CN1222938A - Modified alpha-amylase with altered calcium ion binding properties - Google Patents
Modified alpha-amylase with altered calcium ion binding properties Download PDFInfo
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- CN1222938A CN1222938A CN 97194690 CN97194690A CN1222938A CN 1222938 A CN1222938 A CN 1222938A CN 97194690 CN97194690 CN 97194690 CN 97194690 A CN97194690 A CN 97194690A CN 1222938 A CN1222938 A CN 1222938A
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- amylase
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Abstract
公开了新型α－淀粉酶,其中一种新的钙离子结合位点通过化学或遗传性改造与钙离子结合位点相关的残基加以修饰。此新型α－淀粉酶具有改变了的性能特征,如低pH下的淀粉水解能力、稳定性和活性曲线。
Novel α-amylases are disclosed, wherein a new calcium ion binding site is modified by chemically or genetically modifying residues associated with the calcium ion binding site. This novel α-amylase has altered performance characteristics, such as starch hydrolysis ability at low pH, stability and activity profile.
Description
The present invention relates to have the α-Dian Fenmei of altered calcium ion in conjunction with character.Especially, the present invention relates to have the novel α-Dian Fenmei as the modification of point mutation, modification wherein is intended to change the calcium ion combination at the calcium ion binding site place of a previous the unknown in the molecule.Calcium ion by changing this attachment site can increase the stability of the α-Dian Fenmei of modified in conjunction with character.
α-Dian Fenmei (α-1,4-dextran-4-glucan hydrolase, EC.3.2.1.1) in starch mainly with the inner α-1 of random fashion hydrolysis, the 4-glycosidic link is to produce the Star Dri 5 of small molecular weight.α-Dian Fenmei has considerable commercial value, is applied to the starting stage (liquefaction) of starch processing; Alcohol Production; Clean-out system in the washing matrix and the starch destarch in the textile industry.α-Dian Fenmei comprises that by many different microorganisms genus bacillus (Bacillus) and aspergillus (Asper gillus) produce, and wherein has most the amylase of commercial value to originate from bacterial origin as Bacillus licheniformis (Bacillus licheniformis), bacillus amyloliquefaciens (Bacillus amyloliq uefaciens), subtilis (Bacillus subtilis) or bacstearothermophilus (Bacillus stearothermophilus).In recent years, preferably being used for commercial enzyme is those enzymes from Bacillus licheniformis, because their thermostability and the performance under neutrality and gentle alkaline pH at least.
In U.S. Patent No. 5, in 322,778 by with a kind of as hydrosulphite, xitix or its salt, erythorbic acid antioxidant or join as the phenol antioxidant of butylation oxybenzene methyl ether, butylated hydroxyl toluene or alpha-tocopherol and between pH4.0 and 6.0, to realize liquefaction in the liquefaction slurries.According to this patent, sodium bisulfite must add with the concentration that is higher than 5mM.
In U.S. Patent No. 5,180, in 669, the carbanion of the amount by will be required above buffered soln joins in the ground farinaceous size is realizing liquefaction between the pH5.0 to 6.0.Because it is the adding of carbanion causes the increase of pH value, general by adding a kind of hydrogen ion, for example: resemble in a kind of mineral acid of hydrochloric acid or vitriolic and slurries.
In PCT application No.WO95/10603, disclose that to have improved laundry or wash comprising in the bacillus licheniformis alpha-amylase of dish performance be not alpha-amylase variants in a kind of sudden change of the single sudden change in M197 site.
In PCT application No.WO94/02597, a kind of mutant alpha-amylase with improved oxidative stability has been described, one or more methionine(Met) uses the arbitrary amino acid except half deamination acid or methionine(Met) to replace.
In PCT application No.WO94/18314, described a kind of mutant alpha-amylase with improved oxidative stability, one or more methionine(Met), tryptophane, halfcystine, Histidine or tyrosine residues are with a kind of inoxidizable aminoacid replacement.
In PCT application No.WO91/00353, all handling this α-Dian Fenmei by genetically engineered with liquefy relevant performance characteristics and problem in the wild-type bacillus licheniformis alpha-amylase solves, replace Ala-111-Thr, His-133-Tyr and/or Thr-149-Ile comprising specificity.
Existing many different researchists utilize recombinant DNA technology to carry out detecting which residue to be important for diastatic catalysis activity and/or to detect the Research on effect (people such as Vihinen of the specific amino acids of modifying the avtive spot in different amylase and the glycosylase, journal of biological chemistry (J.Biochem.) vol.107,267-272 (1990)); People such as Holm, protein engineering (ProteinEngineering), Vol.3,181-191 (1990); People such as Takase, biological chemistry and biophysics circular (Biochemica et Biophysica Acta) Vol.1120,281-288 (1992); People such as Matsui, Febs.Letters, Vol.310,216-218 (1992); People such as Matsui, biological chemistry (Biochemistry) Vol.33,451-458 (1992); Sogaard waits the people, journal of biological chemistry (J.Biol.Chem.) vol.268,22480-22484 (1993); People such as Sogaard, carbohydrate (Carbohydrate Polymers) Vol.21,137-146 (1993); Svensson, molecular biology of plants (Plant Mol.Biol.) Vol.25,141-157 (1994); People such as Svensson, biotechnology magazine (J.Biotech.) Vol 29,1-37 (1993)).Which residue the researchist has also studied, and important (Suzuki waits the people, journal of biological chemistry (J.Biol.Chem.) Vol.264,18933-18938 (1989) for thermostability; People such as Watannabe, european journal of biological chemistry (Eur.J.Biochem.) vol.226,277-283 (1994)); And an existing study group makes in this way and introduce sudden change at different histidine residues places in Bacillus licheniformis amylase, its ultimate principle is that known compare the Histidine that Bacillus licheniformis amylase with relatively hot stability contains with other similar bacillus amylase more, therefore points out the replacement of Histidine may influence the thermostability of this enzyme.This work has caused at the affirmation of the stabilization of 133 hyte propylhomoserin residues and 209 alanine residues sudden change (people such as Declerck, journal of biological chemistry (J.Biol.Chem.) vol.265,15481-15488 (1990); FR2,665,178-A1; People such as Joyet, biochemical technology (Bio/Technology), vol10,1579-1583 (1992)).
