CN112342208A - Pullulanase mutant - Google Patents

Pullulanase mutant Download PDF

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CN112342208A
CN112342208A CN202011380782.8A CN202011380782A CN112342208A CN 112342208 A CN112342208 A CN 112342208A CN 202011380782 A CN202011380782 A CN 202011380782A CN 112342208 A CN112342208 A CN 112342208A
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CN112342208B (en
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牛丹丹
王正祥
田康明
申培立
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Senda Tianjin Biotechnology Co ltd
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Tianjin University of Science and Technology
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to combined site-specific mutagenesis of pullulanase molecules, screening and expression preparation of mutants. The invention takes the wild bacillus pullulanase (PulA) of amino acid shown in SEQ ID NO.2 as a basis, obtains the pullulanase mutant with the improved or reduced optimal action temperature, the improved or reduced optimal action pH and the obviously improved level of heterologous expression by directional molecular evolution, thereby improving the application performance of the pullulanase, expanding the application field of the pullulanase and improving the manufacturing efficiency of the pullulanase in industrial scale.

Description

Pullulanase mutant
The technical field is as follows:
the invention belongs to the technical field of enzyme engineering, and particularly relates to combined site-specific mutagenesis of pullulanase molecules, screening and expression preparation of mutants.
Background art:
starch molecules formed in nature by photosynthesis and the like are the most important raw materials for human life. Starch is polymerized from glucose, takes alpha-1, 4 and alpha-1, 6 glucosidic bonds as important polysaccharide substances, can be used as a primary raw material in the industries of food, brewing, pharmacy and the like, and is the most abundant polysaccharide produced by plants besides cellulose. The starch is divided into oligobranched amylose and multi-branched amylopectin, of which 75-80% are amylopectin. The glucose in amylose is linked by alpha-1, 4 glycosidic linkages to form linear molecules, and by alpha-1, 6 glycosidic linkages at the branching points of amylopectin.
Hydrolysis of the alpha-1, 6 glucosidic bonds in starch is the rate-limiting step in starch integrated processing and starch bioavailability. In order to improve the enzymatic hydrolysis efficiency of starch, reduce the usage amount of related enzyme preparations, improve the bioavailability of starch, and produce new products derived from starch and food processing by using starch as raw materials, the alpha-1, 6 glycosidic bond in starch molecule needs to be hydrolyzed in the starch processing and starch biotransformation processes. However, most of the alpha-amylases have alpha-1, 4-glycosidic bond hydrolysis sites, and the saccharifying enzymes have weak alpha-1, 6-glycosidic bond hydrolysis activity, but can only slowly hydrolyze alpha-1, 6 glycosidic bonds in starch molecules, can not efficiently complete the complete hydrolysis of the starch by only using the existing alpha-amylase and saccharifying enzyme, uses the starch debranching enzyme singly, synchronously or step by step, can greatly improve the generation amount of starch to amylose, the saccharification efficiency of starch to glucose or maltose, and remarkably reduce the use amount of saccharifying enzyme, beta-amylase and other enzymes [ van der Maarel M J, van der Veen B, Uitdehaag J C, et al.Properties and applications of static-converting enzymes of the alpha-amylase [ J ]. Journal of Biotechnology, 2002, 94 (2): 137-.
It has been found that various enzyme preparations have professionally hydrolyzed alpha-1, 6 glycosidic linkages in starch molecules, including isoamylases and pullulanases. Wherein the pullulanase comprises pullulanase I, pullulanase II (amylopullulanase), pullulanase I (neopullulanase), pullulanase II (iso-pullulanase) and pullulanase III. Among them, pullulanase type I derived from Bacillus deramificans alone is produced on a large scale and industrially used [ bertholod C, Antranikian g.starch-hydrolizymes from thermophilic archaea and bacteriosis [ J ]. Current Opinion in Chemical Biology, 2002, 6 (2): 151-160. the pullulanases are generally referred to as type I pullulanases.
