coding according at least one functional domain of polypeptide any among SEQ ID NO:2 or the SEQ ID NO:4.To polypeptide according to the present invention this is made a more detailed description below.

Typically; For SEQ ID NO:2; This type of structural domain will comprise the Ala corresponding to the 25th; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; One or more one or more amino acid among the 375th Val or the 377th 's the Val, said position defines with reference to SEQ ID NO:2.

In one embodiment of the invention; The following polypeptide of nucleic acid sequence encoding according to the present invention; Wherein, Said polypeptide is the variant that comprises following aminoacid sequence, and said aminoacid sequence has a place or many places brachymemma and/or at least one place aminoacid replacement, disappearance and/or insertion than the sequence of the 1st to 378 amino acids of SEQID NO:2.This type of polypeptide will typically comprise the 25th Ala; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; One or more among the 375th Val or the 377th 's the Val, said position defines with reference to SEQ ID NO:2.

Typically; For SEQ ID NO:4; This type of structural domain will comprise the Ser corresponding to the 35th; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; One or more one or more amino acid among the 372nd Gly or the 373rd 's the Val, said position defines with reference to SEQID NO:4.

In one embodiment of the invention; The following polypeptide of nucleic acid sequence encoding according to the present invention; Wherein, Said polypeptide is the variant that comprises following aminoacid sequence, and said aminoacid sequence has a place or many places brachymemma and/or at least one place aminoacid replacement, disappearance and/or insertion than the sequence of the 1st to 374 amino acids of SEQID NO:4.This type of polypeptide will typically comprise the 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; One or more among the 372nd Gly or the 373rd 's the Val, said position defines with reference to SEQ ID NO:4.

In another embodiment, the invention provides coding and having at least 3.5, preferably at least 4, the more preferably nucleic acid molecule of the asparaginase of the wide pH field of activity width of at least 5 pH units.A kind of preferred embodiment in, the said nucleic acid molecule that coding has the asparaginase of at least 3.5 pH field of activity width is such nucleic acid, it comprises:

When using in this article, term " gene " and " recombination " refer to following nucleic acid molecule, and it comprises the opening code-reading frame of the variant asparaginase as herein described of encoding, for example, and the variant of wild-type Aspergillus niger asparaginase.Gene can comprise encoding sequence, non-coding sequence, intron and regulating and controlling sequence.That is, when using among this paper, " gene " can be represented like separated nucleic acid molecule defined herein.Therefore, term " gene " not only refers to naturally occurring sequence in the application's context.

Can use to well known to a person skilled in the art standard molecular biological technique, the sequence information that combination this paper provides produces nucleic acid molecule of the present invention.

For example, can use the standard synthetic technology, the nucleic acid molecule that de novo synthesis needs.This type of synthesis technique typically will be an automatic process.This type of technology is well known to a person skilled in the art.

Perhaps, can produce nucleic acid molecule of the present invention through site-directed mutagenesis to existing nucleic acid molecule (for example wild-type nucleic acid molecule).Can use and well known to a person skilled in the art that multiple technologies carry out site-directed mutagenesis.

In a kind of these class methods, only for example, use the oligonucleotide " primer " that contains the sudden change of wanting, at the enterprising performing PCR of plasmid template.In view of primer is the end of new synthetic chain, in first cycle period of synthetic template DNA mispairing will be arranged, after the first round, will equate with original template concentrations based on the chain (containing sudden change) of primer.After the follow-up circulation, it after 25 circulations, will surpass the original not chain of sudden change with exponential growth in the interval of 800 ten thousand :1, be close to uniform amplified fragments through sudden change thereby produce.Can pass through enzymatic digestion, for example use the Restriction Enzyme (for example pn1) that only cuts methylated DNA, remove template DNA.Coming from alkaline bleach liquor cleavage plasmid prepared product and also be destroyed in this step by methylated template thus, but be able to keep through the plasmid of sudden change, because it is external generation, so is unmethylated.

In these class methods, the sudden change above a place can be introduced, in single PCR reaction in nucleic acid molecule for example through using every kind of one or more oligonucleotide that comprise one or more mispairing to realize.Perhaps, can react the sudden change of in nucleic acid molecule, introducing above a place through surpassing a PCR, the each reaction introduced a place or many places sudden change, the feasible nucleic acid of in nucleic acid, introducing change with mode order, repeatedly.

Can use cDNA, mRNA or genomic dna as template, use suitable mispairing Oligonucleolide primers,, produce nucleic acid of the present invention according to side-directed mutagenesis mentioned above.The nucleic acid molecule of Huo Deing can be advanced suitable carriers by the clone by this way, and can come it is characterized through dna sequence analysis.

Than the wild-type sequence of the disclosed wild-type l-asparagine enzyme sequence that obtains from Aspergillus niger of WO 2004/030468 and/or than SEQ ID NO:2 or the SEQ ID NO:4 the sequence shown in any; Nucleic acid molecule of the present invention can comprise a place or many places disappearance; That is breach.Also can use suitable oligonucleotide, utilize site-directed mutagenesis, produce this type of disappearance/breach.The technology that is used to produce this type of disappearance is well known to a person skilled in the art.

In addition, can pass through the standard synthetic technology, for example, use automatic dna synthesizer, prepare corresponding to Nucleotide according to the present invention or can with the oligonucleotide of Nucleotide hybridization according to the present invention.

In addition, the present invention includes the complementary nucleic acid sequence.With another nucleic acid molecule complementary nucleic acid molecule be and the abundant complementary of another nucleotide sequence, thereby, its can with another nucleotide sequence hybridization, form stable disome thus.

One aspect of the present invention (for example relates to coding polypeptide of the present invention or its function equivalent; Bioactive fragment or structural domain) separated nucleic acid molecule; And be enough to be used as the nucleic acid molecule of hybridization probe with the nucleic acid molecule of identification code polypeptide of the present invention; Be the fragment of PCR primer with being suitable for use as with this type of nucleic acid molecule of amplification or mutant nucleic acid molecule (for example, being used to prepare nucleic acid molecule of the present invention).

" separated polynucleotide " or " separated nucleic acid " are such DNA or RNA, one of two (5 ' end of DNA or RNA direct neighbor described in the naturally occurring genome of the organism that itself and said DNA or RNA came from 3 ' of holding) encoding sequence direct neighbor not.Therefore, in one embodiment, separated nucleic acid comprises with the part of 5 ' the non-coding (for example, the promotor) sequence of encoding sequence direct neighbor or all.Therefore this term comprises; For example; Be inserted into the reorganization DNA in the carrier; Be inserted into the reorganization DNA in autonomously replicating plasmid or the virus; Or be inserted into the reorganization DNA in prokaryotic organism or the Eukaryotic genomic dna; Or the recombinant DNA that exists as the independent molecule that does not rely on other sequence (for example, handling cDNA or the genomic DNA fragment that produces) through PCR or restriction enzyme.This term also comprises the ene as hybrid gene (hybrid) a part of reorganization DNA; The other polypeptide of said hybrid gene coding; Described polypeptide has been removed cellular material, viral material or substratum (when producing through recombinant DNA technology) basically, or precursor or other chemical preparations (when chemosynthesis).In addition, " through the nucleic acid fragment that leaves " is not and is natural just to exist as fragment, and it can not be found in state of nature.

When using in this article, term " polynucleotide " or " nucleic acid molecule " are intended to comprise dna molecular (for example cDNA and genome DNA) and RNA molecule (for example mRNA) and DNA or the RNA analogue produced with nucleotide analog.Nucleic acid molecule can be strand or double-stranded, but preferably double-stranded DNA.Can use oligonucleotide analogs or derivative (Trophicardyl or thiophosphoric acid Nucleotide) to come nucleic acid.For example, this class oligonucleotide can be used to prepare the nucleic acid that the base pairing ability changes to some extent or the nuclease resistance is increased.

Another embodiment of the invention provides through isolated nucleic acid molecule, and its antisense for example, has the coding strand of the nucleic acid molecule of the sequence shown in any among SEQ ID NO:1 or the SEQ ID NO:3 in nucleic acid molecule of the present invention.The complementary strand that also comprises the nucleic acid molecule of describing among this paper in the scope of the present invention.

< > Identity and homology <>

Term " homology " or " per-cent identity " are used interchangeably in this article.With regard to the object of the invention; In this definition: be the per-cent identity of confirming two aminoacid sequences or two nucleotide sequences; In line with optimum purpose relatively (for example; Can list the introducing breach at article one aminoacid sequence or nucleotides sequence; To reach best comparison with second aminoacid sequence or nucleotide sequence), said sequence is compared.Then amino-acid residue on corresponding amino acid position or the nucleotide position or Nucleotide are compared.Identical amino acid or the nucleotide residue in corresponding position occupies in the position quilt on article one sequence and second sequence, and these two molecules are identical in this position so.Per-cent identity between two sequences is the function (that is the total x100 in the quantity/position of % identity=same position (being the lap position)) of the quantity of the total same position of said sequence.Preferably, two sequences length is identical.