On primary structure, the considerable difference similar three-dimensional structure is all arranged from different biological α-Dian Fenmei.Fig. 1 shows the structure of bacillus licheniformis alpha-amylase.Though between different α-Dian Fenmei, have the variation of some kind inside, the main structural components of bacillus licheniformis alpha-amylase is commonly considered as the representative of α-Dian Fenmei structure and (is seen people such as Brayer, protein science (Protein Sci.) Vol.4,1730-1742 (1995); People such as Larson, molecular biology magazine (J.Mol.Biol.) Vol.235,1560-1584 (1994); People such as Qian, molecular biology magazine, Vol.231,785-799 (1993)).For example, rite-directed mutagenesis has been confirmed to the catalysis (Svensson that plays an important role, molecular biology of plants, vol.25,141 (1984)) three constant carboxylic acids and two constant Histidines (D231 in the bacillus licheniformis alpha-amylase, E261, D328 and H104 and H327), and a kind of ubiquity mechanism (people such as Mazur, biological chemistry and biophysical studies communication (Biochem.Biophys.Res.Comm.) vol.204,297 (1994)) has been proposed.Characterized and thought the residue that participates in calcium and chloropexia and find that these residue high conservatives in different enzymes (see as people such as Kadziola, molecular biology magazine, vol.239,104 (1994); People such as Qian, the source is the same, people such as Larson, the source is the same; People such as Brayer, the source is the same; People such as Machius, molecular biology, vol.246, people such as 545-559 (1995) and Boel, biological chemistry, vol.29,6244 (1990)).
In addition, between nearly all endo-amylase (scope comprises plant, Mammals and bacterium) that has checked order at present, found homologue (people such as Nakajima, using microbe biotechnology (Appl.Microbiol.Biotechnol.), vol 23,355-360 (1986); Rogers, biological chemistry and biophysical studies communication, vol.128,470-476 (1985); Janecek, european journal of biological chemistry (Eur.J.Biochem.), vol 224,519-524 (19941)).Have 4 zones that extra high homology is arranged in some bacillus amylase, as shown in Figure 5, wherein the zone of underscore is called the height homologous region.Also adopted sequence contrast to indicate relation between genus bacillus endo-amylase people such as (, molecular evolution magazine (J.Molec.Evol.) vol.35,351-360 (1987)) Feng.The correlated series homology is about 66% between bacstearothermophilus and the Bacillus licheniformis amylase, is about 81% between Bacillus licheniformis and the bacillus amyloliquefaciens amylase, as people such as Holm, protein engineering, vol.3, No.3,181-191 (1990) measures.Though sequence homology is important, it is generally acknowledged that structural homology is also very important when comparative starches enzyme or other enzyme.
Derive from the three-dimensional structure similar (except the difference of their primary structures) between the biological different α-Dian Fenmei of difference (and relevant amylolytic enzyme such as Maltose 4-glucosyltransferase and alpha-glucosidase), find its middle body that has a kind of α/β-barrel-shaped one-tenth jointly (structural domain A), arrange (Greek key motif) and at least one additional structural domain as Greece's grain pattern of separated structures territory C, be structural domain B (people such as Machius, the source is the same).Think the binding site of substrate between α/β-bucket and structural domain B, comprise according to kind different and have a beta chain (Machius, the source is the same) of different lengths.Have plenty of demand too altogether, think that it keeps the integrity of structure calcium.Machius discloses a calcium ion binding site, and it relates to corresponding to by N104, D200 in the crystalline structure of α-Dian Fenmei of calcium that has been derived from exhausting of Bacillus licheniformis and the residue of H235.Except the structure of Bacillus licheniformis, aspergillus niger (Aspergillus niger) (people such as Brady, Acta.Crystailog.B have been measured, vol47,527 (1991)), pig pancreas (people such as Qian, molecular biology magazine, vol231,758 (1993); People such as Larson, molecular biology magazine, vol235,1560 (1994)) and human pancreas (people such as Brayer, Prot.Sci.vol.4,1730 (1995)) in diastatic structure.
Except the progress that prior art is obtained, also need to have altered performance (comprising activity and stability) in case starch liquefacation, laundry with and dishwashing detergent, the α-Dian Fenmei that bakes, use in textiles destarch and other the amylase standard purposes.Because under many conditions,, thereby need a kind of amylase under these conditions with altered performance curve that has preferably increased because stability and/or the active problem commerciality amylase that can get can not use.For example: high alkalinity that is associated with washing composition and oxidation (bleaching) property level or the extreme condition that exists in the starch liquefacation process all can cause the instability and the inactivation of α-Dian Fenmei.Therefore, expectation obtains such as the altered performance characteristic of thermostability, pH stability, oxidative stability or Calcium ion stability, compares the amylase of that also changed, that remain stationary or the enzymic activity that increased simultaneously with wild-type or pre-enzyme.Similarly, it is stable to keep that known many α-Dian Fenmei need the interpolation of calcium ion.In some applications since this can increase tooling cost thereby be not expect.
The object of the present invention is to provide a kind of α-Dian Fenmei with altered performance characteristics as: altered pH stability, alkalescence stability, oxidative stability, thermostability or enzymic activity.
Another object of the present invention is to provide a kind of α-Dian Fenmei with altered calcium ion in conjunction with character (for example: the calcium of keeping active required adding reduces).
It is a kind of owing to having increased low pH stability or active (particularly in the process of starch liquefacation) α-Dian Fenmei with performance of improvement that another purpose of the present invention is to provide.
It is a kind of in high temperature or high pH environment or have the α-Dian Fenmei of the performance of improvement when oxygenant or SYNTHETIC OPTICAL WHITNER exist that a further object of the invention is to provide.
A further object of the invention is to provide a kind of because its altered stability or active and have a α-Dian Fenmei of the performance of improvement in textiles destarch or baking.
According to the present invention, a kind of structural domain A that comprises is provided, the α-Dian Fenmei of a domain C and a calcium ion binding site, calcium ion binding site wherein is relevant with domain C with structural domain A and comprise the part residue that is arranged in structural domain A and/or C, wherein the α-Dian Fenmei process is modified the characteristic with change calcium ion binding site, thereby has changed the performance of this α-Dian Fenmei.
In an embodiment preferred, modification comprises a kind of genetically engineered modification, and it causes being equivalent to bacillus licheniformis alpha-amylase amino-acid residue 290-309,339-347,402-411, replacement, deletion or the interpolation at the one or more residues place among 426-436 or the 472-477.In a particularly preferred embodiment, genetically engineered is modified to be included in and is equivalent to bacillus licheniformis alpha-amylase G301, M304, H405, replacement, deletion or the interpolation at the one or more residues place among H406 and/or the K436.
