CN105331593B - A kind of lipase - Google Patents

Abstract

The present invention provides the new polypeptides with lipase active, include the sequence of at least one amino acid substitutions, deletions, or additions it includes the amino acid sequence as shown in SEQ ID NO:1 or in above-mentioned sequence.The present invention also provides the polynucleotides of coding said polypeptide, containing the polynucleotide expression vector and host cells, and the method for production aforementioned polypeptides.In addition, further relating to the purposes of the above-mentioned polypeptide with lipase active.

Description

A kind of lipase

Invention field

The invention belongs to enzyme gene engineerings and enzyme engineering field, and in particular, to polypeptide, its volume with lipase active Code nucleic acid, and expression vector and host cell comprising the code nucleic acid.The invention further relates to the preparation sides of aforementioned polypeptides Method and application thereof.

Background of invention

Lipase is a kind of enzyme with a variety of catalytic capabilities, can be catalyzed triglyceride and be hydrolyzed to glycerol and free rouge Fat acid can also be catalyzed the hydrolysis and transesterification and esters synthetic reaction of other esters.In addition, lipase is also shown pair The enantioselectivity of substrate.The above characteristic imparts lipase in food fats and oils processing, detergent, biodiesel, ester bond chemical combination Extensive use (Abhishek the Kumar Singh, Mausumi of the industry such as the synthesis of object and chiral drug synthesis Mukhopadhyay.Overview of Fungal Lipase:A Review.2012, 166(2):486-520)。

For example, due to the introducing of lipase, grease hydrolysis can carry out at normal temperatures and pressures, therefore in terms of fats and oils processing The biological substances such as highly unsaturated fatty acid and tocopherol will not be made to be denaturalized.In terms of Dairy Processing, using lipase in dairy products Middle progress butterfat hydrolysis, can enhance the flavor of cheese, milk powder, cream, promote the maturation of cheese, improve the quality of dairy products.? Wheaten food processing aspect, add lipase, make wheaten food elasticity improve, improve sense of food, improve bread preservativity (Wang Zishe, After Ma Junyi, Shi Rui Agriculture of Anhui science .2011,39 (7): 3798-3800).In terms of medical treatment, lipase is as diagnosis Disease can be predicted, as lipase can be used for detecting acute pancreatitis and injury of pancreas in serum in tool.Lipase is raw in drug Produce, lose weight etc. also has application.In terms of biodiesel synthesis, enzyme process with extraction and purification process, throw by simple, equipment The advantages that money is less, energy consumption is low, pollution is small, increasingly causes the common concern of people, wherein the Novozym 435 of fabric film immobilization It is the common enzyme for producing biodiesel with Candida sp.99-125.In terms of detergent industry, 1988, Novozymes Company was first It first can effectively introduce to the market fatty enzyme and except the detergent of degreasing, be developed using genetic engineering

Lipase is widely present in animals and plants and microorganism, since not only type is more, breeding is fast, easily loses for microorganism The progress of disease is different, and has pH value more wider than animal, temperature range and Substratspezifitaet, therefore microbial lipase is work The important sources of industry lipase.According to statistics, the microorganism of yielding lipase has 65 categories, is concentrated mainly on aspergillus niger, false silk ferment The bacterial strains such as mother, head mold, pseudomonad, streptomycete and pseudomonas alcaligenes (Wang Zishe, Zhang Ji, Ma Junyi, Shi Rui Agriculture of Anhui section It learns .2011,39 (7): 3798-3800).

Lipase using can produce fragrance, therefore can be applied in cheese manufacture in dairy products.Traditionally, it uses From the ruminant animal fats enzyme preparation such as goat or calf.Preceding gastric tissue of these preparations from these animals, also referred to as Preceding gastric lipase.There are commercial reagents in the marketC, L, KG and K (DSM Food Specialties, lotus It is blue).These lipase are used in a variety of Italy, Spain, Greece and French cheese preparation, these type cheese maturation periods Between the generation of specific fragrance spectrum be largely attributable to effect of the lipase to butter oil.The water of lipase-catalyzed butter oil Solution generates free fatty acid.The fatty acid can have short chain (C₄-C₆Fatty acid, i.e. butyric acid, caproic acid) and in arrive long-chain (C₁₂-C₁₈) fatty acid.Subsequent free fatty acid may participate in chemical reaction, such as perfume compound, as ethyl acetate, beta-keto acid, The formation of methyl ketone, ester and lactone.The conversion of fatty acid in aroma constituent can be with origin from the microbial population in cheese Enzymatic.

The lipase used can influence the type of the free fatty acid discharged in cheese, for example, pungent, strong perfume (or spice) Taste occurs mainly with release short chain fatty acids (C₄-C₆) lipase, and middle long chain fatty acids can then generate saponiform taste. So many researchs are dedicated to for lipase transforming short chain preference type as, manufactured with being applied to cheese.

Summary of the invention

In a first aspect, the polypeptide with lipase active is provided, it includes sequence selected from the following or by selected from following Sequence composition:

(a) amino acid sequence as shown in SEQ ID NO:1, and

(b) sequence that the sequence as described in (a) obtains after replacing, missing or adding at least one amino acid, wherein Lipase active is still kept by the polypeptide variants that (b) is obtained.

In optional embodiment, aforementioned polypeptides and heterologous polypeptide.

In one embodiment, polypeptide of the invention includes amino acid sequence shown in SEQ ID NO:1.Preferred real It applies in scheme, aforementioned polypeptides amino acid sequence shown in SEQ ID NO:1 forms.Herein, as shown in SEQ ID NO:1 Amino acid sequence composition polypeptide be named as FL2.

Second aspect provides the polynucleotides for encoding polypeptide of the invention, it includes sequence selected from the following or by selecting It is formed from sequence below:

(a) nucleotide sequence encodes amino acid sequence shown in SEQ ID NO:1 or in above-mentioned sequence comprising at least The sequence of one amino acid substitutions, deletions, or additions；And

(b) under strict conditions with a) in nucleotide sequence hybridization nucleotide sequence.

In one embodiment, it includes nucleotide sequences shown in SEQ ID NO:2 for polynucleotides of the invention.Excellent In the embodiment of choosing, above-mentioned polynucleotides nucleotide sequence shown in SEQ ID NO:2 is formed.