Can or carry out sequence on quilt two sequences total length relatively on the two sequences fragment relatively.Typically, on by the total length of two sequences relatively, compare.But, can on the zone of for example 20,50,100 or more a plurality of adjacent amino acids residues, confirm sequence identity.

The technician will know have some computer programs can be used to confirm the homology between two sequences.For example, can use mathematical algorithm to accomplish to the comparison of sequence and confirming to per-cent identity between two sequences.A kind of preferred embodiment in, use Needleman and Wunsch(J.Mol.Biol.(48):444-453(1970)) algorithm confirms two per-cent identity between aminoacid sequence, said algorithm be added into the GCG software package (can from < > Ttp ://www.gcg.com <> Acquisition) in the GAP program, wherein use Blossom 62 matrixes or PAM250 matrix, the breach weight is 16,14,12,10,8,6 or 4, and the length weight is 1,2,3,4,5 or 6.The technician will know that all these different parameters will cause having the result of technicality, but the overall percentage identity of two sequences does not have remarkable change when being to use algorithms of different.

Nucleic acid of the present invention and protein sequence also can be used as " search sequence " and carry out the search to public's database, for example, and to identify other member in correlated series or the family.Can use Altschul, the BLASTN of et al.(1990)J.Mol.Biol.215:403-10 and BLASTX program (2.0 version) carry out this type of search.Can use the BLASTP program, with mark=50, word length=3 carries out this type of BLAST albumen search, to obtain and protein molecular homologous aminoacid sequence of the present invention.In line with purpose relatively, for obtaining comparison jaggy, can utilize Altschul et al., the Gapped BLAST that describes among the (1997)Nucleic Acids Res.25(17):3389-3402.When utilizing BLAST and Gapped blast program, can use the default parameter of each program (for example BLASTP and BLASTN).See < > Ttp ://www.ncbi.nlm.nih.gov <>

< > Hybridization <>

Typically, the complementary sequence of sequence shown in any is hybridized with the level that is significantly higher than background among polynucleotide of the present invention and SEQ ID No:1 or the SEQ ID NO:3.Background hybridization possibly take place, for example, because other cDNA that exists in the cDNA library.The signal level that interact to produce between the complementary sequence of any one encoding sequence among polynucleotide of the present invention and SEQ ID No:1 or the SEQ ID NO:3 typically be between sequence any one among other polynucleotide and SEQ ID NO:1 or the SEQ IDNO:3 interactional at least 10 times by force; Preferably, at least 100 times strong.

The term of using among this paper " hybridization " is used to describe the condition of such hybridization and washing; Under the described conditions; About at least 20 of sequence to be compared; For example about at least 50; For example about at least 100; For example about at least 200; When more preferably on the zone of about at least 300 continuous nucleotides or more preferably measuring on the total length; Each other about at least 60%; At least about 70%; Be preferably about at least 80%; More preferably about at least 85%, further more preferably about at least 90%, for example about at least 95% homology; For example about at least each other 98% homology, for example about at least each other 99% homologous nucleotide sequence typically keeps the state of phase mutual cross.

A preferred unrestriced example of this type of hybridization conditions is about 45 ℃ of hybridization down in 6x sodium chloride/sodium citrate (SSC); Subsequently in 1x SSC, 0.1%SDS, carry out the one or many washing under 50 ℃, preferably under 55 ℃; Preferably under 60 ℃, further preferably under 65 ℃.

The height stringent condition comprises: for example, in 5x SSC/5x Denhardt ' s solution/1.0%SDS, hybridizing under 68 ℃, in 0.2x SSC/0.1%SDS, wash under the room temperature.Perhaps, washing can be 42 ℃ of expansion.

Those skilled in the art will know that to be suitable for using tight hybridization conditions and highly tight hybridization conditions under which kind of condition.Be easy to obtain other guidance in this area about this type of condition, for example, at Sambrooket al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, N.Y.; With Ausubel et al.(eds.), 1995, Current Protocols in MolecularBiology, (John Wiley&Sons, N.Y.) in.

Certainly; Only with poly A sequence (for example 3 ' of mRNA terminal poly(A) district) or only with T(or U) polynucleotide of complementary extension of section hybridization of residue will can not be included in the polynucleotide of the present invention that are used for a part of specific hybrid of nucleic acid of the present invention; Because these type of polynucleotide will be hybridized with any nucleic acid molecule or its complementary sequence (for example, being actually the cDNA clone of any two strands) that contains poly (A) extension of section.

Any combination of sequence identity mentioned above and minimum size can be used for defining polynucleotide of the present invention, and more tight combination (that is the higher sequence identity on length) is preferred.

< > Carrier <>

Another aspect of the present invention relates to carrier; Be preferably expression vector; Said carrier contains the nucleic acid of coding variant asparaginase of the present invention (for example, any one l-asparagine zymoprotein or any function equivalent in them among SEQ ID NO:2 or the SEQ ID NO:4).

When using in this article, term " carrier " refers to transport the nucleic acid molecule of another nucleic acid molecule that is connected thereto.A kind of bearer type is " plasmid ", and " plasmid " refers to cyclic double-stranded DNA ring, and other dna segment can be connected on the described ring.The type of another kind of carrier is a virus vector, and wherein, other dna segment can be connected in the viral genome.Some carrier can carry out self-replicating (bacteria carrier and the free Mammals carrier that for example, have the replication orgin of bacterium) in the host cell that it is introduced into.Some other carrier (for example, non-free Mammals carrier) just has been integrated into after being introduced into host cell in the genome of host cell, and they and host genome are together duplicated thus.In addition, some carrier can instruct the expression of gene that is operably connected on it.Examples of such carriers is called as " expression vector " in this article.Generally speaking, the expression vector of in recombinant DNA technology, using is the form of plasmid normally.Term " plasmid " and " carrier " can exchange use in this article, because plasmid is the most frequently used carrier format that arrives.Yet the present invention also will comprise the expression vector of other form, virus vector (for example, replication defect type retrovirus, adenovirus and adeno associated virus) for example, and said carrier can provide the function that is equal to.

Recombinant expression vector of the present invention comprises to be suitable for the nucleic acid of the present invention that form that said nucleic acid expresses exists in host cell; This means that said recombinant expression vector comprises one or more snippets regulating and controlling sequence; Regulating and controlling sequence is based on that the host cell that is used to express selects, and it is operably connected on the nucleotide sequence that will express.In recombinant expression vector; " be operably connected " and be used for expression: the interested nucleotide sequence of people is connected to regulating and controlling sequence; Described connection is carried out with the mode (for example, in in-vitro transcription/translation system or in the host cell that carrier is introduced into) that allows this nucleotide sequence to express.Term " regulating and controlling sequence " is intended to comprise promotor, enhanser and other expression controlling elements (for example, polyadenylation signal).For example, at Goeddel; GeneExpression Technology:Methods in Enzymology 185, Academic ress, SanDiego is described this type of regulating and controlling sequence in CA(1990).Regulating and controlling sequence is included in the host cell of number of different types and instructs the composing type of nucleotide sequence or the regulating and controlling sequence of inducible expression; Also comprise the regulating and controlling sequence (for example, tissue specificity regulating and controlling sequence) that only in some host cell, instructs nucleotide sequence to express.Those skilled in the art will recognize, possibly depend on following factor the design of expression vector: to will be by the selection of transformed host cells, the proteic expression level that expectation obtains etc.Expression vector of the present invention can be introduced into host cell; With the albumen of producing nucleic acid encoding as herein described or peptide (for example; According to l-asparagine enzyme variants any among SEQ ID NO:2 or the SEQ ID NO:4 or any variant in them; For example function equivalent or fragment, or comprise in this type of variant one or more fusion rotein).

Can be directed against the expression of misfolded proteins of the present invention in prokaryotic cell prokaryocyte or eukaryotic cell, and recombinant expression vector of the present invention is designed.For example, albumen of the present invention can be expressed in bacterial cell (for example E.coli), insect cell (use rhabdovirus expression vector), yeast cell or mammalian cell.At Goeddel; Gene Expression Technology:Methods inEnzymology 185, Academic ress, San Diego has carried out further discussion to proper host cell in CA(1990).Perhaps, recombinant expression vector can transcribe and translate external, for example, uses T7 promoter regulation sequence and T7 polysaccharase.