In a product embodiment, the present invention comprises a kind of DNA of the α-Dian Fenmei of the present invention of encoding.In another composition embodiment, the present invention comprises a kind of expression vector that is integrated with coding according to the DNA of α-Dian Fenmei of the present invention, and has used this DNA and/or expression vector transformed host cells.In a method embodiment, be included in DNA or a kind of expression vector that is integrated with this DNA of expressing a kind of α-Dian Fenmei of the present invention of encoding in the host cell.
In another product embodiment, the present invention comprises and a kind ofly mixes according to the laundry of α-Dian Fenmei of the present invention or wash the dish cleaning composition.In another product embodiment, the present invention comprises a kind of textiles destarch composition that mixes according to α-Dian Fenmei of the present invention.In another product embodiment, the present invention comprises a kind of starch liquefacation composition that mixes according to α-Dian Fenmei of the present invention.In another product embodiment, the present invention has comprised a kind of baking that contains with good grounds α-Dian Fenmei of the present invention and has helped material.
In a method embodiment of the present invention, provide a kind of use to mix the method for washing dish washing composition cleaning compositions clothing or cleaning plate according to α-Dian Fenmei of the present invention.In another method embodiment of the present invention, provide a kind of method that makes the textiles destarch according to the composition of α-Dian Fenmei of the present invention of mixing of using.In another method embodiment of the present invention, provide a kind of use to mix method according to the starch liquefacation composition liquefying starch of α-Dian Fenmei of the present invention.In another method embodiment of the present invention, a kind of baking method is provided, it comprises and adds a kind of composition that mixes according to α-Dian Fenmei of the present invention.
To have some according to the α-Dian Fenmei of modified of the present invention and compare important advantage with existing α-Dian Fenmei.For example: in some variants, found in starch liquefacation method commonly used, typically to hang down the active advantage that has increase under pH and the hot conditions.In having the variant of the high pH of increase and oxidative stability, some find to have to be convenient to another advantage that it uses in washing composition.Provide another advantage by the variant that in nothing or low calcium ion concn, has the stability of improvement.Purpose of the present invention and attendant advantages will further be illustrated by the following detailed description and embodiment.
Fig. 1 shows the structure of bacillus licheniformis alpha-amylase, has indicated the position of the calcium ion binding site (CalA) that backbone fold is associated with structural domain A and structural domain B and the position of another calcium ion binding site (CalB) of being associated with structural domain A and domain C.
Fig. 2 show final 2fo-fc disparity map three-dimensional view and with the unusual Fourier's difference of Sm from the structural domain A of the α-Dian Fenmei of the Bacillus licheniformis calcium ion binding site relevant with domain C.
Fig. 3 A-C shows from the dna sequence dna of the gene of the α-Dian Fenmei of Bacillus licheniformis (NCIB8061) and as people such as Gray, bacteriology magazine (J.Bacteriology) vol166, the aminoacid sequence of the supposition of the translation product described in the 635-643 (1986).
Fig. 4 has shown the aminoacid sequence from the sophisticated α-Dian Fenmei of Bacillus licheniformis.
Fig. 5 A-B shows a fragment of the primary structure of three kinds of genus bacillus α-Dian Fenmei.Bacillus licheniformis alpha-amylase (Am-Lich) is as people such as Gray, the bacteriology magazine, and vol.166,635-643, (1986) are described; Bacillus amyloliquefaciens α-Dian Fenmei (Am-Amylo) is as people such as Takkinen, journal of biological chemistry (J.Biol.Chem.) vol.258, and 1007-1013 (1983) is described; Bacstearothermophilus α-Dian Fenmei (Am-Stearo) is as people's journal of biological chemistry (J.Biochem.) vol.98 such as Ihara, and 95-103 (1985) is described.
" AMS " refers to the enzymatic activity of glycosidic bond of a kind of cutting or hydrolysis as the α in starch, amylopectin or amylose polymer-(1-4). AMS used herein comprises AMS and the recombinant alpha-amylases of natural generation. Can be derived from a kind of precursor amylase according to AMS of the present invention. This precursor amylase can be produced by any source that can produce AMS. The suitable source of AMS is protokaryon or eucaryote, comprises fungi, bacterium, plant or animal. Preferably, the precursor AMS is produced by bacillus or fungi, such as the fungi (that is: aspergillus oryzae (A.oryzae) and aspergillus niger) derived from aspergillus. More preferably, precursor is produced by bacillus licheniformis, bacillus amyloliquefaciens or bacillus stearothermophilus; More preferably, the precursor AMS is derived from bacillus licheniformis.
A kind of " modified " AMS be a kind of through heredity or chemical sex modification to change the AMS of its biochemistry, structure or physicochemical properties. " heredity modification " in the AMS refers to encode dna sequence dna natural generation or the precursor AMS through modifying to produce a kind of dna sequence dna of sudden change, one or more amino acid whose replacements in this sequential coding and the AMS sequence that AMS or the precursor α-amylase of natural generation are compared, insertion or deletion.
" expression vector " refers to comprise a kind of DNA construct that can affect the dna sequence dna that this DNA expresses in suitable host, and this dna sequence dna generally operably is connected with suitable control sequence. Such control sequence can comprise the promoter that a kind of impact is transcribed, and the sequence with translation termination is transcribed in a kind of sequence of controlling this selective operon sequence of transcribing, a kind of mRNA ribosome bind site of encoding suitable and control. A kind of preferred promoter is bacillus subtilis aprE promoter. Carrier can be plasmid, phage particle or be intended to affect the DNA that genome inserts (namely integrating). In case be transformed into suitable host, what this carrier can not rely on host genome copies and brings into play function, or can be incorporated in some cases among the genome. Plasmid and carrier are sometimes used instead mutually, because plasmid is the present the most form of carrier commonly used. Yet, the invention is intended to comprise the expression vector that equivalent function is arranged of other form, these carriers are known in the art or know for this area gradually, show particularly including bacteriophage.
" Host Strains " or " host cell " refers to a kind of to encoding according to the suitable host of the expression vector of α of the present invention-diastatic DNA as containing. Be used for host cell of the present invention and be typically protokaryon or eucaryon host, be included in any transformable microorganism that wherein can realize according to the expression of AMS of the present invention. Especially, the host strain of the species or genus identical with the kind that produces AMS is to suit such as Bacillus strain. Preferably, use the bacillus (as: Δ amyE, Δ apr, Δ npr) of the negative Bacillus strain (gene delection) of a kind of AMS and/or a kind of AMS and protease deficiency. Use is transformed or transfection host cell by the carrier that recombinant DNA technology makes up. The carrier of the host cell of this conversion or reproducible coding AMS and its variant (mutant), or the AMS that can express expectation.