In one embodiment, polynucleotides of the invention are generated by artificial synthesized generation or by recombination , preferably synthetic generation.

The third aspect provides expression vector, and it includes at least one above-mentioned polynucleotides.

In certain embodiments, expression vector of the invention also includes the regulating and controlling sequence for adjusting polynucleotides expression, Middle polynucleotides are operably connected with regulating and controlling sequence.In preferred embodiments, the expression vector is pET24a (+).

Fourth aspect provides the host cell comprising polynucleotides or expression vector of the invention.Preferably implementing In scheme, host cell is e. coli bl21 (DE3).

5th aspect, provides the method for preparing polypeptide of the invention comprising:

1) polynucleotides described in any one of claim 3-5 are cloned on expression vector,

2) expression vector is converted or is transduceed into suitable host cell,

3) host cell in suitable culture medium, is cultivated,

4) it is separated from the host cell or culture medium and purifies the polypeptide.

In preferred embodiments, the polypeptide that preparation amino acid sequence shown in SEQ ID NO:1 forms is provided Method comprising: the nucleotide sequence of amino acid sequence shown in SEQ ID NO:1 will be encoded and be cloned in plasmid expression vector In, and the plasmid expression vector with the polynucleotide sequence is converted to Escherichia coli and carries out inducing expression, then from big It is separated in enterobacteria and purifies FL2 polypeptide.

6th aspect, provides polypeptide of the invention and is preparing the purposes in dairy products.In one embodiment, above-mentioned cream Product is cheese.

Polypeptide of the invention has short chain Preference and/or one or more characteristics below: within the scope of extensive pH With good enzymatic activity and stability, there is certain surfactant tolerance, and shown in the presence of potassium ion High lipase active.

Brief Description Of Drawings

Fig. 1 shows the gel electrophoresis figure of FL2 polypeptide.1st swimming lane is molecular weight marker, and the 2nd swimming lane is FL2 polypeptide.

When Fig. 2 is shown respectively using pNPP and pNPB as substrate, the enzyme activity of FL2 at different temperatures.

When Fig. 3 is shown using pNPP as substrate, enzyme activity of the FL2 at different pH.

When Fig. 4 is shown using pNPP as substrate, the stability of lipase active of the FL2 at different pH.

Fig. 5 is shown respectively with 4- Nitrophenyl butyrate (pNPB), 4- nitrobenzophenone caprylate (pNPO), 4- nitro Phenyl laurate (pNPD), 4- nitrobenzophenone myristinate (pNPM), 4- nitrobenzophenone palmitate (pNPP) and 4- nitre When base phenyl stearic acid ester (pNPS) is used as substrate, Hydrolytic catalyzing of the FL2 as lipase.

When Fig. 6 is shown respectively using pNPP and pNPB as substrate, influence of the metal ion to FL2 lipase activity.It is right It is to add water in the reaction system according to group, remaining group adds ZnSO respectively in the reaction system₄、MnCl₂、CoCl₂、CaCl₂、 MgSO₄、CuSO₄、KCl、(NH4)₂SO₄、 NaCl、NiSO4、FeCl₃Liquid is stored with EDTA inorganic salts.

When Fig. 7 is shown respectively using pNPP and pNPB as substrate, shadow of the surfactant for FL2 lipase activity It rings.Control group adds water in the reaction system, remaining group adds 0.5% cation surface activating respectively in the reaction system CTAB, anionic surfactant SDS, nonionic surfactant Tween80, AEO-9 and Triton X-100.

Specific embodiment

Polypeptide of the invention

This application provides the polypeptides with lipase active, it includes sequence selected from the following or by sequence selected from the following Column composition:

(a) amino acid sequence as shown in SEQ ID NO:1, and

(b) sequence that the sequence as described in (a) obtains after replacing, missing or adding at least one amino acid, wherein Lipase active is still kept by the polypeptide variants that (b) is obtained.

In certain embodiments, the number of above-mentioned amino acid substitutions, deletions, or additions is 1-30, preferably 1-20 It is a, more preferably 1-10, wherein the polypeptide variants obtained are kept substantially the lipase active of unchanged albumen.Preferred Embodiment in, aforementioned polypeptides variant differs about 1 with amino acid sequence shown in SEQ ID NO:1,2,3,4,5,6,7,8, Substitution, the deletion and/or addition of 9 or 10 amino acid.In a more preferred embodiment, aforementioned polypeptides variant and SEQ ID Amino acid sequence shown in NO:1 differs replacing, missing or adding for about 1,2,3,4 or 5 amino acid.

In one embodiment, polypeptide of the invention includes amino acid sequence shown in SEQ ID NO:1.Preferred real It applies in scheme, aforementioned polypeptides amino acid sequence shown in SEQ ID NO:1 forms.Herein, as shown in SEQ ID NO:1 Amino acid sequence composition polypeptide be named as FL2.

In optional embodiment, aforementioned polypeptides and heterologous polypeptide form fusion protein.

As used herein, term " amino acid " indicates naturally occurring and non-naturally occurring amino acid and amino acid Analog and analogies.Naturally occurring amino acid include protein biology synthesis used in 20 kinds of (L)-amino acid and other Amino acid, such as 4-Hydroxyproline, hydroxylysine, desmosine, isodensmosine, homocysteine, citrulling and ornithine.Non- day So existing amino acid includes such as (D)-amino acid, nor-leucine, norvaline, p- fluorophenylalanine, ethionine etc., These are known to the skilled in the art.Amino acid analogue includes the modification shape of natural and non-naturally occurring amino acid Formula.This modification may include the derivatization of chemical group on such as substituted amino acid and part or amino acid.Amino acid Analogies include the organic structure for for example showing functionally similarity, and the charge and charge of the property such as amino acid are empty Between characteristic.For example, the organic structure of simulation arginine (Arg or R) have be located at similar molecule space and with and naturally deposit Arg amino acid side chain e- amino same degree ambulant positive charge part.Analogies further include restraining structure To maintain the optimal spatial and charge interaction of amino acid or amino acid functional group.Those skilled in the art can determine anything Structure constitutes functionally equivalent amino acid analogue and amino acid simulant.