In the starch debranching process, the enzymological properties of pullulanase, such as optimum action temperature, optimum action pH and enzymolysis catalysis efficiency, are main factors influencing the application value of pullulanase. For example, when preparing glucose by an amylase method, the pullulanase can greatly improve the formation ratio and the formation rate of the glucose in the sugar preparation process by the amylase method and can obviously reduce the dosage of the saccharifying enzyme; the consistency with the saccharification reaction conditions (pH4.5, 60-62 ℃) of the saccharifying enzyme can greatly simplify the saccharification process and the engineering control. For another example, when preparing maltose by the amylase method, under the condition of beta-amylase saccharification reaction (pH5.5, 55-60 ℃), the yield of the maltose can be improved by more than 20 percent by using the beta-amylase and the pullulanase in a composite way. However, the optimum action pH of the existing pullulanase is 5.0, the optimum action temperature is 55 ℃, the optimum action condition range is narrow, the catalytic action of the pullulanase is not favorably exerted, and the starch debranching action of the pullulanase cannot be well exerted even under the condition that the using amount of the pullulanase is remarkably increased.
The biochemical property and the catalytic property of a specific enzyme molecule can be effectively improved through the in vitro evolution technology of the enzyme molecule, such as the change of the optimal action temperature, the optimal action pH, the specific enzyme activity and the like. For example, deletion of the N-terminal or C-terminal part of pullulanase can significantly improve its thermostability and improve its heterologous expression level [ Qin, Xienizhong, Caoshao, etc. ], molecular modification and enzymatic property research of Bacillus nanoganoensis pullulanase gene [ J ]. Biotechnology 2014(2) ]; a linker sequence between functional domains in pullulanase molecules is modified to significantly improve its thermal stability [ Xuguo Duan, Jian Chen, Ju Wu. Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans cellulase by Site-Directed Mutagenesis [ J ]. Applied and Environmental Microbiology,2013 ].
However, through directed molecular evolution, mutation or combined mutation is carried out on specific amino acid sites of the pullulanase (PulA) of Bacillus nanoscopic (Bacillus nanoensis), the optimal action temperature of the pullulanase is improved or reduced, the activity of the pullulanase at relatively low or high pH (pH4.5 or below or pH5.5 or above) is improved, the expression level of the pullulanase in common host cells is improved, the application effect of the pullulanase in starch processing is improved, the industrial preparation level of the pullulanase is improved, the production cost is reduced, and the method has important significance for optimizing and expanding the industrial application value of the pullulanase.
The invention content is as follows:
the invention aims to obtain the pullulanase mutant with the optimal action temperature increased or reduced, the optimal action pH increased or reduced and the heterologous expression level obviously increased by directed molecular evolution, thereby improving the application performance of the pullulanase, expanding the application field of the pullulanase and improving the manufacturing efficiency of the pullulanase in industrial scale.