" liquefaction " or " liquefaction " refers to a kind of process that starch is transformed into short chain and low viscosity dextrin. When usually, this process comprises the starch gel gel or add afterwards AMS.
" calcium binding site " refer to be suitable in the AMS and when free calcium ions exists in conjunction with the effective zone of calcium ion. It is generally acknowledged that calcium ion is essential to the structural intergrity of keeping AMS under many conditions, and the amino acid residue that has confirmed in different enzymes to participate in calcium binding is the high conservative (people such as Machius, the molecular biology magazine, vol246,545-559 (1995)). According to the present invention, thereby compare the performance that the characteristic that has changed the calcium binding site has changed AMS with wild type or precursor AMS. The change in calcium binding site can comprise the affinity that reduces or increase this site and calcium binding. Be intended to refer to stability (such as oxidation or heat endurance) or the activity (such as speed or the efficient of α-amylasehydrolysis starch substrates) of this enzyme in different application by changing its performance.
" part residue " or " calcium ion part " refers to the one or more amino acid residues in AMS, and it forms a part with calcium binding among the calcium binding site. Just by the applicant, the calcium binding site among the AMS of discovery has confirmed to have thought 5 amino acid ligands of calcium ion part effect. Calcium ion part residue comprise with bacillus licheniformis alpha-amylase in G300, Y302, H406, the amino acid residue of D407 and D430 equivalence. Especially, with regard to calcium ion parts that these are determined, think G300, the ketonic oxygen of Y302 and H406 and D407 and D430 side chain have participated in calcium binding.
According to the present invention, provide and comprised domain A, a kind of AMS in domain C and a calcium binding site, calcium binding site wherein is relevant with domain C with domain A and comprise part residue in domain A and/or the domain C, thus the modified performance that changes this AMS with the characteristic that changes the calcium binding site of AMS wherein.
The present invention also provides a kind of nucleic acid molecules (DNA) of the amino acid sequence of at least a portion that coding contains AMS provided by the invention, be integrated with the expression system that this DNA comprises carrier and bacteriophage, the antisense strand of the host cell that transforms with this DNA and the DNA corresponding with the dna molecular of this amino acid sequence of coding. Similarly, the present invention includes a kind of by expressing in the method that the DNA on a kind of expression system produces a kind of AMS that is incorporated into that is transformed in the host cell.
Can be by it being linked to each other with expression control sequenc in the suitable expression vector and transforming a suitable host to express this dna sequence dna according to known technology with this expression vector. The combination of different host's surface systems all can be used for expressing dna sequence dna of the present invention in a large number. Useful expression is carried all, for example, comprises chromosome, achromosomal fragment and synthetic dna sequence dna, such as the different plasmid that becomes known for this purpose and bacteriophages. In addition, any in a large amount of different expression control sequencs usually all can be used for these carriers. For example, the applicant has found that the preferred expression control sequenc for the bacillus transformant is the aprE signal peptide derived from bacillus subtilis. In addition, phage display system also is applicable to the present invention.
Many host cells also can be used for expressing dna sequence dna of the present invention and also take at this. These hosts can comprise known eucaryon and prokaryotic hosts, bacterial strain, yeast and zooblast such as Escherichia coli, Pseudomonas (Pseudomonas), bacillus, streptomyces (Streptomyces), different fungies such as trichoderma (Trichoderma) or aspergillus. Preferably, the host expresses born of the same parents with AMS of the present invention and is beneficial to purifying and Downstream processing outward. Expression and the purifying of the AMS of the present invention's sudden change can be undertaken by method of carrying out this generic operation known in the art.
α-Dian Fenmei according to the present invention comprises a kind of aminoacid sequence of the aminoacid sequence derived from a kind of precursor α-Dian Fenmei.This precursor α-Dian Fenmei comprises the α-Dian Fenmei and the recombinant alpha-amylases of natural generation.The aminoacid sequence of alpha-amylase mutant by the precursor aminoacid sequence one or more amino acid whose replacements, deletion or insertion and derived from precursor α-Dian Fenmei aminoacid sequence.This modification usually is to the precursor dna sequence of the aminoacid sequence of coding precursor α-Dian Fenmei rather than to the operation of preceding α-Dian Fenmei itself.It is described by prior art in accordance with the method for the enzyme product that gene produced of sudden change to modify alpha-amylase gene (that is: by the oligonucleotide sudden change of fixing a point) and conversion, expression and secretion, comprise the open text No.WO95/10603 (NovoNordisk) of PCT, the open text No.WO94/02597 (Novo Nordisk) of PCT, open text No.WO94/18314 (the Genencor International of PCT, company) and the open text No.WO91/00353 (Gist Brocades) of PCT, introduce herein that these are open as a reference.The method that is suitable for the precursor dna series of operations in addition comprises U.S. Patent No. 4,760,025 and 5,185,258 disclosed herein and that own together, quotes as a reference herein.
Main structural element comprises that newfound CalB site disclosed herein and the change of carrying out for the performance that changes α-Dian Fenmei are described with the general word that is applicable to most of α-Dian Fenmei below.As shown in Figure 1, clear and definite three main structural domains: structural domain A, B and C and two calcium ion binding site CalA and CalB.Structural domain A comprise the middle body of this molecule and confirmed as a α/β or TIM barrel-shaped.This α/β bucket is made of with the inner beta chain that links to each other of alpha-helix series of parallel.At the carboxyl terminal of enzyme, the side of structural domain A be a zone of forming by antiparallel β-bucket (being called " Greece's grain pattern ") (referring to as: people such as Richardson, high protein chemistry (Advan.Protein Chem.), vol.34,167-339 (1981); People such as Braden, protein structure introduction (Introduction to Protein Structure), Garland publishing company, New York (1991)).This structural domain has been confirmed as domain C.In a structural domain A side relative with domain C (N-end) is an additional structural domain, and it comprises some, and the different beta chain of length is called structural domain B with the kind difference.Found structural domain B alterable height and often comprise the ring of extension between different types of α-Dian Fenmei.Think in the crack of substrate binding site between structural domain A and structural domain B, and avtive spot also the molecule in zone is relevant therewith.The CalA binding site is arranged in the crack of separation structure territory A and structural domain B, thinks that it can stablize this zone.CalB binding site disclosed herein is positioned at the zone of structural domain A and domain C boundary.