In some embodiments, the variant of amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:1 have extremely Few 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology.Preferred Embodiment in, sequence shown in polypeptide variants and SEQ ID NO:1 has 99% or more homology.

" homology " as described herein is defined as by sequence alignment and after introducing vacancy, amino acid or nucleotide sequence The percentage of identical residue in variant, if it is desired, reach the homology of largest percentage.Method and calculating for comparison Machine program is well known in the art.

" polypeptide " and " albumen " of the invention used interchangeably herein, refer to amino acid residue polymer and its variant and Synthesis and naturally occurring analog.Therefore, these terms are suitable for naturally occurring amino acid polymer and its naturally deposit Chemical derivative and wherein one or more amino acid residues be synthesis non-naturally occurring amino acid (such as phase The chemical analog for the naturally occurring amino acid answered) amino acid polymer.This analog derivative includes such as posttranslational modification And catabolite, the phosphorylation including polypeptide fragment shown in SEQ ID NO:1, glycosylated, oxidation, isomerization sum Deaminated variant.

In preferred embodiments, the sequence of FL2 polypeptide variants is in the amino acid sequence shown in SEQ ID NO:1 Comprising the sequence that one or several conservative amino acids replace, wherein the sequence after being substituted still keeps catalytic activity of lipase.

Certain amino acid substitutions of referred to as " conserved amino acid substitution " can frequently occur in protein, but not change The conformation or function of the protein, this is established rule in protein chemistry.

It includes but is not limited to use glycine (G), alanine (A), isoleucine that conserved amino acid in the present invention, which replaces, (I), any of valine (V) and leucine (L) replace these aliphatic amino acids it is any another；Use serine (S) replace threonine (T), vice versa；Replace glutamic acid (E) with aspartic acid (D), vice versa；With glutamine (Q) Replace asparagine (N), vice versa；Replace arginine (R) with lysine (K), vice versa；With phenylalanine (F), junket ammonia Sour (Y) and tryptophan (W) replace in these aromatic amino acids it is any another；And replace half with methionine (M) Cystine (C), vice versa.Others replace can also be considered to be conservative, this depend on specific amino acid environment and Its effect in protein three-dimensional structure.For example, glycine (G) and alanine (A) often can be interchanged, such as alanine (A) It can be interchanged with valine (V).The methionine (M) of relative hydrophobic can be exchanged often with leucine and isoleucine, and Sometimes it is exchanged with valine.Lysine (K) and arginine (R) are often in following location swap: wherein amino acid residue is important It is characterized in that the different pK of its charge and both amino acid residues are not obvious.Under particular circumstances, still have some other Variation can be considered as " conservative " (see, e.g., BIOCHEMISTRY at pp.13-15,2^nd ed.Lubert Stryer ed.(Stanford University)；Henikoff et al.,Proc.Nat'l Acad.Sci.USA(1992) 89:10915-10919；Lei et al.,J.Biol.Chem.(1995)270(20):11882-11886).

Hereinafter, by amino acid residue by substitutive residue classification citing, but substitutive amino acid residue be not limited to The residue of lower record:

A group: leucine, isoleucine, nor-leucine, valine, norvaline, alanine, 2-amino-butyric acid, egg ammonia Acid, O- methyl serine, t-butylglycine, t-butylglycine and Cyclohexylalanine；

B group: aspartic acid, glutamic acid, different aspartic acid, isoglutamic acid, 2-aminoadipate and 2- amino suberic acid；

C group: asparagine and glutamine；

D group: lysine, arginine, ornithine, 2,4- diaminobutyric acid, that is, 2,3- diaminopropionic acid；

E group: proline, 3- hydroxy-proline and 4- hydroxy-proline；

F group: serine, threonine and homoserine；

G group: phenylalanine and tyrosine.

For example, present inventor has found, be located at FL2 polypeptide the 5th, 7,8,9,11,18,19,21,21,23,28,30, 35、38、59、62、68、69、70、71、75、80、 82、87、88、90、91、92、93、95、96、97、100、105、108、109、 114、 115、132、133、137、138、140、143、147、151、152、153、157、158、 160、161、164、165、 166, the polypeptide after 167,168,169,172,173, and/or 174 amino acids generation conservative replaces is kept substantially FL2's Lipase active.

In other specific embodiments, the C-terminal of FL2 polypeptide or N-terminal region can also be truncated about 1,2,3,4,5, 6,7,8,9,10,11,12,13,14,15,20,25 or more amino acid, and still with the lipase active of FL2.

In a further embodiment, can also the C-terminal of FL2 polypeptide or N-terminal region addition 1,2,3,4,5,6,7,8, 9,10,11,12,13,14,15,20,25 or more amino acid, obtained FL2 variant still have catalytic activity of lipase.

Further, it is also possible to other than the C-terminal of FL2 polypeptide or N-terminal region addition or missing 1,2,3,4,5,6,7,8,9, 10,11,12,13,14,15,20,25 or more amino acid, as long as the polypeptide after changing is kept substantially the lipase activity of FL2 Property.

In certain embodiments, polypeptide of the invention, such as FL2 polypeptide or its variant and heterologous polypeptide.One In a little embodiments, FL2 fusion protein is kept substantially the lipase active of FL2.In certain embodiments, heterologous polypeptide with The N-terminal of FL2 polypeptide connects.In certain embodiments, heterologous polypeptide is connect with the C-terminal of FL2 polypeptide.In these embodiments In, heterologous polypeptide (such as can wrap selected from purification tag (such as can include but is not limited to: GST, MBP), epitope tag Include but be not limited to: Myc, FLAG), targeting sequence, signal peptide etc..

In a particular embodiment, fusion protein includes FL2 polypeptide and label, the end C- of the label and FL2 polypeptide Or the end N- combines, usually peptide tag.The label usually can be used for separating and purifying the fusion protein peptide or Amino acid sequence.Therefore, the label can be with one or more ligand bindings, for example, such as chromatographic supports or Gao Qinhe One or more ligands of the affinity substrate of power magnetic bead.The example of the label is can be with high-affinity and nickel (Ni²⁺) column or Cobalt (Co²⁺) histidine tag (His- label or HT) that combines of column, such as the mark comprising 6 histidine residues (His6 or H6) Label.Other example tags for being used for isolated or purified fusion protein include Arg- label, FLAG- label, Strep- label etc..