In order to achieve the purpose, the technical scheme provided by the invention is that based on the nucleotide (obtained after codon optimization) shown in a sequence table SEQ ID NO.1 or the amino acid of the wild bacillus pullulanase (PulA) shown in a sequence table SEQ ID NO.2, a mutant is prepared through site-specific mutagenesis, a pullulanase mutant with excellent performance is obtained through the expression and directional screening of the mutant, and the high-efficiency preparation of the pullulanase mutant is realized through high-efficiency expression;
the pullulanase mutants described are those in which a mutation or a combination of mutations at amino acid residues S99, E100, Q108, S112, a146, a235, a272, N317, T238, N322, K327, N342, a347, T355, a356, S357, G358, T385, a414, N450, K453, K460, K463, N467, K469, K476, G479, N492, N521, N523, N587, a591, K626, S630, a656, K665, G666, K669, N709, G723, T698, T730, S731, N734, G758, G769, G775, G776, N870, N879, N888, G900 or G904 has occurred at an amino acid residue position in the amino acid sequence shown in SEQ ID No.2, with a similarity of more than 90%, or more than 85%, more than 95% or more than 98% similarity of the amino acid sequence 2, or with a single point mutation or a combination of the amino acid residues of the amino acid sequence indicated above 95, more than 95%, or more than 95% similarity of the amino acid sequence indicated in SEQ ID No.2, or more than 95% of the amino acid sequence indicated above;
further, the pullulanase mutants described are mutations at amino acid residues S99, E100, Q108, S112, a146, a235, a272, N317, T238, N322, K327, N342, a347, T355, a356, S357, G358, T385, a414, N450, K453, K460, K463, N467, K469, K476, G479, N492, N521, N523, N587, a591, K626, S630, a656, K665, G666, K669, N709, G723, T698, T730, S731, N734, G758, G769, G775, G776, N864, N870, N879, N888, G900 or G904 shown in SEQ ID No.2, and can be arbitrarily replaced by the remaining 19 amino acid residues, i.e: serine residues can be replaced with alanine (Ala, a) [ mutations are: s99 → a99 or S99A, and so on, arginine (Arg, R), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), histidine (His, H), isoleucine (Ile, I), glycine (Gly, G), asparagine (Asn, N), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), or valine (Val, V); the glutamic acid residue (Glu, E) may be replaced with alanine (Ala, a), arginine (Arg, R), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), histidine (His, H), isoleucine (Ile, I), glycine (Gly, G), asparagine (Asn, N) leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), or valine (Val, V). Mutation of other amino acid sites, and so on.
Furthermore, combined mutation can be obtained on the basis of single site mutation to form a pullulanase mutant combined by multi-site mutation. The multi-site combined mutation is beneficial to the improvement of the enzymatic properties and the catalytic efficiency of the pullulanase, and the labeling mode is as follows: the mutation of the 99 th serine residue and the 100 th glutamic acid residue occurs on the basis of the amino acid sequence shown in SEQ ID NO.2, and can be marked as "S99 + E100"; furthermore, the 99 th serine residue is mutated to alanine, while the 100 th glutamic acid residue is mutated to alanine, which can be labeled as: "S99 → A99, E100 → A100" or "S99A + E100A", and so on;
further, the pullulanase mutants described are mutations at amino acid residues S99, E100, Q108, S112, a146, a235, a272, N317, T238, N322, K327, N342, a347, T355, a356, S357, G358, T385, a414, N450, K453, K460, K463, N467, K469, K476, G479, N492, N521, N523, N587, a591, K626, S630, a656, K665, G666, K669, N709, G723, T698, T730, S731, N734, G758, G769, G775, G776, N864, N870, N879, N888, G900 or G904 in the amino acid sequence shown in SEQ ID No.2, preferably substituted with the following amino acid residues: wherein the serine (S) residue may be mutated to V, L, I, V, M, F, W, Y, R, E, C, Q, K, N, D, P, T, A, G, M or H; the glutamic acid (E) residue may be mutated to L, I, M, V, Y, C, H, F, P, R, W, A, Q, S or G; the glutamine (Q) residue may be mutated to L, I, C, K, R, E, Y, V, M, F, W, H, N, P, T or D; asparagine (N) residue can be mutated to V, L, I, M, P, T, E, Y, C, F, W, D, R, K, G, S, A, Q or H; lysine (K) residues may be mutated to Y, V, L, P, F, C, Q, D, M, T, E, N, W, S, R, I, G, A or H; the alanine (a) residue can be replaced with V, L, I, C, P, M, E, D, F, S or W; the glycine (G) residue may be replaced with E, V, L, I, M, Y, A, P, S, C or E; the threonine (T) residue can be replaced with Y, V, I, M, N, K, L, F, P, E, C, D, S or W;
further, the pullulanase mutants described are combined mutations at two or more positions on S99, E100, Q108, S112, a146, a235, a272, N317, T238, N322, K327, N342, a347, T355, a356, S357, G358, T385, a414, N450, K453, K460, K463, N467, K469, K476, G479, N492, N521, N523, N587, a591, K626, S630, a656, K665, G666, K669, N709, G723, T698, T730, S731, N734, G758, G769, G775, G776, N864, N870, N879, N888, G900 or G904 at the amino acid residues in the amino acid sequence shown in SEQ ID No. 2. The similarity of the amino acid sequence shown in SEQ ID NO.2 is more than 85%, or more than 90%, or more than 95%, or more than 96%, or more than 97%, or more than 98%, or more than 99%, but the similarity is lower than 100%.