Make the applicant can develop a kind of mutant alpha-amylase by the CalB binding site in the genus bacillus α-Dian Fenmei of applicant's discovery, and have altered especially stability with altered performance.For example: the rule that is used for the stabilizing protein structure can be applied to zone on every side, CalB site.In addition, being in particular the calcium ion of raising in the CalB site also can adopt to increase the stability of this enzyme in conjunction with the scheme that designs.Preferably, within this 15A that is modified at the calcium ion central authorities that are attached to the CalB binding site, more preferably within 10 of the calcium ion central authorities that are attached to the CalB site.
Also determined to be used for the residue of the α-Dian Fenmei of deleting or replacing herein.Therefore, special residue discussed herein is meant with reference to a kind of amino acid whose location number that is shown in the number of distributing to ripe bacillus licheniformis alpha-amylase sequence among Fig. 4.Yet the present invention is not limited to the sudden change of specific ripe bacillus licheniformis alpha-amylase, and may extend into non-Bacillus licheniformis precursor α-Dian Fenmei, contains the amino acid with the specific residue equivalent locations of bacillus licheniformis alpha-amylase in these enzymes.A kind of residue of precursor α-Dian Fenmei is equivalent to the residue of bacillus licheniformis alpha-amylase, if its with bacillus licheniformis alpha-amylase in specific residue or portion homologous (that is: corresponding to or the elementary or position of quaternary structure) or similar (that is: have same or analogously combine, the function of reaction or chemical or functional interaction).
In order to make up the homologue of primary structure, directly the aminoacid sequence of precursor α-Dian Fenmei and bacillus licheniformis alpha-amylase primary sequence are particularly compared with constant series of residues in all α-Dian Fenmei (its sequence is known) (opinion) as Fig. 7.Also can be by pig pancreas α-Dian Fenmei (people such as Buisson, EMBO J.vol6, the 3909-3916 (1987) that has reported; People such as Qian, biological chemistry (Biochemistry), vol.33,6284-6294 (1994); People such as Larson, molecular biology magazine, vol335,1560-1584 (1994)); Derive from the Taka-amylase A (people such as Matsuura of aspergillus oryzae, biological chemistry (J.Biochem) (Tokyo), vol95,697-702 (1984)) with from a kind of acid alpha-amylase (people such as Boel, the biological chemistry (Biochemistry) of having of aspergillus niger of preceding two structural similitudies, vol, 29,6244-6249 (1990)) and barley α-Dian Fenmei (people such as Vallee, molecular biology magazine, vol.236,368-371 (1994); Kadziola, molecular biology magazine, vol.239,104-121 (1994)) the quaternary structure analysis of crystalline structure determine residue of equal value.Though some preliminary researchs open (people such as Suzuki, journal of biological chemistry (J.Biochem.), vol.108,379-381 (1990) are arranged; People such as Lee, biological chemistry and biophysics document (Arch.Biochem.Biophys.) vol291,255-257 (1991); People such as Chang, molecular biology, vol229,235-238 (1993); People such as Mizuno, molecular biology, vol.234,1282-1283 (1993)), but only relevant for the disclosed structure (people such as Machius, molecular biology, vol.246,545-549 (1995)) of bacillus licheniformis alpha-amylase.Yet, some researchists have predicted dextranase (people such as MacGregor, journal of biological chemistry (Biochem.J.) vol.259,145-152 (1989)) and α-Dian Fenmei is inner and other starch metabolism enzymes (Jaspersen, protein chemistry magazine (J.Prot.Chem.) vol.12,791-805 (1993); MacGregor, Starke, vol.45,232-237 (1993)) between the common supersecondary structure; And sequence similarity (Janecek, FEBS Letters, vol.316,23-26 (1993) between the enzyme of similar supersecondary structure are arranged to α-Dian Fenmei; People such as Janecek, protein chemistry magazine, vol.12,509-514, (1993)).A kind of structure of bacstearothermophilus enzyme is made model (people such as Holm, protein engineering, Vol.3,181-191 (1990)) by Taka-amylase A.4 high conservative zones shown in Fig. 7 comprise manyly thinks that (Matsuura waits the people to the residue that belongs to avtive spot, journal of biological chemistry (J.Biochem.) (Tokyo), vol.95,697-702 (1984); People such as Buisson, EMBO J., vol.6,3909-3916 (1987); People such as Vihinen, journal of biological chemistry (J.Biochem.) vol.107,267-272 (1990)), it comprises by the His105 in the Bacillus licheniformis number system; Arg+229; ASP+231; His+235; Glu+261 and Asp+328.
The α-Dian Fenmei polypeptide chain fragment that contains the CalB binding site comprises residue 290-309,339-347,402-411,426-436 and 472-477.These polypeptide fragments contain the CalB binding site.Correspondingly, the regiospecificity random mutation that can be expected in these zones will produce by adjust the variant of α-Dian Fenmei stability in the adjustment of these site calcium ion avidities.
Other more particularly scheme is provided below:
(1) increases the stability that the entropy that does not fold main chain can increase enzyme.For example, proline residue introduced on 2 of the N-of alpha-helix and ring texture end corner can obviously stablize this protein by the entropy that increases folded chain not and (see as people such as Watanabe, european journal of biological chemistry, vol.226,277-283 (1994)).Similarly, glycine residue does not have beta carbon, thereby has obviously bigger bone framework image degree of freedom than other residue.This can cause the high flexibility that causes weak stability.G299 in being equivalent to bacillus licheniformis alpha-amylase, G410, G433, G474, L-Ala is preferably used in the replacement of one or more glycine residues of G475, can reduce flexibility and increases stability.In addition, also can increase stability by shortening extended loop.Observe the protein of the thermophilic generation of high temperature and compared extended loop short (see as: people such as Russel, modern viewpoint (the Current Opinions in Biotechnolog) vol.6 of biotechnology, 370-374 (1995)) with warm counterpart wherein.The introducing of disulfide linkage is also effective to stable unique to each other quaternary structure.To change the segmental stability of 290-309 in the modification at G301 place by restriction or the conformational change that increases glycine.Especially, consideration is in the replacement of this residue aspartic acid or proline(Pro).By replacing and to increase stability because of introducing a C β, therefore reduce its conformational freedom in the modification of G474 with other residue.