Polynucleotides

This application provides the polynucleotides of encoding such polypeptides, it includes sequence selected from the following or by selected from the following Sequence composition:

(a) nucleotide sequence encodes amino acid sequence shown in SEQ ID NO:1 or in above-mentioned sequence comprising at least The sequence of one amino acid substitutions, deletions, or additions；And

(b) under strict conditions with a) in nucleotide sequence hybridization nucleotide sequence.

In certain specific embodiments, polynucleotide encoding FL2 polypeptide and its function equivalent modifications of the invention.? In one embodiment, polynucleotides of the invention and coding FL2 and its function equivalent modifications polynucleotides have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology.

In certain embodiments, polynucleotides of the invention include to have with nucleotide sequence shown in SEQ ID NO:2 There is the homology of at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% Nucleotide sequence.In preferred embodiments, polynucleotides of the invention include nucleotides sequence shown in SEQ ID NO:2 Column.

In preferred embodiments, polynucleotides of the invention with nucleotide sequence shown in SEQ ID NO:2 as having There is the nucleotide sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology Composition.In a more preferred embodiment, the polynucleotides of the invention nucleotide sequence shown in SEQ ID NO:2 forms.

Terms used herein " polynucleotides " or " nucleic acid " refer to mRNA, RNA, cRNA, cDNA or DNA, including it is single-stranded and The DNA of double-stranded form.The term is often referred to the nucleotide Multimeric forms of at least ten bases longs, and the nucleotide is ribose core The modified forms of thuja acid or deoxynucleotide or any type of nucleotide.

In certain embodiments, polynucleotides of the invention include and coding FL2 polypeptide and its function equivalent modifications The nucleotide sequence that nucleotide sequence hybridizes under strict conditions, or by with coding FL2 polypeptide and its function equivalent modifications Nucleotide sequence specific hybrid and the nucleotide sequence composition for encoding the polypeptide being functionally equal with FL2 polypeptide.

Those skilled in the art can be with the stringent condition of conventional selection DNA hybridization.Usually longer probe needs higher Temperature, to carry out annealing appropriate, and shorter probe needs lower temperature.Hybridization is generally depended on when complementary strand is in Lower than the reannealing ability of the environment time variation DNA of its melting temperature.It probe and can get over degree of homology between hybridization sequences Height, the relative temperature that can be used are higher.Then, higher relative temperature often makes reaction condition tightened up, and lower At a temperature of, then stringency is lower.About the detailed description of hybridization reaction stringent condition, Ausubel etc., Current are seen Protocols in Molecular Biology,Wiley Interscience Publishers,(1995)。

In certain embodiments, using low ionic strength and height when the stringent condition that DNA hybridization uses includes: 1) washing 0.015M sodium chloride/0.0015M sodium citrate/0.1% lauryl sodium sulfate at temperature, such as 50 DEG C；2) it is used when hybridizing (v/v) formamide adds 0.1% bovine serum albumin(BSA)/0.1%Ficoll/ 50% at the denaturants such as formamide, such as 42 DEG C 0.1% polydiene pyrrolidones/pH6.5 50mM sodium phosphate buffer and 750mM sodium chloride, 75mM sodium citrate；Or (3) overnight hybridization at 42 DEG C, hybridization solution contain 50% formamide, 5x SSC (0.75M sodium chloride, the rotten 1 lemon acid of 0.075M Sodium), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5xDenhardt ' s solution, ultrasonication salmon sperm dna (50.mu.g/ml), 0.1%SDS and 10% dextran sulfate, then in 42 in 0.2x SSC (sodium chloride/sodium citrate) DEG C washing 10 minutes, then with the 0.1x SSC containing EDTA in 55 DEG C of progress high stringency degree washings.Medium stringency condition can be pressed Sambrook etc., Molecular Cloning:A Laboratory Manual, NewYork:Cold Spring Harbor Press, the description in 1989 are determined.Medium stringency condition include using stringency lower than washing solution described above with Hybridization conditions (such as temperature, ionic strength and SDS percentage).For example, medium stringency condition includes at least about 16%v/v to extremely The formamide and at least about 0.5M of few about 30%v/v hybridizes at least about salt of 0.9M at 42 DEG C, and is arrived at least about 0.1M At least about 0.2M salt is washed at 55 DEG C.Medium stringency condition can also include with 1% bovine serum albumin(BSA) (BSA), 1mM EDTA, 0.5M NaHPO4 (pH7.2), 7%SDS hybridize at 65 DEG C, and with (i) 2 × SSC, 0.1%SDS；Or (ii) 0.5%BSA, 1mM EDTA, 40mM NaHPO4 (pH47.2), 5%SDS are washed at 60-65 DEG C.Professional will according to probe length etc. because Element adjusts temperature, ionic strength etc..Stringency when hybrid nucleic acid depends on length of nucleic acid molecule and complementarity and other Variable well known in the art.Similitude or homology between two nucleotide sequences is bigger, then contains these sequences Nucleic acid hybrid Tm it is bigger.The relative stability (corresponding to higher Tm) of nucleic acid hybridization successively decreases in the following order: RNA: RNA,DNA:RNA,DNA:DNA.Preferably, can the minimum length of hybrid nucleic acid be at least about 12 nucleotide, preferably at least about For 16, more preferably at least about 24, most preferably at least about 36 nucleotide.

Polynucleotides of the invention can be combined with other DNA sequence dnas, other described DNA sequence dnas such as promoter, poly- gland Glycosidation signal, other restriction enzyme sites, multiple cloning sites, other encoding segments etc., show their total length It writes different.Therefore consideration can use the polynucleotide passage of almost random length；Total length is preferably by expected recombinant DNA side The limitation of the convenience made and used in case.