Can be used for expression vector of the present invention and comprise the carrier that is derived from karyomit(e), episome and virus; For example be derived from the carrier of bacterial plasmid, phage, yeast episome, yeast chromosomal element; Be derived from virus; For example baculovirus, papova viruses (papova virus), vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retroviral carrier and be derived from the carrier of the combination of above-mentioned substance; For example be derived from the carrier of plasmid and phage genetic elements, for example clay and phagemid (phagemid).

The DNA that inserts should be operably connected on the suitable promotor; For example: lac promotor, trp promotor and the tac promotor of λ PL promotor of phage, the promotor of the early promoter of SV40 and late promoter and retrovirus LTR etc.The technician knows the promotor that other is suitable.In a kind of concrete embodiment, it is preferred instructing asparaginase promotor of high level expression in filamentous fungus.This type of promotor is known in the art.Expression construct can contain and is useful on transcription initiation and terminated site, and can contain the ribosome bind site that is useful on translation transcribing the zone.The encoding part of the ripe transcript of being expressed by construct comprises AUG that is used for initial translation that is in starting point and the termination codon that is positioned polypeptide end to be translated rightly.

Can carrier DNA be introduced prokaryotic cell prokaryocyte or eukaryotic cell through traditional conversion or rotaring dyeing technology.When using in this article; Term " conversion " and " transfection " refer to a series of technology that are used for external nucleic acid (for example DNA) is incorporated into host cell known in the art, comprise transfection or electroporation that calcium phosphate or calcium chloride co-precipitation, the transfection of EAE-dextran mediation, transduction, infection, lipofection, cationic lipid mediate.Be used for to host cell transform with the appropriate method of transfection can be at Sambrook, et al.(Molecular Cloning:A Laboratory Manual, 2 < > Nd <> , ed.ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press SpringHarbor, NY, 1989) et al., Basic Methods in Molecular Biology(1986) and other laboratory manual in find.

As far as the stable transfection of mammalian cell, people are known, only have a fraction of cell foreign DNA can be incorporated in its genome, and this depends on used expression vector and rotaring dyeing technology.For differentiating and select above-mentioned intasome that the selected marker thing of will encoding usually (for example to antibiotic resistance) gene is incorporated in the host cell with the interested gene of people.Preferred selected marker thing comprises the marker of giving drug resistance, and said medicine for example is G418, Totomycin and methotrexate.The nucleic acid of coding selected marker thing can be transferred in the host cell inventing on the identical carrier of proteic carrier with code book, perhaps, can on other carrier, be transferred into.Can identify nucleic acid stability cells transfected (for example, the cell of having incorporated selected marker thing gene into will be survived, and other cell then can be dead) through medicament selection through introducing.

The expression of albumen in prokaryotic organism normally carried out in E.coli, wherein uses the carrier that contains composing type or inducible promoter, to instruct fusion rotein or non-Expression of Fusion Protein.Fusion vector joins a plurality of amino acid on the albumen that is encoded, and for example, joins the N-terminal of recombinant protein.Typically, this type of fusion vector is used as three kinds of purposes: 1) to strengthen Recombinant Protein Expression; 2) to increase the solubleness of recombinant protein; With 3) pass through in affinity purification as the purifying of part with the assistance recombinant protein.Usually, in fusion expression vector, introduce the proteolytic cleavage site, separate with the fusion part after fusion rotein is by purifying, can make recombinant protein to the junction of merging part and recombinant protein.

As pointed, expression vector will preferably contain the selected marker thing.This type of marker comprises: be used for Tetrahydrofolate dehydrogenase or neomycin resistance that eukaryotic cell is cultivated, and be used for tetracyclin resistance or the amicillin resistance cultivated on E.coli and other bacterium.The representative example of appropriate host comprises: bacterial cell, the for example kind of E.coli, Streptomyces Salmonella typhimurium and some Bacillus; Fungal cell, the for example kind of Aspergillus, for example A.niger, A.oryzae and A.nidulans, for example yeast, for example Kluyveromyces, for example K.lactis and/or Puchia, for example P.pastoris; Insect cell, for example Drosophila S2 and SpodopteraSf9; Zooblast, for example CHO, COS and Bowes melanoma; And vegetable cell.The suitable medium and the culture condition that are used for above-mentioned host cell all are known in the art.

The carrier that is preferred for bacterium is for example to incorporate this paper by reference at WO-A1-2004/074468() in those disclosed.Other carrier that is fit to will be apparent to those skilled in the art.

Be applicable to that known bacterium promotor of the present invention comprises that WO-A1-2004/074468(incorporates this paper by reference into) middle those disclosed.

Can come DNA the transcribing in higher eucaryote of enhance encoding variant of the present invention through enhancer sequence is inserted in the carrier.Enhanser is the cis-acting elements of DNA, it typically is 10bp to 300bp, and it plays a role in given host cell type, to increase the transcriptional activity of promotor.The example of enhanser comprise be in replication orgin side (late side in late period one) the SV40 enhanser at 100bp to 70bp place, the enhanser of cytomegalovirus early promoter, be in the polyomavirus enhanser and the adenovirus enhanser of replication orgin side in late period one.

Can be secreted into for the albumen that makes translation in chamber, kytoplasm gap or the born of the same parents' external environment of endoplasmic reticulum, suitable secretion signal can be inserted in the polypeptide expressed.Described signal can be endogenous as far as polypeptide, also can be the allos signal.

Variant of the present invention can be with following formal representation, and said form makes it can comprise other allos functional area, for example, and secretion signal.Variant of the present invention also can comprise; For example; Join the zone of terminal other amino acid (particularly charged amino acid) of polypeptide N-, be increased in the host cell, during purifying or at subsequently operation and the stability and the persistence (persistence of duration of storage).In addition, the part of peptide can also be joined on the said polypeptide,, for example realize through adding histidine residues or T7 label to assist to carry out purifying.

< > According to polypeptide of the present invention <>

The invention provides following asparaginase, it is:

A) polypeptide, it has the aminoacid sequence shown in any among SEQ ID NO:2 or the SEQ ID NO:4;

B) polypeptide, its with have at least 85% homology according to a) polypeptide;

C) polypeptide, its can through replace, lack and/or insert one or more amino acid and from a) or b) polypeptide of definition obtains;

D) polypeptide, its be the encoding mature polypeptide SEQ ID NO:1 or SEQ ID NO:3 nucleotide sequence or have its subsequence codings of at least 100 Nucleotide.

Typically; For SEQ ID NO:2; According to b); C) or polypeptide d) comprise the 25th Ala; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; One or more among the 375th Val or the 377th 's the Val, said position defines with reference to SEQ ID NO:2.

Polypeptide of the present invention can comprise the 25th Ala; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; Two or more among the 375th Val or the 377th 's the Val, said position defines with reference to SEQ ID NO:2.More preferably, polypeptide of the present invention can comprise all these amino acid of above-mentioned position, and said position defines with reference to SEQ IDNO:2.

For SEQ ID NO:4; Can comprise the 35th Ser according to polypeptide of the present invention; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; One or more among the 372nd Gly or the 373rd 's the Val, said position defines with reference to SEQ ID NO:4.

Polypeptide of the present invention can comprise the 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; Two or more among the 372nd Gly or the 373rd 's the Val, said position defines with reference to SEQ ID NO:4.

More preferably, polypeptide of the present invention can comprise these amino acid of all above-mentioned positions, and said position defines with reference to SEQ ID NO:4.

In another embodiment, the present invention also provides at least 3.5, and preferably at least 4, the more preferably asparaginase of the wide pH field of activity width of at least 5 pH units.A kind of preferred embodiment in, the asparaginase with pH field of activity width of at least 3.5 is such asparaginase, it is:

In addition, peptide or the polypeptide that comprises the function equivalent of aforementioned polypeptides is also contained in the present invention.Aforementioned polypeptides is included in term " according to polypeptide of the present invention " or " according to the variant of the present invention " jointly.

(as usually confessed) term " peptide " and " oligopeptides " are considered to synonym, and when it may be noted that at least two amino acid whose chains that get up through the peptide bond coupling in the context, two terms can exchange use." polypeptide " this speech is used to refer to contain the chain above the seven amino acid residue in this article.Among this paper the molecular formula of all oligopeptides and polypeptide or sequence all be from left to right and the direction from the aminoterminal to the carboxyl terminal write.The single-letter amino acid code that uses among this paper is known in the art; It can be at Sambrook al.(Molecular Cloning:A LaboratoryManual Spring Harbor Laboratory, Cold Spring HarborLaboratory ress, Cold Spring Harbor; NY, 989) find in.

" separated " polypeptide or albumen are represented the polypeptide or the albumen that from its physical environment, are shifted out.For example, for purposes of the present invention, the polypeptide and the albumen through recombinant production of in host cell, expressing are considered to through isolating; Natural or the recombinant polypeptide that has carried out basic purifying with any suitable technique also is considered to through isolating, and said technology for example is at Smith and Johnson, Gene67:31-40(1988) in disclosed single step purification.