Further, the optimum action temperature of the pullulanase mutant is 60 ℃ or above or 50 ℃ or below;
further, the optimum action pH of the pullulanase mutant is pH4.5 or below or pH5.5 or above;
further, compared with the pullulanase of the amino acid sequence 2, the expression level of the described pullulanase mutant in a common expression host cell is improved by at least more than 5%, or is improved by at least more than 10%, or is further improved by more than 20%, or is further improved by more than 30%, or is improved by more than 50%, or is improved by more than 100%, or is improved by more than 200%, or is improved by more than 300%, or is improved by more than 400%, or is improved by more than 500%;
further, the pullulanase mutants described are combined mutations of amino acid residues S99, E100, Q108, S112, a146, a235, a272, N317, T238, N322, K327, N342, a347, T355, a356, S357, G358, T385, a414, N450, K453, K460, K463, N467, K469, K476, G479, N492, N521, N523, N587, a591, K626, S630, a656, K665, G666, K669, N709, G723, T698, T730, S731, N734, G758, G769, G775, G776, N864, N870, N879, N888, G900 or G904 in the amino acid sequence shown in SEQ ID No.2, such combined mutations facilitate increased expression levels of pullulanase heterologous alterations. Compared with pullulanase shown in SEQ ID NO.2, the hydrolytic activity of the pullulanase is improved by at least 5 percent, or improved by at least 10 percent, or further improved by more than 20 percent, or further improved by more than 30 percent, or further improved by more than 50 percent, or further improved by more than 100 percent, or further improved by more than 200 percent, or further improved by more than 300 percent, or further improved by more than 400 percent, or further improved by more than 500 percent;
wherein, the pullulanase combination mutation which is beneficial to improving the starch debranching hydrolysis activity at pH4.5 and/or 60 ℃ is as follows:
N467+N709
N467+N492+N709
N492+S630+N709+N734
N492+N734
N467+N492+S731
N467+N492+N709+T730
N467+N492+N709+A591+G723
N467+N492+N709+A591+T730；
further, the pullulanase combinatorial mutations that facilitate the enhancement of starch debranching hydrolysis activity at ph4.5 and/or 60 ℃ are:
N467G+N709R
N467G+N492A+N709R
N467G+N492A+S731C
N467G+N492A+N709R+T730S
N467G+N492A+N709R+A591S+G723S
N467G+N492A+N709R+A591S+T730S；
wherein, the pullulanase combined mutation which is beneficial to improving the starch debranching hydrolysis activity at pH5.5 and 55 ℃ is as follows:
N467+N492
N467+N492+G723
N467+N492+N709+S731
N467+N492+N709+G723+S731
N467+N492+N709+G723+T730
E100+A317+N450+N523+T730
E100+K460
E100+A317+N450+K463+N523+N864
E100+A317+N450+N523+G758+N846；
further, the pullulanase combinatorial mutations that contribute to the enhancement of starch debranching hydrolysis activity at ph5.5 and 55 ℃ are:
N467G+N492A
N467G+N492A+G723S
N467G+N492A+N709R+S731C
N467G+N492A+N709R+G723S+S731C
N467G+N492A+N709R+G723S+T730S；
the pullulanase combined mutation which is beneficial to improving the starch debranching hydrolysis activity at lower pH (pH4.5) or lower temperature (45-50 ℃) is as follows:
N467+A591+N709
N467+T730
N467+A591
N467+S731
N492+S630+N734
N467+A591+G723
N467+A591+T730
E100+K460+N734+N870
N467G+N492+A591；
further, the pullulanase combinatorial mutations that facilitate the enhancement of starch debranching hydrolysis activity at lower pH (pH4.5) or lower temperature (45-50 ℃) are:
N467G+A591S+N709R
N467G+T730S
N467G+A591S
N467G+A591S+G723S
N467G+A591S+T730S
N467G+N492A+A591S
N467G+S731C；