Any one of known in the art and obtainable a variety of mature technologies be can use to prepare, manipulate And/or express polynucleotides and its fusions.For example, encoding the polynucleotide sequence of polypeptide or its variant of the invention can use To instruct polypeptide to express in host cell appropriate in recombinant DNA molecules.Due to the intrinsic degeneracy of genetic codon, compile Other DNA sequence dnas of the substantially the same or functionally equivalent amino acid sequence of code can be used in the present invention, and this A little sequences can be used for cloning and expressing given polypeptide.

In addition it is possible to use polynucleotide sequence of the invention is transformed in method well known in the art, including but not limited to Clone, processing, expression and/or the active change of gene product.

In certain embodiments, polynucleotides of the invention pass through artificial synthesized generation, such as direct chemical synthesis Or enzymatic synthesis.In alternative embodiments, above-mentioned polynucleotides are generated by recombinant technique.

It in certain embodiments, can be by conventional method preferably such as dideoxy chain termination (Sanger et Al.PNAS, 1977,74:5463-5467) measurement polynucleotides obtained sequence.This kind of polynucleotide sequence measurement can also It is completed with the sequencing kit of purchase.In order to obtain the cDNA sequence of overall length, sequencing need to be repeated.Sometimes for measurement The cDNA sequence of multiple clones can just be spliced into the cDNA sequence of overall length.

Expression vector

This application provides the expression vectors comprising polynucleotides of the invention.

" expression vector " of the present invention is the nucleic acid construct recombinantly or synthetically generated, special with a series of permissions The specified nucleic acid elements that fixed nucleic acid is transcribed in host cell.Expression vector of the invention can be such as pET-24a The plasmid of (+), pIRES2-EGFP, pcDNA3.1, pCI-neo, pDC516, pVAC, pcDNA4.0, pGEM-T, pDC315 carry Body, or such as adenovirus, adeno-associated virus, retrovirus, semliki forest virus (sFv) carrier viral vectors, Either other carriers well known in the art.

In certain embodiments, it encodes FL2 polypeptide and its polynucleotide sequence of variant is cloned into carrier, with structure At the recombinant vector for containing polynucleotides of the present invention.

In preferred embodiments, the expression vector for cloning polynucleotides is plasmid vector.Preferred real It applies in scheme, the plasmid vector is pET-24a (+).

In specific embodiments, above-mentioned expression vector also includes the regulating and controlling sequence for adjusting polynucleotides expression, wherein The polynucleotides are operably connected with the regulating and controlling sequence.

The term as used herein " regulating and controlling sequence " refers to the polynucleotides for realizing that coded sequence expression connected to it is required Sequence.The property of this kind of regulating and controlling sequence changes with host organism.In prokaryotes, this kind of regulating and controlling sequence generally comprises starting Son, ribosome bind site and terminator；In eucaryote, this kind of regulating and controlling sequence generally comprise promoter, terminator and Enhancer in some cases.Therefore, term " regulating and controlling sequence " include its exist to the expression of target gene be it is required most The all sequences of lower bound degree also may include that its presence is advantageous other sequences, such as leader sequence to destination gene expression.

The term as used herein " being operably connected " refers to following situation: involved sequence be in allow they with Among the relationship that desired mode works.Thus, for example the regulating and controlling sequence of " being operably connected " to a coded sequence makes The expression of the coded sequence is realized under conditions of compatible with the regulating and controlling sequence.

In certain embodiments, using method well-known to those having ordinary skill in the art building comprising coding FL2 polypeptide and The nucleotide sequence of its variant and suitable transcription/translational control element expression vector.These methods include extracorporeal recombinant DNA (Sambroook, et the al.Molecular Cloning, a such as technology, DNA synthetic technology, In vivo recombination technology Laboratory Manual,cold Spring Harbor Laboratory.New York,1989).Nucleotide sequence can be grasped It is connected in the appropriate promoter in expression vector with making, to instruct mRNA to synthesize.The representative example of these promoters includes: Lac the or trp promoter of Escherichia coli；The PL promoter of λ bacteriophage；Eukaryotic promoter include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retrovirus LTRs and some other is known controllable The promoter that gene is expressed in prokaryotic cell or eukaryocyte or its virus.Expression vector further includes the ribose of translation initiation Body binding site and transcription terminator etc..Insertion enhancer sequence will make its transcription in higher eucaryotic cells in the carrier Enhanced.Enhancer is the cis-acting factors of DNA expression, generally about has 10 to 300 base-pairs, acts on starting Son is to enhance the transcription of gene.Example includes enhancing in the SV40 of 100 to 270 base-pairs of replication origin advanced stage side Son, in the polyoma enhancer of replication origin advanced stage side and adenovirus cancers etc..

In addition, expression vector preferably includes one or more selected markers, to provide for selecting conversion The phenotypic character of host cell, such as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescence egg White (GFP), or tetracycline for Escherichia coli or amicillin resistance etc..

Host cell

This application provides the host cells comprising polynucleotides or expression vector of the invention.

In certain embodiments, the polynucleotides or containing the polynucleotide table of FL2 polypeptide and its variant will be encoded It is transformed or transduced into host cell up to carrier, obtains the genetically engineered host cell containing the polynucleotides or expression vector.

Host cell used herein can be any host cell well known to those skilled in the art, including protokaryon is thin Born of the same parents, eukaryocyte, such as bacterial cell, fungal cell, yeast cells, mammalian cell, insect cell or plant cell etc.. Illustrative bacterial cell includes Escherichia, Bacillus, streptomyces, Salmonella, pseudomonas and grape ball Any kind in Pseudomonas, including such as Escherichia coli, Lactococcus, bacillus subtilis, wax printing fabric, mouse typhus sramana Salmonella, Pseudomonas fluorescens.Illustrative fungal cell includes any kind of aspergillus.Illustrative yeast cells is red including finishing Any kind in saccharomyces, brewer's yeast Pseudomonas, Schizosaccharomyces or Blastocystis, including Pichia pastoris, brewer's yeast or split Grow yeast.Illustrative insect cell includes any kind in prodenia litura or drosophila, including drosophila S2 and prodenia litura Sf9. Illustrative zooblast includes CHO, COS or melanoma or any mouse or human cell line.Select suitable host at this In the limit of power of field technical staff.