(2) reduce the stability that inner cavity can change a kind of enzyme by increasing the side chain hydrophobicity.Thereby reducing the quantity of internal cavities and volume can (see as Mattews, Ann.Rew.biochem.vol.62,139-160 (1993) by maximization hydrophobic interaction and the stability that reduces filling defect increase enzyme; People such as Burley, science, vol.229,23-29 (1985); Zuber, Biophys.Chem., vol29,171-179 (1988); People such as Kellis, nature, vol.333,784-786 (1988)).Knownly often have a liking for hydrophobicity subunit interface that big superficial complementarity is arranged in warm nature counterpart people such as (, the source is the same) Russel. than it from the proteic multimeric protein of thermophilic.The applicant thinks that this principle may be used on the structural domain surface of single protein subunit matter.Specificity is replaced and can be comprised that Methionin changes arginine into, Serine changes L-Ala into and Threonine changes L-Ala (Russel waits the people, and the source is the same) into by increasing hydrophobicity to increase stability.Can increase the size of side chain by replacing to L-Ala or proline(Pro) in the modification at G301 place, thereby cause cavity to reduce, the better and increase hydrophobicity of fillibility.In addition, the cavity of the intersection in the CalB calmodulin binding domain CaM between structural domain A and the domain C is with Y302, and M304, L307, F343, L427 and I428 are the border.The replacement that reduces the complementarity of cavity size, increase hydrophobicity and raising structural domain A-domain C intersection can improve the stability of enzyme.Especially, can be improved performance with the different residue that is selected from phenylalanine, tryptophane, tyrosine, leucine and the Isoleucine to modifying at the specificity residue in these sites.The other replacement that comes in handy is at V409 and F403, preferably replaces at V409 to comprise Isoleucine or leucine, and comprises tyrosine or tryptophane at F403.
(3) electric charge in the balance rigidity secondary structure (that is: alpha-helix and β-corner) can improve stability.For example, with in the negative charge on the aspartic acid and the part positive charge of spiral N-end can stablize this structure (see as, people such as Eriksson, science, vol.255,178-183 (1992)).Similarly, with in the positive charge and spiral C-end on negative charge can increase stability.Remove with β-corner in the N-of peptide hold interactional positive charge can effectively give quaternary structure stability.With the residue of non-positive charge replace H405 can from the 405-408 corner the D407 amide nitrogen remove disadvantageous positive charge in interacting.
(4) introducing salt bridge and hydrogen bond is effective to stablize quaternary structure.For example, ion pair interacts, as: between aspartic acid or L-glutamic acid and Methionin, arginine or the Histidine, thereby can cause the strong stability effect and can be used to attach the raising that different quaternary structure elements causes thermostability.In addition, the number of charged residue/non-charged residue hydrogen bond and the increase of hydrogen bond number usually can improve thermostability (see as people such as Tanner, biological chemistry (Biochemistry), vol.35,2597-2609).With aspartic acid, l-asparagine, L-glutamic acid or glutamine replace that H405 can introduce and framework amide D407 between hydrogen bond, thereby stablized the 405-408 corner.Replacing K436 with arginine can improve with the salt bridge of D404 and introduce the skeleton carbonyl of a kind of H key to I408.
(5) avoid heat labile residue generally can increase thermostability.For example, l-asparagine and glutamine are easy to deacylated tRNA amine and halfcystine is easy to oxidized under the high temperature.The thermostability that can cause the number that reduces these residues at sensitive position improves people such as (, the source is the same) Russel.Replace or deletion Q291 by the arbitrary amino acid except glutamine or halfcystine, Q298, N309, Q340 or N473 can increase stability by having avoided heat labile residue.
(6) introduce the 6th aglucon from protein and can improve the stability of bonded calcium ion, thereby stablize this enzyme to calcium ion.Replace the avidity that H406 can increase calcium ion with aspartic acid, l-asparagine, L-glutamic acid or glutamine.
(7) the stable of existing calcium ion aglucon also can increase the stability of bonded calcium ion among the CalB, thereby stablizes this enzyme.For example, M304 can replace to introduce aromatic side chain/aspartic acid side chain stability, the wherein favourable interaction of part positive charge that the oxygen of carboxylation can be relevant with benzyl rings, thereby the stability of increase D407 and D430 with phenylalanine or tyrosine.Replace H405 with phenylalanine or tyrosine and near D407, introduce a hydrophobic group because of, the stability of the C-b that can be by the D407 side chain and favourable Van der Waals interactional formation the increasing D407 of C-g atom.Can remove side chain H key with Q291 with phenylalanine in the replacement of G300.
(8) electronegativity that increases any one calcium ion part can strengthen the calcium ion combination.For example, replacing M304 with phenylalanine or tyrosine can increase the electronegativity of D407 and D430 to the shielding of solvent by improving, thereby improves the calcium ion combination.
(9) can disturb calcium ion bonded positive charge can improve the stability of calcium ion binding site similarly from removing near the calcium ion.For example, replace the H405 or the H406 of next-door neighbour's bonded calcium ion, these residues can have the positive charge that may produce disadvantageous electric charge-coulombic interaction with the calcium ion of positively charged and can with the electronegative competitive electric charge-coulombic interaction of calcium ion part.Therefore, use the replacement of a suitable non-positive charge residue can increase calcium ion avidity and protein stability.
(10) the stable combination that also can improve at the calcium ion in this site of the CalB binding site of the residue by introducing negative charge in its vicinity (is for example seen people such as Pantoliano, biological chemistry (Biochemisry) vol.27,8311-8317 (1988); Bryan, " the stable B portion of pharmaceutical grade protein: the strategy of external degradation approach and protein stabilization " (Stability of Protein Pharmaceuticals Part B:In vitro Pathwaysfor Degradation and Strategies for Protein Stabilization) (Ahern and Manning compile), 147-181 (1992); Fagain, Biochim.Biophys.Acta, vol.1252,1-14 (1995)).For example, replace Q291 with electronegative aspartic acid or L-glutamic acid, Q298, N309, Q304, H405, H406, N473 and/or G474 can increase the net negative charge in calcium ion district and can increase the avidity of calcium ion, thereby increase the stability of enzyme.
Can present altered performance according to α-Dian Fenmei of the present invention, if these expectations with unexpected effect in the different application of usually using α-Dian Fenmei, be useful.For example, α-Dian Fenmei according to the present invention presents altered performance characteristics under low pH, comprises thermostability, pH stability and/or the oxidative stability improved, and it just can be used for the starch liquefacation of low pH.The enhanced thermostability can be used for prolonging the quality guaranteed period of the product that mixes this enzyme.Enhanced oxidative stability or the performance of having improved need in cleaning product especially, and can be used for prolonging the quality guaranteed period of the α-Dian Fenmei of the peracid that contains SYNTHETIC OPTICAL WHITNER, perborate, percarbonate or be used for these cleaning product.On the contrary, low thermal stability or oxidative stability needing can be used for the commercial run of the rapidly efficient inactivation of starch hydrolytic activity.In addition, favourable when visible sequester component exists usually in washing composition to the low needs or the strong avidity of calcium ion, as: the material (washing assistant) that increases cleaning action.