Can from the reconstitution cell culture, reclaim and purifying variant polypeptide of the present invention through known method.Most preferably, use ion-exchange chromatography or high performance liquid chromatography (HPLC) carry out purifying.

Polypeptide of the present invention comprises the product of chemosynthesis flow process and the product of from protokaryon or eucaryon host, producing through recombinant technology, and said host comprises for example bacterium, yeast, higher plant, insect and Mammals.Polypeptide of the present invention can be to have passed through glycosylatedly, perhaps can be not pass through glycosylatedly, and this depends on employed host in the recombinant production flow process.In addition, polypeptide of the present invention can also comprise initial methionine residues through modifying, and this is the result of the process that mediates of host in some cases.

< > Protein fragments <>

The invention still further relates to bioactive fragment according to polypeptide of the present invention.Term " variant of the present invention " comprises this type of fragment.

The bioactive fragment of polypeptide of the present invention comprises the polypeptide that comprises following aminoacid sequence; The aminoacid sequence (the for example aminoacid sequence of ID NO:2 or SEQ ID NO:4) of said aminoacid sequence and misfolded proteins of the present invention is fully identical or come from aminoacid sequence (the aminoacid sequence) of ID NO:2 or SEQ ID NO:4 for example of misfolded proteins of the present invention; The amino acid that said fragment comprises will lack than full-length proteins, but demonstrates at least a biological activity of corresponding full-length proteins.Typically, bioactive fragment comprises at least a bioactive structural domain or the primitive (motif with corresponding full-length proteins).Preferably, bioactive fragment will have asparaginase activity at least.Proteic bioactive fragment of the present invention can be a polypeptide, and its length for example is 10,25,50,100 or more a plurality of amino acid.In addition, can prepare other biologically-active moiety, be removed said proteic other zone in the said part, and can be directed against one or more biological activitys of the natural form of polypeptide of the present invention, said part is assessed through recombinant technology.

Typically; For SEQ ID NO:2; Protein fragments of the present invention will comprise the 25th Ala; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; One or more among the 375th Val or the 377th 's the Val, said position defines with reference to SEQ ID NO:2.

Protein fragments of the present invention can comprise the 25th Ala; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; Two or more among the 375th Val or the 377th 's the Val, said position defines with reference to SEQ ID NO:2.More preferably, protein fragments of the present invention can comprise all these amino acid of above-mentioned position, and said position defines with reference to SEQID NO:2.

Typically; For SEQ ID NO:4; Protein fragments of the present invention will comprise the 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; One or more among the 372nd Gly or the 373rd 's the Val, said position defines with reference to SEQ ID NO:4.

Protein fragments of the present invention can comprise the 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; Two or more among the 372nd Gly or the 373rd 's the Val, said position defines with reference to SEQ ID NO:4.More preferably, protein fragments of the present invention can comprise these amino acid of all above-mentioned positions, and said position defines with reference to SEQ ID NO:4.

The invention still further relates to the nucleic acid fragment (said bioactive fragment self is a variant of the present invention) of the above-mentioned bioactive fragment of coding.

< > Fusion rotein <>

Variant of the present invention, albumen for example of the present invention or its function equivalent, for example its biologically-active moiety and fragment, can be operably connected to not is according on the polypeptide of the present invention (for example, the allogeneic amino acid sequence), to form fusion rotein.Be not polypeptide according to the present invention context have corresponding to the basic proteic aminoacid sequence of homologous of variant asparaginase of the present invention.

In fusion rotein, variant of the present invention can be corresponding to the full length sequence of the bioactive fragment of polypeptide of the present invention.A kind of preferred embodiment in, fusion rotein of the present invention comprises at least two biologically-active moieties.In fusion rotein, term " be operably connected " be used for the expression: variant polypeptide be not that polypeptide according to the present invention merges with the mode that meets reading frame each other.Be not to be fused on the N-end or C-end of variant polypeptide according to polypeptide of the present invention.

For example, in one embodiment, fusion rotein is such fusion rotein, and wherein, the variant sequence is fused on the C-end of GST sequence.This type of fusion rotein can be assisted the purifying to reorganization variant according to the present invention.In another embodiment, described fusion rotein is the variant of the present invention that contains the allos signal sequence at its N-end.Can increase expression and/or the secretion of variant of the present invention in some host cell (for example, mammalian host cell and yeast host cell) through using the allos signal sequence.

In another example, the gp67 secretion sequence of baculovirus envelope protein can be used as allos signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., john wiley & sons, 992).Other example of eucaryon allos signal sequence comprises the secretion sequence of mellitin and the secretion sequence (Stratagene of human placenta's alkaline phosphatase; La Jolla, California).In another example, useful protokaryon allos signal sequence comprises phoA secretion signal (aforesaid Sambrook et al.) and egg matter A secretion signal (Pharmacia Biotech; Piscataway, NewJersey).

Signal sequence can be used to assist the secretion and the separation of variant of the present invention.Typically, being characterized as of signal sequence has the hydrophobic amino acid core, between the secretory phase, in one or many cutting incident, said signal sequence can be cut away from maturation protein usually.This type of signal peptide contains the processing site, allows signal sequence in Secretory Pathway, being cut away from maturation protein.Signal sequence can instruct the secretion of variant, for example from the eucaryon host that transforms expression vector, secretes, and signal sequence is cut subsequently or simultaneously.Then by means commonly known in the art, can outside born of the same parents, be purified into variant of the present invention at an easy rate the substratum.Perhaps, can use the sequence that to assist purifying, for example signal sequence is connected on the interested variant of people with the GST structural domain.Therefore, for example, the sequence of code book invention variant can with marker sequence (for example coding can promote the sequence of peptide of the purifying of fusion variant of the present invention) fusion.This on the one hand some of the present invention preferred embodiment in, the marker sequence is six Histidine peptides, pQE carrier (Qiagen for example, the label that provides in nc.); A lot of marker sequences all can buy.For example, Proc.Natl.Acad.Sci.USA86:821-824(1989) said like Gentz et al, six Histidines are that fusion rotein provides purifying easily.The HA label is the peptide that another kind helps purifying, and it is corresponding to the epi-position from influenza hemagglutinin protein, and this is for example by Wilson et al., Cell 37:767(1984) be described.

Fusion rotein of the present invention can produce through the recombinant DNA technology of standard.For example,, the dna fragmentation of the different peptide sequences of coding is connected together with the mode that meets reading frame, for example, be used for connecting through adopting flat end or stagger end according to conventional art; Adopt Restriction Enzyme digestion so that suitable end to be provided, or fill and lead up sticking terminal as required; Adopt alkaline phosphatase treatment, connect to avoid undesired combination of people or enzyme.In another embodiment, can synthesize fusion gene through conventional art (comprising automatic dna synthesizer).Perhaps; Can use anchor primer (anchor primer) come gene fragment is carried out pcr amplification; Said primer can produce complementary overhang (overhang) between two sections successive gene fragments; Can anneal subsequently and amplification once more; (for example to produce chimeric gene sequence; See Current Protocols in MolecularBiology, eds.Ausubel et al.John Wiley&Sons:1992).In addition, can also commercial sources obtaining has much encoded merges the expression vector of part (for example, gst polypeptide).The nucleic acid of coding variant can be advanced examples of such carriers by the clone, couples together with mode and the said variant that meets reading frame so that merge part.

< > Function equivalent <>

Term " function equivalent " and " functional variant " can exchange use in this article.Function equivalent according to the present invention is through the separated DNA fragment, and said fragment coding shows the polypeptide of specific function of the Aspergillus niger l-asparagine enzyme variants of this paper definition.Function equivalent according to polypeptide of the present invention is the polypeptide that shows at least a function of the Aspergillus niger asparaginase that defines among this paper.Therefore, function equivalent also comprises the fragment of biologically active, and they self are also included within the scope of term " variant " of the present invention.

Preferably; The present invention comprises the 25th Ala about the function equivalent polypeptide of SEQ ID NO:2; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd 11e; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; One or more among the 375th Val or the 377th 's the Val, said position defines with reference to SEQ ID NO:2.