Can use any technology known in the art by expression vector import host cell in, including conversion, transduction, turn The gene transfer that dye, virus infection, particle gun or Ti- are mediated.Specific method includes calcium phosphate transfection, DEAE- glucan Jie (Davis, L., Dibner, M., Battey, I., Basic the Methods in such as transfection, fat transfection or electroporation for leading Molecular Biology,(1986)).As an example, when host is prokaryotes such as Escherichia coli, it can be in exponential growth Competent cell is harvested after phase, with CaCl well known in the art₂Method is converted.

In specific embodiments, the host cell used in the present invention is Escherichia coli.In preferred embodiments, The expression vector for carrying polynucleotide sequence of the present invention is converted to e. coli bl21 (DE3) and carries out inducing expression.

The preparation method of polypeptide of the invention

Polypeptide of the invention can be prepared by any suitable method known to those skilled in the art, such as by recombinating skill Art generates or chemical synthesis.The method for generating recombinant peptide is known in the art.The chemical synthesis process of peptide is also this field Known to technical staff, for example, polypeptide and its change of the invention can be generated by using the orientation peptide synthesis of solid phase technique Body (Merrifield, J.Am.Chem.Soc.85:2149-2154 (1963)).It can be carried out with either manually or by automation Albumen synthesis.For example, can realize automation with the 431A peptide synthesizer (Perkin Elmer) of Applied Biosystems Synthesis.Selectively, different piece degree can be synthesized respectively by chemical mode and is combined using chemical method to prepare Required molecule.

Fusion protein caused by polypeptide and heterologous polypeptide of the invention can also be obtained by conventional means, such as Nucleotides sequence by making encoding said fusion protein, which is listed in suitable yeast cells, carries out gene expression.If desired, energy It is enough to carry out fusion protein described in isolated or purified using whole label (eventual tag).

In specific embodiments, the method for preparing polypeptide of the invention is provided comprising:

1) polynucleotides for encoding polypeptide of the present invention are cloned on expression vector,

2) expression vector is converted or is transduceed into suitable host cell,

3) host cell in suitable culture medium, is cultivated,

4) it is separated from the host cell or culture medium and purifies the polypeptide

Suitable host cell refers to the host cell suitable for expression vector or polynucleotide of interest expression.Suitable culture Base refers to the culture medium for carrying out suitable for host cell growth or to it inducing expression.

In certain embodiments, according to host cell used, various conventional mediums be can choose.It is being suitable for host It is cultivated under conditions of cell growth.Preferably, the host cell of engineering can be suitable for activating promoter being modified It is cultivated in conventional nutrient culture, to screen transformant or expand polynucleotides of the invention.It is thin to convert suitable host Born of the same parents and after host cell growth is to cell density appropriate, are induced with suitable method (such as temperature transition or chemical induction) and are selected Cell is further cultured for a period of time by the promoter selected, to allow it to produce desired polypeptides or its segment.

In certain embodiments, host cell is harvested by centrifugation, by either physically or chemically smudge cells, and Obtained crude extract is preserved for being further purified.Microbial cell for protein expression can pass through any convenient side Method is crushed, including Frozen-thawed cycled, ultrasound, Mechanical Crushing or use cell cracking agent.These methods are those skilled in the art Known to member.

In certain embodiments, the recombinant polypeptide of host cell production can be coated in the intracellular or table on cell membrane It reaches or is secreted into extracellular.If desired, various separation methods point can be passed through using its physics, chemical and other characteristics From the albumen with purifying recombination.For example, the polypeptide of expression or its segment can be by following methods well known in the art from recombination Recycle and purify in cell culture: conventional renaturation process, protein precipitant processing (salting-out method), centrifugation, permeate broken bacterium, Ultrasonication, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and the combination of various other liquid chromatography technologies and these methods.As exemplary illustration, in the end C- or the end N- The affinity chromatography purifying of albumen comprising peptide tag (such as His- label etc.) is for obtaining the normal of high-purity polypeptide formulations Rule method.

In preferred embodiments, the polypeptide that preparation amino acid sequence shown in SEQ ID NO:1 forms is provided Method comprising: the polynucleotides that the nucleotide sequence shown in SEQ ID NO:2 forms are cloned in expression vector In pET24a (+), and the pET24a (+) with the polynucleotide sequence is converted to e. coli bl21 (DE3) and is lured Expression is led, then separated from e. coli bl21 (DE3) and purifies FL2 polypeptide.

The application of polypeptide with lipase active

Polypeptide of the invention is the polypeptide with lipase active, can be catalyzed the hydrolysis of grease, especially catalysis butterfat The hydrolysis of fat.More specifically, polypeptide of the invention being capable of the sour ester bond between glycerol hydroxy groups of hydrolyzed fat.

In some embodiments, polypeptide of the invention be the lipase with short chain Preference, can be used in generate and/ Or strengthen the fragrance of dairy products.

Therefore, this application provides polypeptides of the invention to prepare the purposes in dairy products.In preferred embodiments, Above-mentioned dairy products are cheese.

In the specification and claims, word "include", "comprise" and " containing " mean " including but not limited to ", and It is not intended to exclude other parts, additive, component or step.

It should be understood that the feature described in certain aspects of the present disclosure, embodiment or embodiment, characteristic, component or Step is applicable to any other aspect, embodiment or embodiment described herein, unless contradiction therewith.

Above disclosure generally describes the present invention, passes through the further example present invention of the following examples.Description These embodiments only to illustrate the invention, are not intended to limit the scope of the invention.Although be used herein special term and Value, these terms and value are equally understood to illustratively, not delimit the scope of the invention.Unless specifically stated otherwise, this explanation Experimental method and technology in book methods and techniques known to those skilled in the art.

Embodiment

The expression and purifying of embodiment 1:FL2 polypeptide

Nucleotide sequence shown in SEQ ID NO.:2 (its coded sequence polypeptide as shown in SEQ ID No.:1) is by giving birth to The synthesis of work bioengineering (Shanghai) limited liability company, and be cloned on expression vector pET24a (+) by the said firm.