α-Dian Fenmei of the present invention is used in particular in starch processing and the particularly starch liquefacation.Characteristic condition at the liquefaction process of commercialization expectation comprises: low pH, and high temperature and possible oxidizing condition, this just requires α-Dian Fenmei to present the low pH performance of improvement, the thermostability of improvement and the oxidative stability of improvement.Correspondingly, according to of the present invention in liquefaction process useful especially α-Dian Fenmei be lower than about pH6, be preferably lower than about pH5.5, the performance that more preferably between about pH5.0 and 5.5, presents improvement, in addition,, between the about 80-120 of temperature ℃, preferably between about 100-110 ℃, present the thermostability of increase and having the α-Dian Fenmei of the stability that increases in the presence of the oxygenant particularly useful according to of the present invention.Preferably, the α-Dian Fenmei that is used to liquefy according to the present invention also is included in M15, V128, H133, W138, N188, the disappearance of one or more positions of A209 and/or M197 or replacement.
In another embodiment of the present invention, provide the detergent composition of the liquid, gel or the particle form that comprise according to α-Dian Fenmei of the present invention.These detergent composition will be from according to being benefited the interpolation of a kind of α-Dian Fenmei of the present invention, the thermostability that these α-Dian Fenmei have increase can extend the shelf life or increase oxidative stability, thereby this α-Dian Fenmei has the enhanced resistance to SYNTHETIC OPTICAL WHITNER or the peracid compound that usually exists in the washing composition.Therefore, can be had powder, liquid or gel-type washing composition between the pH about 6.5 to 12.0 by favourable making according to α-Dian Fenmei of the present invention.An embodiment preferred of the present invention also is included in M15, V128, H133, W138, N188, the disappearance of one or more positions of A209 and/or M197 or replacement.The detergent composition that contains with good grounds α-Dian Fenmei of the present invention can further comprise as endoglycosidase, cellulase, proteolytic enzyme, lipase or other diastatic other enzyme, for example, is the known amylase that is derived from bacstearothermophilus in this area.
Embodiment of the present invention comprise the binding substances according to α-Dian Fenmei of the present invention and proteolytic enzyme, preferably include oxidation-stabilized proteolytic enzyme, as at US.Re34, the enzyme of describing in 606 (herein quoting as a reference) and the commerciality enzyme that can get are as DURAZYM (NovoNordisk), MAXAPEM (Gist brocades) and PURAFECT
OxP (Genencor International company).Make these protease mutants (oxidative stability proteolytic enzyme) and particularly have in the method that is equivalent to the mutant that the methionine(Met) of separating M222 position in the starch Bacillus licheniformis replaces and see U.S, Re34 is described in 606.
Below provide and do not constitute restriction with by way of example the claim scope.Abbreviation used herein, particularly about amino acid whose trigram or single-letter notion such as Dale, J.W. bacteria molecule genetics (Molecular Genetics of Bacteria) John Wiley﹠amp; Described in Sons (1989) the appendix B.Experimental section
Embodiment
The preparation of bacillus licheniformis α-Dian Fenmei crystalline
Crystal growth is carried out in the hanging drop of 10 μ l, wherein contains 1.6-1.8M Li
2SO
4, 1mMCaCl
2, 50mM NaCl is buffered in pH6.5 with 200mM bistrispropane.Crystal grew into rectangular prism-shaped at 7-14 days, the about 1.5mm of maximum dimension, and its spacer is P2
12
12
1, unit cell parameters is: a=118.3 , b=119.0 , c=84.9 .Suppose to contain 2 molecules in the asymmetry unit that then Matthews value (seeing Matthews, molecular biology, vol.33,409 (1968)) is 3.01, is within the normal range.RAXIS II imaging plate system is used in data gathering, is fixed in the CuK α radiation that RU-200B changes the graphite monochromatization of target X source generation.(The Woodlands, Texas) software of system disposition carries out data processing and is reduced to amplitude for this reason to use Molecular Structures company.Definite employing of phase angle information is many to be aided with anomalous scattering data (MIRAS) to the isomorphous replacement(method) (MIR), and density modification method subsequently.Heavy atom derivative prepares with traditional infusion method, SmCl
3Except the derivative, it prepares with the eutectic method.Adopt the difference Patterson method (Pattersons) and the phase difference value Fuli leaf method of reporting to the leadship after accomplishing a task to determine the heavy atom position.SmCl
3Derivative provides fabulous anomalous scattering data, is used to determine correct chirality, and determines the common initial point for all heavy atoms.Use Xheavy program people such as (, Acta Crystallog.A, vol.46,377 (1990)) Zhang to revise the heavy atom position and calculated the MIRAS phase angle.Phase angle further adopts the solvent filter method to improve, and the program of using is SQUASH (McRee, J., Mol.Graph., vol.10,44 (1992)), obtained the electron density map of 3 resolving power thus, most of secondary structure element of two molecules can both be identified on it.It is average to have carried out model building, real space correction and symmetry with Xfit program (Zhang, the source is the same).In two molecules, the C alpha position of the C-terminal in the βZhe Die in α/β barrel shaped structure territory and the C alpha position of α spiral and all beta structure territories is all identified.With Aspergillus α-Dian Fenmei (PDB code name 6TAA) (people such as Swift, Acta Crystallog.B, vol.47,535 (1991)) the TIM barrel mould in is covered on the C α track of two molecules in the asymmetry unit approx, accurately locatees with the real space correction of whole unmodified structural domain then.So just can accurately measure local symmetry operator, it is average that the latter is used to the non-crystallographic symmetry of electron density map.The electronics that the has obtained remarkable improvement thus figure that conspires, but structural domain B is still bad.At this moment, only have a molecule to be built in the average figure of symmetry, another molecule generates with local symmetrical operator.The C alpha position is identified, and main chain constructs with the method for pentamer of coinciding, pentamer take from a database through the structure of fine correction (Zhang, the source is the same; People such as Jones, EMBO, vol.5,819 (1986)).At structural domain B, the major part of electron density map can not explain that all only residue 105-116 and 133-169 can be built.Those are not had the residue of obvious side chain density, replace with the L-Ala model.Use Xplor program (people such as Brunger, ActaCrystallog.A, vol.45,50 (1989)) to this initial model with the slow temperature-fall period of simulated annealing (initial temperature=3000K), revise with traditional method of least squares subsequently, use the data (F＞=3 σ F) between the 15-3.0 , apply the non-crystallographic symmetry constraint.Model converges on the R factor 0.28.In conjunction with the phase angle of MIRAS and model, calculated the electron density map of 2.2 with sigmaA method (Read, Acta Crystallog.A, Vol.42,140 (1986)).The residue of disappearance is built out, and the rest part to structure has carried out sizable manual setting simultaneously, and (initial temperature=1000K) revise is used the data (F＞=3 σ F) between the 8.0-2.2A to use simulated annealing then.Model converges on the R factor 0.245.The restricted isotropy B factor correction of carrying out subsequently provides the R factor 0.225.Adopt the phase angle meter that calculates to calculate the 2fo-fc and the fo-fc electron density map of sigmaA weighting, and be used to differentiate mistake, and the location calcium ion.After obtaining the parent data of 1.9 , fo-fc and 2fo-fc difference electron density map are used to locate residual mistake and the orderly water molecules of identification, succeeded by the B factor correction of Powell energy minimization and stereochemistry constraint.