Therefore; Variant l-asparagine enzyme polypeptide of the present invention can comprise the 25th Ala; The 28th Thr; The 30th Tyr; The 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 131st Ser; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 210th Ala; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 240th Tyr; The 250th Thr; The 251st Thr; The 252nd Val; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 273rd Gln; The 279th Ser; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 307th Asp; The 309th Ile; The 310th Ala; The 311st Ser; The 312nd Lys; The 313rd His; The 314th Ser; The 317th Ile; The 319th Leu; The 321st Thr; The 324th Gly; The 330th Thr; The 345th Phe; The 351st Ala; The 360th Ala; The 361st Glu; The 364th Gly; The 365th Phe; The 366th Lys; The 369th Arg; The 370th Glu; The 374th Lys; Among the 375th Val or the 377th 's the Val two; Three; Four; Five; For example; At least 10; At least 15; At least 20; For example at least 25; At least 30; At least 40; At least 50; At least 60; At least 70; At least 80 or whole, said position defines with reference to SEQ ID NO:2.

Preferably; The present invention comprises the 35th Ser about the function equivalent polypeptide of SEQ ID NO:4; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; One or more among the 372nd Gly or the 373rd 's the Val, said position defines with reference to SEQ ID NO:4.

Therefore; Variant l-asparagine enzyme polypeptide of the present invention can comprise the 35th Ser; The 53rd Leu; The 63rd Ser; The 64th Ala; The 65th Asp; The 66th Asn; The 74th Ala; The 77th Ile; The 79th Ile; The 80th Gln; The 81st Thr; The 88th Glu; The 106th Pro; The 108th Val; The 111st Ser; The 114th Leu; The 117th Ala; The 119th Thr; The 122nd Glu; The 126th Asp; The 161st Val; The 168th Ala; The 181st Gln; The 189th Pro; The 190th Ala; The 197th Leu; The 205th Val; The 208th Phe; The 211st Ser; The 224th Val; The 228th Asn; The 231st Ala; The 232nd Ile; The 233rd Val; The 236th Lys; The 238th Tyr; The 250th Thr; The 251st Thr; The 254th Val; The 255th Arg; The 259th Ser; The 267th Tyr; The 270th Gln; The 279th Asp; The 282nd Asp; The 283rd Asn; The 286th Lys; The 293rd Ser; The 297th Ser; The 299th Ser; The 300th Ser; The 301st Gly; The 303rd Ser; The 304th Asp; The 309th Ile; The 311st Ser; The 312nd Thr; The 313rd His; The 314th Ser; The 317th Ile; The 321st Thr; The 324th Gly; The 330th Pro; The 333rd Glu; The 336th Gln; The 341st Phe; The 356th Ala; The 360th Gly; The 362nd Glu; The 365th Arg; The 366th Glu; The 370th Lys; The 371st Val; Among the 372nd Gly or the 373rd 's the Val two; Three; Four; Five; For example; At least 10; At least 15; At least 20; For example at least 25; At least 30; At least 40; At least 50; At least 60; At least 70 or whole, said position defines with reference to SEQID NO:4.

Albumen or polypeptide function equivalent can contain to one or more amino acid whose replacement any among SEQ ID NO:2 or the SEQ ID NO:4 or to non-key amino acid whose replacement, insertion or disappearance.Therefore, non-key amino acid is can reformed residue in any one among SEQ ID NO:2 or the SEQ ID NO:4, and said change or not biological function basically.In addition, conservative amino acid unlikely is easy to be changed between albumen according to the present invention and other asparaginase.

Can among SEQ ID NO:2 or the SEQ ID NO:4 any one carry out aminoacid replacement, for example, from 1,2 or 3 to about 10, about 20, about 30 or more many places replace, so that functional variant of the present invention to be provided.

Typically, the nucleic acid function equivalent can contain silent mutation or not change the sudden change of the biological function of coded polypeptide.Therefore, the invention provides the proteic nucleic acid molecule of coding variant asparaginase, it contains the change of non-essential amino-acid residue concerning particular organisms is active.Aminoacid sequence any one among this type of misfolded proteins and SEQ ID NO:2 or the SEQ ID NO:4 is different, but still keeps its at least a biological activity.In one embodiment; Separated nucleic acid molecule comprises the following proteic nucleic acid molecule of coding; Wherein, said albumen comprise with SEQ ID NO:2 or SEQ ID NO:4 in aminoacid sequence about at least 85%, 90%, 95%, 96%, 97%, 98%, 99% shown in any or the basic homologous aminoacid sequence of homologous more.

Can through following method make the coding with according to albumen homology any among SEQ ID NO:2 or the SEQ ID NO:4 (typically; The separated nucleic acid molecule of variant of the present invention basic homology): in coding nucleotide sequence according to the present invention, introduce one or more Nucleotide and replace, increase or disappearance, thereby in the albumen that is encoded, introduce a place or many places aminoacid replacement, disappearance or insertion.Can introduce this type of sudden change through standard technique, for example site-directed mutagenesis and PCR mediated mutagenesis.

As defined herein; Term " basic homology " refers to: that article one amino acid or nucleotide sequence and second amino acid or nucleotide sequence contain enough numbers or minimum requirements number identical or (for example be equal to; Have similar side chain) amino acid or Nucleotide; Thereby described article one and second amino acid or nucleotide sequence have the common structure territory.For example; Contain the common structure territory; And have about 60%; Be preferably 65%; More preferably 70%; Further be preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or the aminoacid sequence or the nucleotide sequence of higher identity, just be defined as fully identical in this article.

Can be based on the homology of itself and the disclosed nucleic acid of the present invention of this paper and separated corresponding to the nucleic acid molecule of (that is, coding) variant asparaginase of the present invention, wherein use the disclosed cDNA of this paper.

The technician will know, can introduce change through the sudden change in nucleotide sequence according to the present invention, in the proteic aminoacid sequence that obtains, produce thus to change, and not influence this type of proteic function basically.

In another aspect of this invention, improved asparaginase misfolded proteins is provided.Improved asparaginase misfolded proteins is such albumen, and wherein, at least a biologic activity is modified, for example, and than the wild-type asparaginase, for example, from A.niger's.

Especially; Misfolded proteins of the present invention can have improved specific activity than wild-type asparaginase (for example the SEQ ID NO:3 of WO2004/030468 is disclosed from A.niger's) under given pH; Perhaps; Can have improved pH than wild-type asparaginase (for example the SEQ ID NO:3 of WO2004/030468 is disclosed from A.niger's) replys; For example, more pro-alkaline or parent are acid.For example, when measuring under the same conditions, misfolded proteins of the present invention can have discloseder from the high specific activity of the wild-type asparaginase of A.niger than the SEQ IDNO:3 of WO2004/030468 under at least 5 pH.For example, the disclosed wild-type asparaginase from A.niger of the SEQ ID NO:3 of WO2004/030468 has the pH optimum value of pH 4 to pH 5.Misfolded proteins of the present invention possibly promptly, for example can have the pH optimum value of pH 6 to pH 7 than this type of wild-type enzyme pro-alkaline more.Certainly more the parent is acid for the also comparable wild-type asparaginase of variant.

In another embodiment, misfolded proteins of the present invention can have at least 3.5, and preferably at least 4, more preferably at least 5 pH field of activity width that pH unit is wide.

Can obtain this proteinoid through on encoding sequence of the present invention all or part of, introducing sudden change at random, for example introduce sudden change through saturation mutagenesis.Then, the mutant that obtains can be by recombinant expressed, and to biological activity it is screened.For example, this area provides the standard test of measuring the asparaginase enzymatic activity, can easily select improved albumen thus.

A kind of preferred embodiment in, l-asparagine enzyme variants of the present invention has according to any one aminoacid sequence among SEQID NO:2 or the SEQ ID NO:4.In another embodiment; Variant of the present invention with according to the basic homology of aminoacid sequence any among SEQ ID NO:2 or the SEQ ID NO:4; And typically; Also remain with biological activity according to polypeptide any among SEQ ID NO:2 or the SEQID NO:4, but because mutagenesis mentioned above and different on aminoacid sequence.

Another preferred embodiment in, l-asparagine enzyme variants of the present invention have can with nucleic acid hybridization according to the present invention (preferably, under the tight degree hybridization conditions of height) through the separating acid fragment amino acid sequence coded.

Therefore; The preferably such albumen of l-asparagine enzyme variants of the present invention; It comprises with according to about at least 85% of aminoacid sequence shown in any among SEQ ID NO:2 or the SEQ ID NO:4,95%, 96%, 99% homology or homologous aminoacid sequence more, and keep at least a functionally active according to polypeptide any among SEQ ID NO:2 or the SEQ ID NO:4.

The proteic function equivalent of the present invention can also be identified that for example through being directed against asparaginase activity, to the proteic mutant of the present invention, for example the combinatorial library of truncated mutant screens in several ways.In one embodiment, produced the variegated library (variegated library of variant through the combinatorial mutagenesis on the nucleic acid level).The variegated library of variant can produce in several ways; For example; Through enzymatic the synthetic oligonucleotide mixture is connected on the gene order; Make that a cover possibly be that proteic degenerate sequence can be used as one expression of polypeptides; Perhaps; As the bigger expressing fusion protein (when for example, being used for phage display) of a cover.Having a variety of methods to can be used for from oligonucleotide sequence production of degeneracy possibly be the library of polypeptide variants of the present invention.The method that is used for synthetic degenerate oligonucleotide be well known in the art (see: for example, Narang(1983)Tetrahedron39:3; Itakura et al.(1984)Annu.Rev.Biochem.53:323; Itakura et al.(1984)Science 198:1056; Ike et al.(1983)Nucleic Acid Res.11:477).