among the pullulanase combinatorial mutations that contribute to the enhancement of heterologous expression levels in common microbial expression systems are:
N467+N492
N467+A591
N467+N709
N467+G723
N467+T730
N467+N492+A591
N467+N492+N709
N467+N492+T730
N467+N492+S731
N467+A591+N709
N467+A591+G723
N467+A591+T730
N467+A591+S731
N467+N492+N709+G723
N467+N492+N709+T730
N467+N492+N709+S731
N467+N492+N709+A591
N467+N492+N709+A591+G723
N467+N492+N709+A591+T730
N467+N492+N709+G723+T730
N467+N492+N709+G723+S731
N467+N492+N709+A591+T730+S731
A317+N467+G476+N709
A317+N467+S630+S731+G900
E100+K460+N734+N870
E100+K460+A591+N734+G758；
further, the pullulanase combinatorial mutations that facilitate the enhancement of heterologous expression levels in common microbial expression systems are:
N467G+N492A
N467G+A591S
N467G+N709R
N467G+G723S
N467G+T730S
N467G+N492A+A591S
N467G+N492A+N709R
N467G+N492A+T730S
N467G+N492A+S731C
N467G+A591S+N709R
N467G+A591S+G723S
N467G+A591S+T730S
N467G+A591S+S731C
N467G+N492A+N709R+A591S
N467G+N492A+N709R+G723S
N467G+N492A+N709R+T730S
N467G+N492A+N709R+S731C
N467G+N492A+A591S+N709R+G723S
N467G+N492A+N709R+A591S+T730S
N467G+N492A+N709R+G723S+T730S
N467G+N492A+N709R+G723S+S731C
N467G+N492A+N709R+A591S+T730S+S731C；
the described pullulanase mutant encoding gene sequence is a sequence obtained by artificial synthesis after codon optimization according to the nucleotide sequence shown in SEQ ID NO.1, the amino acid shown in SEQ ID NO.2 and the codon change of corresponding mutation sites, and has the similarity of more than 80%, or more than 90%, or more than 95%, or more than 96%, or more than 97%, or more than 98%, or more than 99% with the nucleotide sequence shown in SEQ ID NO.1, but the similarity is lower than 100%. But not limited to, this sequence can be easily obtained by nucleotide sequence design and chemical synthesis according to codon bias of different expression hosts.
The invention also provides a recombinant vector or a recombinant bacterium containing the pullulanase mutant coding gene, so that the described pullulanase mutant can obtain high-efficiency secretion expression in the recombinant bacterium through genetic recombination and molecular cloning technologies;
preferably, the recombinant vector uses expression vectors including, but not limited to, pHY-WZX, pHY300plk, pUB110, pE194, pHT1469 plasmid (MoBiTec);
more preferably, the recombinant vector adopts an expression vector which is integrated with a signal peptide derived from a Bacillus licheniformis amylase promoter PamyL or a Bacillus subtilis promoter P43 or a Bacillus thuringiensis insecticidal protein promoter Pcry and a Bacillus licheniformis amylase amyL signal peptide, and an alkaline protease aprE signal peptide or a Bacillus subtilis amylase amyE signal peptide;
preferably, the recombinant bacteria for high-efficiency expression of the pullulanase mutant refer to recombinant bacteria taking escherichia coli, bacillus subtilis, bacillus licheniformis, bacillus pumilus, bacillus megaterium, saccharomyces cerevisiae, pichia pastoris, aspergillus niger, aspergillus oryzae, trichoderma reesei or the like as host cells;
more preferably, the recombinant bacterium for high-efficiency expression of the pullulanase mutant takes bacillus subtilis or bacillus licheniformis as a host cell;
more preferably, the recombinant bacterium for high-efficiency expression of the pullulanase mutant takes bacillus licheniformis as a host cell;
more preferably, the recombinant strain adopts an expression host of Bacillus licheniformis CBB3008 (number CCTCC NO. M208236).