The R factor of model is 0.19 (15-1.9 , F＞=3 σ F) at present, comprises 7914 non-hydrogen atoms and comprises 630 water Sauerstoffatoms and 3 calcium atoms.Its geometry quality is fine, is respectively 0.012 and 1.35 ° with the r.m.s deviation of desirable bond distance's bond angle.The Ramachandron figure at φ and ψ angle shows that residue 150 is unique non-glycine residues that significantly depart from the permission district.
The bacillus licheniformis α-Dian Fenmei comprises 483 residues.The residue disappearance of 3 residues of N end and C end in present model, the residue of disappearance also has: the residue 181-195 of molecule 1, the residue 181-193 of molecule 2.The data that this example is derived are listed in table 1.
Table 1
| Data set | Resolving power | Rmerge | ????R-deriv | Number of sites | Phasing?power | Anomalous scattering |
| Parent SmCl 3????PtI 6????PtCl 4??Hg(Ac) 2????IrCl 6????HgI 3???Me 3PbI | ????????? ?50-1.8 ?50-2.2 ?50-3.0 ?50-3.0 ?50-3.0 ?50-3.0 ?50-2.2 ?50-2.2 | ?0.09 | ?????- ????0.073 ????0.259 ????0.249 ????0.124 ????0.226 ????0.133 ????0.186 | ????- ????4 ????5 ????5 ????4 ????4 ????12 1????2 | ?????- ????1.45 ????1.22 ????1.29 ????1.33 ????1.01 ????1.48 ????1.29 | ????- ????Y ????N ????N ????N ????N ????Y ????Y |
Claims (14)
1. one kind comprises structural domain A, the α-Dian Fenmei of domain C and calcium ion binding site, wherein this calcium ion binding site is relevant with described domain C with described structural domain A, it comprises the part residue that is arranged in described structural domain A and/or domain C, thereby wherein said α-Dian Fenmei is modified the character that changes described α-Dian Fenmei with the characteristic that changes this calcium ion binding site.
2. according to the α-Dian Fenmei of claim 1, wherein said modification to this α-Dian Fenmei is included in this α-Dian Fenmei and replaces, adds or delete the genetically engineered of one or more amino-acid residues and modifies.
3. according to the α-Dian Fenmei of claim 2, wherein said α-Dian Fenmei is produced by genus bacillus or its bacterium that derives.
4. according to the α-Dian Fenmei of claim 3, wherein said α-Dian Fenmei is produced by Bacillus licheniformis, bacillus amyloliquefaciens or bacstearothermophilus or its bacterium that derives.
5. according to the α-Dian Fenmei of claim 2, wherein said genetically engineered is modified to be included in and is equivalent to 290-309 in the Bacillus licheniformis, 339-347, and 402-411 replaces, adds or deletes a kind of amino-acid residue on the position of 426-436 and/or 472-477.
6. according to the α-Dian Fenmei of claim 5, wherein said genetically engineered is modified and is included in Q291 in the Bacillus licheniformis, Q298, G299, G301, Y302, M304, L307, N309, Q340, F343, F403, H405, H406, D407, V409, G410, L427, I428, D430, G433, K436, N473, replace place among G474 and the G475 or many places.
7. washing composition that contains the α-Dian Fenmei of with good grounds claim 1.
8. starch liquefacation composition that contains the α-Dian Fenmei of with good grounds claim 1.
9. according to the α-Dian Fenmei of claim 1, wherein said α-Dian Fenmei also is included in and is equivalent to M15 in the Bacillus licheniformis, V128, H133, W138, N188, the replacement of one or more residues of A209 and/or M197 or deletion.
10. the method for a liquefying starch, it may further comprise the steps: will join according to a kind of α-Dian Fenmei of claim 1 in the aqueous solution of starch, and hatch the suitable time with this amidin that liquefies under appropriate condition.
11. a coding is according to the DNA of the α-Dian Fenmei of claim 1.
12. an expression vector, wherein the DNA of claim 11 operably is connected to the expression control sequenc that can be used to express this DNA.
13. expression vector transformed host cells with claim 12.
14. method of in host cell, expressing and/or secreting the DNA of claim 11.
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| US8343747B2 (en) | 2006-12-21 | 2013-01-01 | Verenium Corporation | Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them |
| US10100299B2 (en) | 2006-12-21 | 2018-10-16 | Basf Enzymes Llc | Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them |
| CN102378813A (en) * | 2009-04-01 | 2012-03-14 | 丹尼斯科美国公司 | Compositions and methods comprising alpha-amylase variants with altered properties |
| CN102378813B (en) * | 2009-04-01 | 2014-05-14 | 丹尼斯科美国公司 | Compositions and methods comprising alpha-amylase variants with altered properties |
| EP3121270A3 (en) * | 2011-06-30 | 2017-03-08 | The Procter & Gamble Company | Cleaning compositions comprising amylase variants reference to a sequence listing |
| CN103667095B (en) * | 2013-03-12 | 2015-05-13 | 江南大学 | Alpha-amylase high-yield bacterial strain and method for producing amylase by fermenting |
| CN103667095A (en) * | 2013-03-12 | 2014-03-26 | 江南大学 | Alpha-amylase high-yield bacterial strain and method for producing amylase by fermenting |
| US12173260B2 (en) | 2017-02-01 | 2024-12-24 | The Procter & Gamble Company | Cleaning compositions comprising amylase variants |
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