In addition, the segmental library of the sequence of code book invention polypeptide also can be used for making the variegated crowd (population of polypeptide), to screen in the selection to variant subsequently.For example, can make the segmental library of encoding sequence through following method: as far as each molecule, otch (nick) only occur handling the double-stranded PCR fragment of the interested encoding sequence of people with nuclease under the condition approximately once; Double-stranded DNA is carried out sex change; DNA is carried out renaturation, and to form double-stranded DNA, it is right that said double-stranded DNA possibly comprise from the synonym/antisense of different incisions product; Handle the part of from the duplex that forms again, removing strand through the S1 nuclease; The fragment library and the expression vector that obtain are coupled together.Through this kind method, can obtain encode the N-terminal fragment of the big or small proteins of interest of multiple difference and the expression library of interior segments.

Multiple technologies as known in the art all can be used for the gene product of screening the combinatorial library of making through the brachymemma point mutation to the gene product with selected attribute and screening cDNA library.Typically, most popularly be used for screening big gene library and be applicable to that the technology of high throughput analysis comprises: said gene library is cloned into reproducible expression vector; Vector library with obtaining removes to transform suitable cell; Express described combination gene under certain condition, under said expression condition, the detection that the activity of wanting to people is carried out helps to isolate the carrier of the gene that its product of coding is detected.The overall mutagenesis (recursive of recurrence ensemble mutagenesis; REM) technology can enhancement function the frequency that in the library, occurs of mutant; It can use with screening test, to identify the proteic variant (Arkin of the present invention and Yourvan(1992)Proc.Natl.Acad.Sci.USA89:7811-7815; Delgrave et al.(1993)Protein Engineering 6(3):327-331).

Fragment according to polynucleotide of the present invention can also comprise not many nucleic acids of encoding function polypeptide.These type of polynucleotide can be as probe or the primer that reacts as PCR.

As far as nucleic acid of the present invention,, all can be used as the primer of hybridization probe or polymerase chain reaction (PCR) regardless of its encoding function polypeptide or coding NOT-function polypeptide.The do not encode purposes of nucleic acid molecule of the present invention of polypeptide with asparaginase activity; Comprise :(1) like Verma et al. Chromosomes:a Manual of Basic Techniques Press) as described in; To Metaphase Chromosome expansion (spreads) in situ hybridization (for example ISH) carried out, so that the accurate chromosomal localization to the l-asparagine enzyme coding gene to be provided; (2)Northern is hybridized engram analysis, is used for detecting the expression of asparaginase mRNA at particular organization and/or cell; (3) can be used as primer and the probe that diagnostic tool uses, can be in given biology (for example tissue) sample whether exist with the nucleic acid of this type of probe or primer hybridization to analyze.

The method that also comprises the function equivalent that obtains polynucleotide of the present invention among the present invention.These class methods need: obtain the probe through mark, said probe comprises separated nucleic acid, and said nucleic acid encoding is all or part of according to protein sequence any among SEQ ID NO:2 or the SEQ ID NO:4; Use probe that the nucleic acid fragment library is screened through mark; Screening is under the condition that allows the hybridization of probe and library amplifying nucleic acid fragment, to carry out; Thereby formation nucleic acid duplex; And prepare full-length gene order by any nucleic acid fragment in the duplex of mark, to obtain the gene relevant with polynucleotide of the present invention.

< > Host cell <>

In another embodiment, the present invention relates to contain the cell of the nucleic acid that the present invention includes, for example through transformed host cells or recombinant host cell." through cell transformed " or " reconstitution cell " is through recombinant DNA technology, introduced the cell of nucleic acid of the present invention to wherein (or its for generations in).In prokaryotic cell prokaryocyte and eukaryotic cell all are included in, for example, bacterium, fungi, yeast etc., especially preferred is cell, particularly Aspergillus niger from filamentous fungus.

Can select host cell, select the host cell that to regulate host cell that insertion sequence expresses or can modify or process gene product with mode specific, desirable.Can promote the proteic optimum function that is encoded to this type of modification (for example glycosylation) of protein product and processing (for example cutting).

Multiple host cell all has distinctive and specific mechanism, is used for translation post-treatment and modification to albumen and gene product.Suitable clone or the host system that can select the technician in molecular biology and/or microbiology field to be familiar with are to guarantee that modification and processing to expressed foreign protein are that make us expecting and correct.For reaching this target, can use eukaryotic host cell with processing mechanism in the born of the same parents, said mechanism be used for to primary transcribe correct processing, to the glycosylation and the phosphorylation of gene product.This type of host cell is as known in the art.

Host cell also includes but not limited to: mammal cell line, for example CHO, VERO, BHK, HeLa, COS, MDCK, 293,3T3, WI38 and choroid plexus cell system.

If necessary, can be through producing variant of the present invention through the clone of stable transfection.The a large amount of carriers that are suitable for mammalian cell is carried out stable transfection all are that the public is obtainable, and the method that makes up this type of clone is also known for public institute, for example (among the aforesaid)Ausubel et al..

< > The purposes of variant asparaginase of the present invention in industrial technology <>

The invention also discloses the composition that comprises according to asparaginase of the present invention.Said composition is optional can to comprise other composition, for example, and other enzyme.Can be used for producing foodstuff products according to l-asparagine enzyme variants of the present invention or the composition that comprises said asparaginase.In one embodiment of the invention, can be used for reducing amount according to l-asparagine enzyme variants of the present invention or the composition that comprises said asparaginase based on the raw-material acrylamide that in hot worked foodstuff products, forms that contains l-asparagine.They can for example be used for the technology of following production foodstuff products; Said technology relates at least one heating steps; Be included in the said production technique one or more asparaginases are added in the intermediate product form of said foodstuff products, the amount of the level of the asparaginase that before said heating steps, enzyme is existed in the said intermediate product form that can effectively reduce said foodstuff products adds thus.This type of process quilt is disclosed among the WO04/030468, and said technology and all prioritys thereof are incorporated this paper by reference into.Also described among the WO04/026043 and wherein can use appropriate process according to asparaginase of the present invention.Disclosed method and all prioritys thereof are incorporated this paper by reference among the WO04/026043.

The intermediate product form of foodstuff products is defined as any form that before the final form that obtains foodstuff products, occurs during the production technique in this article.The intermediate product form can comprise each starting material and/or its mixture of use and/or with the mixture of additive and/or processing aid or it is through the form with post-treatment.For example; For foodstuff products bread; The intermediate product form comprises; For example, the initial mixture of wheat, whole meal flour, itself and other bread composition (for example water, salt, yeast and bread improve composition), mixing dough, through the dough of pugging mullering, through the dough of fermentation with through the part baked dough.For example, for some kinds based on for the product of potato, dehydrated potato sheet or particle are intermediate products, as far as maize crisps, cornmeal mush is an intermediate product.

Foodstuff products can be from least a plant origin (for example potato, tobacco, coffee, cocoa, paddy rice, cereal, for example wheat, naked barley, corn, barley, oat grain (groats), buckwheat and oat) starting material make.Wheat comprises all known kinds that Triticum belongs to herein and hereinafter, for example, and aestivum, urum and/or spelta.Be also included within the scope of the present invention from surpassing the foodstuff products that a kind of starting material or intermediate product make, for example comprise the two foodstuff products of wheat (flour and/or starch) and potato.

Example according to the suitable foodstuff products of technology of the present invention is any product based on flour; For example; Bread, cake, cake, cracknel, bagel, Dutch honey cake, cookies, ginger bread, gingerbread and crispbread; And any product based on potato; For example, French fries, pommesfrites, potato chips, fried pork balls.

The known l-asparagine that contains suitable content of starting material mentioned above, it participates in the formation of acrylamide during the heating steps of production technique.Perhaps, l-asparagine can be from being not said raw-material other source, for example, from protein hydrolysate, yeast extract for example, soya hydrolysate, caseic hydrolysate etc. (in foodstuffs production technology, being used as additive).A kind of preferred production technique is bread and curing from other baked product of the flour in whole meal flour and/or other cereal source.Another preferred production technique is from the potato slices potato chips.