PET24a (+) conversion with the nucleotide sequence is subjected to inducing expression (tool into e. coli bl21 (DE3) Body is referring to the Molecular Cloning:A Laboratory guide third edition, Science Press 2002 [beauty] J. Pehanorm Brooker, D.W Russell writes, yellow Training hall etc. is translated), conversion condition is as follows: heat shock 50 seconds at 42 DEG C, ice bath 2 minutes, applies LB plate, picking transformant is seeded in LB training It supports in base (peptone 10g/L, yeast extract 5g/L and sodium chloride 10g/L), carries out staying overnight seed culture in 37 DEG C, by 1% Inoculum concentration, by seed culture fluid be inoculated into expression culture medium in, cultivate in 37 DEG C, 220rpm to OD600=0.6-0.8.

After being cooled to 20 DEG C, isopropyl-beta D-thio galactopyranoside (IPTG) 0.1mM is added and is induced, in 20 DEG C, 190rpm expresses overnight.After inducing expression, thalline were collected by centrifugation.With lysis buffer (50mM sodium dihydrogen phosphate, 300mM sodium chloride, 10mM imidazoles, pH8) thallus is resuspended, wherein every gram of cell adds 5 milliliters of lysis buffers.

The thallus of resuspension is carried out low temperature ultrasonic smudge cells, and (50% voltage output, ultrasonication in 2 seconds are spaced, altogether for 9 seconds Ultrasound 20 minutes), it is sample ice bath is cooling with protected protein when operation.After clasmatosis, 20 minutes are centrifuged simultaneously with 14000rpm Collect supernatant.

1 milliliter of Ni-NTA resin ice bath is added in every 100 milliliters of supernatants to vibrate 30 minutes.By clasmatosis supernatant Ni tree Rouge is transferred in Ni-NTA Agarose chromatographic column (Qiagen, Cat. No.30210), all passes through Ni to clasmatosis supernatant After resin, first with the elution buffer 1 of 20 column volumes (50mM sodium dihydrogen phosphate, 300mM sodium chloride, 20mM imidazoles, pH8) Cleaning, then it is clear with the elution buffer 2 (50mM sodium dihydrogen phosphate, 300 mM sodium chloride, 50mM imidazoles, pH8) of 20 column volumes It washes, it is finally clear with the elution buffer 3 of 5 column volumes (50mM sodium dihydrogen phosphate, 300mM sodium chloride, 250mM imidazoles, pH8) It washes, and collects eluent, by eluent in 4 DEG C of dialysed overnights, wherein the formula of dialyzate used are as follows: 150mM sodium chloride, 20mM Tris-HCl, 10mM zinc sulfate, 1mM dithiothreitol (DTT), pH8.Pass through gel electrophoresis (10%SDS-PAGE, 100V, 2 Hour) testing result as shown in Fig. 1.According to Fig. 1 as a result, acquired solution is the FL2 protein solution of purifying.

Embodiment 2: the zymologic property of lipase FL2

The measuring method of lipase activity

Using colorimetric method for determining low-temperature lipase vigor.With p-nitrophenyl palmitate (pNPP) or p-nitrophenyl fourth Acid esters (pNPB) is substrate, digests the production quantity for generating p-nitrophenol (pNP) within the unit time with the enzyme solution of unit volume Carry out the calculating of enzyme activity.The specific method is as follows: be pre-configured with substrate and buffer, substrate: (isopropanol is molten by 6mg/mL pNPP Solution) (same configuration 6mg/mL pNPB), buffer: 0.05M Tris (pH8.0,0.1% Arabic gum).By substrate and buffering Liquid is made into reaction mixture with 1:9 (v/v).Take two 2mL centrifuge tubes, respectively control tube and sample cell.400 μ are added respectively L reaction mixture is to two centrifuge tubes, in suitable reaction temperature (such as 35 DEG C) pre- warm bath 5min.It is added into sample cell certain The dilution enzyme solution of amount continues warm bath 15min after mixing.1.5mL ethyl alcohol is added and is terminated to above-mentioned two centrifuge tube and reacts, and The dilution enzyme solution of same amount is added to control tube.It is centrifuged 2min with 12000rpm, takes supernatant, surveys the light absorption value at 405nm.

Enzyme activity unit is defined as: 1 unit refers to the pNP of 1 μm of ol of catalysis release per minute under standard laboratory conditions Required enzyme amount.According to enzyme activity calculation formula obtained by standard curve are as follows: A=- ([A1-A0] × 0.7885-0.0118) × V1 ×n/(V2×t).A: sample enzyme activity (U/mL) A1: the OD405 of sample enzyme solution, A0: compares the OD405 of enzyme solution, V1: overall reaction The volume (mL) of liquid, n: the extension rate of enzyme solution, V2: the volume (mL) of enzyme solution, t: reaction time (min).

Optimal reactive temperature

According to the lipase activity of above-mentioned pNPP method measurement FL2 at different temperature (0-50 DEG C), to measure vigor most Enzyme activity when high is 100%, calculates the enzyme activity (%) at a temperature of other.It is measured by the same way using pNPB the bottom of as Optimum temperature when object.By Fig. 2 as it can be seen that the zymetology vigor of FL2 with temperature raising present first rise after downward trend, When using pNPP as substrate, wherein 40 DEG C or so enzyme activity highests, are its optimum temperature；When using pNPB as substrate, most suitable work It is 30 DEG C or so with temperature.

Optimal reaction pH

Respectively in the buffer of different pH (3.0-10.0), 35 DEG C at a temperature of by above-mentioned pNPP method measure FL2 enzyme The lipase activity of liquid.To measure enzyme activity when vigor highest as 100%, the relative activity (%) of enzyme under other pH is calculated. As seen from Figure 3, downward trend after the enzyme activity of FL2 first rises as the raising of pH value is presented, enzyme activity is close to zero when pH < 7 (not showing data), wherein enzyme activity highest when pH9.5.Therefore, the optimal pH of FL2 is 9.5 or so.Thus infer, the present invention The lipase is alkaline lipase.