Preferred heating steps is such, wherein, at least a portion of intermediate product foodstuff products, for example, the surface of foodstuff products is exposed to the temperature that promotes that acrylamide forms, for example, and 110 ℃ or higher, 120 ℃ or higher temperature.Can in baking box, carry out according to the heating steps in the technology of the present invention, for example, under the temperature between 180-220 ℃, carry out, for example, be used to cure bread and other baked product, perhaps in oil, carry out, for example potato crisps for example carries out under 160-190 ℃.

In yet another aspect, the invention provides can be through described technology of the present invention of preamble or the foodstuff products through using the described l-asparagine that is used to produce the novelty of foodstuff products of preamble to obtain.These foodstuff productss are characterised in that than can having significantly reduced levels of acrylamide through the foodstuff products that following production technique obtains, and said production technique does not comprise can effectively reduce the amount of participating in the amino acid levels that acrylamide forms during the said heating steps and adds one or more enzymes.Technology according to the present invention can be used for making the acrylamide content of the foodstuff products of production to reduce than the foodstuff products that obtains with traditional technology and preferably surpasses 50%, more preferably surpasses 20%, and further more preferably 10%, most preferably surpass 5%.

Another Application according to l-asparagine enzyme variants of the present invention is the oncotherapy that is used for the animal and human.The metabolism of tumour cell needs altheine, and it can be degraded by asparaginase fast.Also can be used as adjuvant according to asparaginase of the present invention and be used to treat some leukemia.In laboratory animal and people, using asparaginase makes some lymphoma and leukemia disappear.Therefore, in one embodiment, the present invention relates to be used as medicine according to asparaginase of the present invention or composition, for example, be used for animal and human's oncotherapy, for example, be used for lymphoma or leukemic treatment.

Can in microorganism, produce easily according to l-asparagine enzyme variants of the present invention.In above-mentioned technology, advantageously, use the asparaginase that obtains through recombinant DNA technology.This type of recombinase has dramatic benefit, for example, with low-cost, high yield production, and does not contain contaminative reagent, for example bacterium or virus, and do not contain other enzymic activity of bacteriotoxin or contaminative.

Hereinafter set forth the present invention through following non-limiting example.

Embodiment

< > Material and method <>

< > The asparaginase check is to measure the pH dependency in pH=4 to the pH=9 scope <>

Use altheine to measure asparaginase activity as substrate.React the amount of measuring the ammonia that discharges through the enzyme effect according to Berthelot.Obtain the general (phenolnitroprusside of ready-made available reagent phenol nitre from Sigma) and alkaline chlorinated lime (alkaline hypoclorite).In the mixture of the damping fluid with the pH that wants of 50mM Hydrocerol A and 50mM trisodium phosphate, 100 μ l enzyme samples are mixed with 2000 μ l 100mM altheines.After 37 ℃ hatch 30 minutes, come termination reaction through adding 400 μ l, 25% trichoroacetic acid(TCA), add 2500 μ l water afterwards.Between incubation period, except as otherwise noted, temperature is fixed as 37 ℃.

It will be understood by those skilled in the art that the dosage that should select enzyme by following requirement: make the signal that obtains to be significantly higher than the signal of background after hatching under these conditions but obtain still with the amount proportional band of the enzyme that adds in.Preferably, reaction is the (zero order of zero level).

After the termination reaction, 4 μ l mixtures incubated are added in the 156 μ l water.Subsequently, add the general solution (Sigma of 34 μ l phenol/nitre P6994) and 34 μ l alkalescence Eusol (SigmaA1727).37 ℃ hatch 676 seconds after, measure delustring at 600nm.Through comprising into appropriate blank, proofread and correct reading to background signal.The sample of using the enzyme with (TCA) inactivation is as blank.Check is at automatic analyser, for example Konelab Arena 30(Thermo Scientific) on carry out.Use the compensation line of the known ammonium sulfate concentrations mapping of standard series being made through the absorbancy of measuring with the 600nm place, measure activity.Active in unit representation, wherein, unit is defined in per minute under the condition for surveys of definition discharges the required enzyme of 1 micromole's ammonia from altheine amount.

< > The asparaginase check is to measure the pH dependency in pH4 to the pH8 scope <>

According to carrying out present method with the same mode of active pH dependency that is used to measure in pH=4 to the pH=9 scope mentioned above, in the 50mM phosphoric acid salt/citrate buffer that is 100 μ l enzyme samples and 2000 μ l 100mM altheines are had the pH that wants, the mixing of difference.

In all checks, the activity of asparaginase sample is all represented with unit/ml.

< > Embodiment 1 is according to fermentation, separation and the purifying of asparaginase of the present invention <>

The expression plasmid that contains the dna sequence dna of code book invention asparaginase through structure; Transform Aspergillus niger bacterial strain and, obtain asparaginase of the present invention with this plasmid according to the said cultivation of WO2004/030468 Aspergillus niger bacterial strain.

Cultivation contains after the Aspergillus niger of correct expression plasmid, through 4 ℃ with 5000xg centrifugal 30 minutes to fermented liquid, prepare not celliferous supernatant liquor.If necessary; Again supernatant is further filtered Miracloth filter (Calbiochemcat#475855) and GF/AWhatmannGlass microfilter (150mm

) respectively, to remove any solid.For removing any fungal material, available 4N KOH is adjusted to pH=5 with supernatant liquor, and at suction (suction) under, at the bottom of 2 μ m(-and the top) the strainer filtration sterilization.Supernatant liquor is preserved in 4 ℃,, perhaps if necessary, be frozen in-20 ℃ up to use.

Under impurity surpassed the situation of 60%w/w, through the anionic ion exchange chromatography, never celliferous supernatant liquor began to asparaginase purifying in addition, perhaps via D-10 post (AmershamBiosciences) carry out the ccUF desalination.To be applied to through the material of desalination at 20mM histidine buffering liquid (pH 5.96) on the balance Mono-Q or Q-Sepharose post that cross.After the thorough washing, use the gradient of 0 to 1M NaCl, wash-out asparaginase from the post.

Through analytical size exclusion chromatogram (HP-SEC: efficient size exclusion chromatogram, TSKgel3000SW-XL, post 300*7.8mm; MW scope 10-300kDa, 100mM phosphate buffered saline buffer pH7 and pH5.96), check the purity that the supernatant liquor level that contains asparaginase activity is divided or purified asparaginase level is divided (measuring) with mg albumen/ml.All flow velocitys all are 1ml/ minute (except the sample injection on Q-Sepharose, it are 5ml/ minute).Proteic detection to wash-out is carried out at the 80nm place.Use 10240M < >-1 <> .cm < >-1 <> Mole delustring constant (A280 < > 1cm, 1mg/ml <>=0.28, wherein, A280 < > 1cm, 1mg/ml <> Be to use the optical path length of 1cm, under the pure protein concentration of 1mg/ml, the delustring at 80nm place), from the delustring (A280 of 80nm) calculate the concentration of the Aspergillus niger asparaginase of wash-out.At Uvikon XL Secomamf spectrophotometer (Beun de onde, Abcoude carries out in TheNetherlands) to the measurement of A280.For asparaginase (ASN002 corresponding to SEQ ID NO:4) and corresponding to the asparaginase (ASN001 of SEQ ID NO:2) for, 960M used respectively < >-1 <> .cm < >-1 <> (A280 < > 1cm, 1mg/ml <>=0.25) and 11520M < >-1 <> .cm < >-1 <> (A280 < > 1cm, 1mg/ml <>=0.31).Under the situation that 80nm place impurity absorbs, the ratio of the total area of area and 80nm place absorption peak is taken advantage of under the A280 through the asparaginase sample that will record and the asparaginase peak, based on the HP-SEC chromatogram, proofreaies and correct the l-asparagine enzyme concn.Can not know under the situation of separating with other peak when the peak of asparagus fern Ntn hydrolase, replace peak area with peak heights.

< > Embodiment 2 is according to the performance of asparaginase of the present invention <>

< > Specific activity as the pH function <>

Under 37 ℃, in 50mM phosphoric acid salt/citrate buffer, use not celliferous supernatant liquor, when pH=4, pH=5, pH=6, pH=7, pH=8, measure the specific activity of l-asparagine enzyme variants.Specific activity is defined as the activity to the enzyme sample determination in this article, the enzyme polypeptide that its unit of being expressed as/mg is pure.Therefore, the activity of representing to the asparaginase sample determination, with the pure l-asparagine enzyme polypeptide of unit/mg than asparaginase activity.

	?pH＝4(％)	?pH＝5(％)	?pH＝6(％)	?pH＝7(％)	?pH＝8(％)
						wt	100	100	100	100	100
ASN002	14	125	226	374	1033
						ASN001	55	246	418	697	1758

Table 1: the regulation pH value under, the use l-asparagine as substrate, variant is with respect to wild-type (wt)A.niger asparaginase (WO2004/030468) specific activity.For each pH, the wild-type specific activity is set to 100%.Active in 37 ℃ of mensuration.

Be to measure the specific activity of asparaginase, measure the concentration that obtains the supernatant liquor from A280, wherein according to described in " material and method " based on the HP-SEC chromatogram, proofread and correct to any impurity.

For wild-type, the pH optimum value of variant ASN002 and ASN001 is moved to pH=6 from pH=4.In addition, the pH optimum value becomes wideer, particularly alkaline range.To the pH value more than the pH=4 of all measurements, variant ASN002 and ASN001 demonstrate than the high many specific activities of wild-type.

< > PH-activity dependent enzymes and pH optimum value <>

In 50mM phosphoric acid salt/citrate buffer, to the pH scope of pH=4 to pH=8, use not celliferous supernatant liquor, measure the pH dependency of asparaginase activity.PH when observing high reactivity to variant is called as the pH optimum value of said variant.In table 2, be set to 100% to the observed maximum activity of variant, the activity of said variant under other pH value recently shows as the percentage to the observed maximum activity of said variant.

	?pH＝4(％)	?pH＝5(％)	?pH＝6(％)	?pH＝7(％)	?pH＝8(％)
						wt	100	98	71	42	15
ASN002	9	76	100	95	90
						ASN001	19	81	100	95	83

Table 2: than wild-type (wt)A.niger asparaginase (WO2004/030468), the pH dependency of the asparaginase activity of variant.Be set to 100% to every kind of observed high reactivity of asparaginase.Active in 37 ℃ of mensuration.

Table 2 shows that the pH optimum value is moved to higher pH.In addition, the activity concerning variant under the alkaline pH significantly improves, and this makes these variants to need be very useful in the application of the asparaginase activity under the alkaline condition more.

< > The width of the pH field of activity of variant <>

The notable attribute of ASN002 and ASN001 is that these two kinds of asparaginases show extremely wide pH field of activity between the most useful pH scope pH=4 to pH=9, and this is former did not all observe to any asparaginase.For setting up the width of pH field of activity, use Hydrocerol A/pyrophosphate salt damping fluid, on pH=4 to pH=9, measure active.The width of pH field of activity is with pH unit is calculated, wherein enzyme shows its maximum activity 50% to 100% pH scope width.Through being drawn to pH, activity measures pH field of activity width.Show the width of the pH scope that at least 50% pH unit of its maximum activity calculates with enzyme, confirmed the width of the pH field of activity of said enzyme.The straight line parallel with the pH axle drawn at half place at maximum activity, and the pH field of activity intersects at two pH value places with this line.Two intersection pH values (one is the acid ramose to the pH field of activity, and one is alkaline ramose) have been confirmed the width of pH field of activity.In middle pH value, activity is at least 50% of a maximum activity.Maximum activity is defined as observed high reactivity when drawing active figure to pH.Corresponding to the most highly active pH is the pH optimum value of said enzyme.

Asparaginase	50% active acid branch	50% active alkaline branch	50% the pH field of activity width that show to surpass maximum activity
				A.niger	ND	pH＝6.7	Undetermined (ND)
ASN002	pH＝4.6	pH＝9.0	4.4 individual pH unit
				ASN001	pH＝4.5	pH＝8.6	4.1 individual pH unit
A.oryzae	pH＝5.2	pH＝8.2	3.0 individual pH unit

Table 3:ASN002 and ASN001 are than wild-type A.niger(WO2004/030468) and wild-type A.oryzae(WO2004/032648) width of pH field of activity of asparaginase.Active in 37 ℃ of mensuration.Use that " material and method " chapters and sections are described, 50mM Hydrocerol A with the pH that wants and the altheine mixture of 50mM sodium pyrophosphate buffer solution, measure the activity on the pH scope of pH=4 to pH=9.The enzyme sample that uses is in the scope of 1.5-12 unit/ml.

Table 4 has shown the complete pH activity relationship of measuring to the pH scope of pH=4 to pH=9.

	ASN002	ASN001	Aspergillus?niger	Aspergillus?oryzae
					pH	Relative reactivity (%)	Relative reactivity (%)	Relative reactivity (%)	Relative reactivity (%)
4	10％	18％	100％	4％
					5	75％	76％	97％	50％
6	100％	100％	69％	95％
					7	95％	94％	44％	100％
8	80％	72％	15％	63％
					9	49％	35％	0％	30％

Table 4:ASN002 and ASN001 are than wild-type A.niger(WO2004/030468) and wild-type A.oryzae(WO2004/032648) the pH activity relationship of asparaginase.To every kind of asparaginase, high reactivity is set to 100%.Active in 37 ℃ of mensuration.Use that " material and method " chapters and sections are described, 50mM Hydrocerol A with the pH that wants and the altheine mixture of 50mM sodium pyrophosphate buffer solution, measure the activity on the pH scope of pH=4 to pH=9.The enzyme sample that uses is in the scope of 1.5-12 unit/ml.

< > The stability of variant <>

The activity under given pH, the performance of enzyme also depends critically upon its thermostability between transition phase.For checking has more the thermostability of active variant, under 60 ℃, carry out the activity check.In a kind of check, stop enzyme reaction after 10 minutes, in second kind of check, 30 minutes termination reactions.30 minutes the check in enzyme dosage be 10 minutes the check in dosage 1/3.If under condition of application, enzyme is stable, and observed activity should be similar.Under the situation that inactivation takes place, estimate that activity will reduce after longer proving time.The result is shown in Table 5.

Table 5: the stability of variant.Check is carried out at 60 ℃, the enzyme dosage in the check in 30 minutes be in the check in 10 minutes dosage 1/3, the hatching of the incubation time of corresponding respectively 10 minutes and 30 minutes.

Table 5 shows that the stability of variant and wild-type Aspergillus niger asparaginase are closely similar.When pH=6, pH=7, pH=8, variant ASN002 and ASN001 have active completely at 60 ℃, and hatch the very little reduction of the active only demonstration in back at 30 minutes.Therefore, the activity of increase can be used for the conversion of l-asparagine to aspartic acid fully.Especially, under the pH value in neutrality of measuring and alkalescence zone, the performance of variant is significantly improved.

Claims

1. asparaginase, it is:

A) polypeptide, said polypeptide have the aminoacid sequence shown in any among SEQ ID NO:2 or the SEQ ID NO:4;

B) polypeptide, said polypeptide are according to nucleic acid sequence encoding any among the SEQ ID NO:1 of encoding mature polypeptide or the SEQ IDNO:3.

2. the asparaginase of claim 1; It has the pH optimum value between 6 to 7; And/or, when under similarity condition, measuring, the higher specific activity of specific activity of disclosed wild-type asparaginase from A.niger during it has than WO 2004/030468 under being at least 5 pH.

3. claim 1 or 2 asparaginase, it has at least 3.5 pH field of activity width that pH unit is wide.

4. the asparaginase of claim 3, it has at least 4 pH field of activity width that pH unit is wide.

5. the asparaginase of claim 4, it has at least 5 pH field of activity width that pH unit is wide.

6. the nucleic acid of any one asparaginase in the claim 3 to 5 of encoding.

7. nucleic acid, it comprises:

Dna sequence dna, said dna sequence encoding is according to any one asparaginase among SEQ ID NO:2 or the SEQ ID NO:4.

8. nucleic acid construct, it comprises claim 6 or 7 described nucleic acid, and said nucleic acid is operably connected with one or more control sequence that can instruct asparaginase in suitable expressive host, to express.

9. recombinant expression vector, it comprises the nucleic acid construct of claim 8.

10. recombinant host cell, it comprises the expression vector of claim 9.

11. produce the method for asparaginase, said method is included in is of value to the host cell of cultivating claim 10 under the condition of producing asparaginase, and reclaims said asparaginase.

12. composition, it comprises according to any described asparaginase in the claim 1 to 5 or the asparaginase that obtains according to the method for claim 11.

13. the purposes of composition in producing foodstuff products according to any described asparaginase or claim 12 in the claim 1 to 5.

14. be used for reducing purposes based on the amount of the raw-material acrylamide that forms through the hot-work foodstuff products that contains l-asparagine according to the composition of any described asparaginase or claim 12 in the claim 1 to 5.

15. produce the method for foodstuff products; Wherein relate at least one heating steps; Said method is included in the said working method; In the intermediate product form of said foodstuff products, add composition according to any described asparaginase or claim 12 in the claim 1 to 5; Thus; Before said heating steps, add enzyme with the amount of the level of the asparaginase that exists in the said intermediate product form that can effectively reduce said foodstuff products.

16. be used to make the purposes of the medicine of treating tumour according to the composition of any described asparaginase or claim 12 in the claim 1 to 5.