PH stability

By FL2 enzyme solution respectively in the buffer system of different pH (3.0-11.0) in 4 DEG C keep the temperature 24 hours, then at 35 DEG C, By the vigor of pNPP method measurement lipase under pH8.0.Measured enzyme activity is 100% in the highest buffer of enzyme activity, Calculate the enzyme activity under other pH.From fig. 4, it can be seen that FL2 polypeptide (5.0-10.0) in wider pH range keeps stablizing, enzyme Vigor changes in the section 80%-100%.In pH < 5.0 and pH > 10.0, lipase activity decline is very fast, such as pH is When 11.0, lipase activity is decreased to less than 20%.

The specificity of lipase hydrolysis substrate-fatty acid specificity

Prepare 6mg/mL4- Nitrophenyl butyrate (pNPB), 4- nitrobenzophenone caprylate (pNPO), the 4- nitrobenzophenone moon Cinnamic acid ester (pNPD), 4- nitrobenzophenone myristinate (pNPM), 4- nitrobenzophenone palmitate (pNPP) and 4- nitrobenzophenone Stearate (pNPS), is dissolved with isopropanol, and the vigor of lipase is detected according to standard pNPP method (being changed to corresponding substrate), with Enzyme activity measured by the highest substrate of enzyme activity determination is 100%, calculates the enzyme activity for hydrolyzing other substrates.It can by Fig. 5 See, FL2 is optimal for the hydrolysis effect of 4- Nitrophenyl butyrate, and with fatty acid carbons chain growth, enzyme activity sharply declines.

Influence of the metal ion to lipase activity

Prepare ZnSO₄、MnCl₂、CoCl₂、CaCl₂、MgSO₄、CuSO₄、KCl、(NH4)₂SO₄、 NaCl、NiSO4、FeCl₃ Liquid is stored with inorganic salts such as EDTA.According to above-mentioned pNPP method, first preparation reaction mixture, dispensed into each reaction tube 400 μ L mixed liquors, and the inorganic salts storage liquid of final concentration of 5mmol/L is added, enzymatic activity is measured according still further to the above method.Control Group addition water replaces inorganic salts to store liquid, and enzyme activity calculates opposite enzyme activity of remaining group relative to control group as 100% Power.Substrate is changed to pNPB, measures the influence of metal ion by the same way.As shown in fig. 6, in addition to KCl, with pNPP and It is distinguished when pNPB tests influence of other ions for FL2 lipase activity respectively as substrate, between pNPP and pNPB little. But when using pNPB as substrate, KCl influences less enzyme activity, and when using pNPP as substrate, lipase activity is improved to twice, is said Bright KCl has facilitation to the hydrolysis of pNPP.In addition, as Fig. 6's the results show that FL2 does not have dependence for divalent ion.

Influence of the surfactant for lipase activity

Cation surface activating CTAB, the anionic surfactant SDS, nonionic surfactant of configuration 10% Tween80, AEO-9 and Triton X-100 etc. stores liquid.It is added according to pNPP standard method, while in the reaction system 0.5% above-mentioned surfactant measures lipase activity.Control group adds water and replaces surfactant, enzyme activity conduct 100%, calculate enzyme activity of remaining group relative to control group.Substrate is replaced with into pNPB, measures table by the same way The influence of face activating agent.As shown in fig. 7, the vigor of SDS and CTAB strong inhibition lipase, Tween80, AEO-9 and Triton Influence of the X-100 to lipase is different according to the difference of reaction substrate, when using pNPP as substrate, Triton X-100, AEO-9 Improve enzyme activity to a certain extent, Tween80 inhibits enzyme activity, result when using pNPB as substrate on the contrary, Triton X-100, AEO-9 inhibits enzyme activity, and Tween80 improves enzyme activity.

As can be known from the above results, polypeptide of the invention has lipase active, and/or has following at least one beneficial Characteristic:

1) short chain Preference, as shown in figure 5, FL2 shows highest when using 4- Nitrophenyl butyrate as substrate Enzymatic activity.

2) there is good enzyme activity and stability within the scope of very extensive pH, as shown in figure 4, FL2 polypeptide is wider (5.0-10.0) keeps stablizing within the scope of pH, and enzyme activity is about 80% or more.

3) there is certain surfactant tolerance, as shown in fig. 7, when using pNPP as substrate, Triton X-100, In the presence of AEO-9, the enzyme activity of FL2 polypeptide is improved；When using pNPB as substrate, in the presence of Tween80, the enzyme activity of FL2 polypeptide Even power is basically unchanged increase.

4) shown in the presence of potassium ion compared with high enzymatic activity, as shown in fig. 6, in the presence of KCl, FL2 is catalyzed pNPP About 1 times of vigor increase or more of hydrolysis.

It is appreciated that the application is not limited to show in this specification although the application is illustrated with some form The content shown and described.It should be apparent to those skilled in the art that under the premise of without departing from scope of the present application also It can make a variety of changes.These variation all this application claims in the range of.

Claims (12)

1. the polypeptide with lipase active, the amino acid sequence shown in SEQ ID NO:1 form.

2. encoding the polynucleotides of polypeptide described in claim 1.

3. polynucleotides as claimed in claim 2, it includes nucleotide sequences shown in SEQ ID NO:2.

4. polynucleotides as claimed in claim 2, the nucleotide sequence shown in SEQ ID NO:2 is formed.

5. the polynucleotides as described in any one of claim 2-4 are generated by artificial synthesized generation or recombination.

6. expression vector, it includes the polynucleotides described in any one of at least one claim 2-5.

7. expression vector as claimed in claim 6 also includes the regulating and controlling sequence for adjusting the polynucleotides expression, wherein institute Polynucleotides are stated to be operably connected with the regulating and controlling sequence.

8. expression vector as claimed in claim 7, wherein the expression vector is pET24a (+).

9. host cell, it includes described in any one of claim 2-5 polynucleotides or any one of claim 6-8 institute The expression vector stated.

10. host cell as claimed in claim 9 is e. coli bl21 (DE3).

11. the method for preparing polypeptide described in claim 1 comprising:

1) polynucleotides described in any one of claim 2-5 are cloned on expression vector,

2) expression vector is converted or is transduceed into suitable host cell,

3) host cell in suitable culture medium, is cultivated,

4) it is separated from the host cell or culture medium and purifies the polypeptide.

12. polypeptide described in claim 1 is preparing the purposes in dairy products.