More preferably, the recombinant bacterium is used for carrying out recombinant expression on the pullulanase mutant encoding gene by adopting an expression vector pHY-WZX in a Bacillus licheniformis CBB3008 host cell.
The invention also provides a method for producing the pullulanase mutant by fermenting the recombinant bacteria, which comprises the following steps:
fermenting in a fermentation tank to produce pullulanase mutants: inoculating the strain into a fermentation tank according to the inoculation amount of 5% -10%; in the fermentation process, the fermentation temperature is 33-45 ℃, the dissolved oxygen is controlled to be 0.1-20%, the pH is 6.0-7.8, 30-60% (w/w) of maltose syrup is fed-batch, and the content of reducing sugar is maintained to be 0.1-5%; the fermentation lasts for 90-120h, the sampling analysis is carried out at regular time in the fermentation process, and the fermentation end point is controlled to be that the enzyme activity increase value is less than 5-25U/h within 4 hours of fermentation.
The fermentation medium comprises the following components: 1-5% of maltose syrup, 0-5% of cottonseed meal, 0-4% of corn steep liquor, 0.5-5% of bean cake meal, 0.1-5% of ammonium sulfate and the balance of water, wherein the pH value is 6.0-8.0.
After fermentation, the enzyme activity of the fermentation liquid of the pullulanase mutant fermented in the fermentation tank can reach 2000-4600U/mL.
Has the advantages that:
the pullulanase mutant is obtained by directed molecular evolution and has the advantages of higher/lower optimal action temperature, lower pH/higher optimal action pH and higher heterologous expression level. The pullulanase mutant which catalyzes the debranching of starch molecules at higher temperature and lower pH can be used together with saccharifying enzyme in the production of glucose prepared from starch; the pullulanase mutant which catalyzes the debranching of starch molecules under higher pH can be used together with beta-amylase or fungal amylase in the production of maltose prepared from starch, and also can be used as an additive of animal feed; the pullulanase mutant which can catalyze the debranching of starch molecules at a lower action temperature can be used in food processing such as baking, pre-fermented dough, fine dried noodles and the like, and can also be used in alcohol synchronous saccharification and fermentation. The pullulanase of the invention is a pullulanase mutant with obviously improved heterologous expression level, is beneficial to the high-efficiency and low-cost fermentation preparation of the pullulanase and has enzyme preparation cost.
Description of the drawings:
FIG. 1 optimal pH for representative pullulanase mutants
Wherein, ●: combination mutant N467G + N492A + N709R;
solid content: combination mutant N467G + N492A + a591S + N709R + G723S;
a tangle-solidup: combination mutant N467G + N492A + N709R + G723S + S731C;
□: wild-type pullulanase;
FIG. 2 optimal temperature for a representative pullulanase mutant
Wherein, ●: combination mutant N467G + N492A + N709R;
solid content: combination mutant N467G + N492A + a591S + N709R + G723S;
a tangle-solidup: combination mutant N467G + N492A + N709R + G723S + S731C;
□: wild-type pullulanase.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.
The wild pullulanase adopted by the invention is pullulanase (PulA) (sequence 1 (obtained by chemical synthesis after codon optimization) and sequence 2) derived from bacillus longissimus (B.nanogeneris) ATCC 53909.
The invention relates to a method for constructing pullulanase mutant, which is characterized in that an oligonucleotide sequence with a changed specific codon obtained by chemical synthesis is used for carrying out mutation preparation on specific amino acid residues in pullulanase moleculesThe corresponding codons of the gene are directionally altered by the technique of overlapping PCR (Crossover PCR) commonly used in laboratories or by using commercially available mutation kits (e.g., those of BioLab, New England)
Figure BDA0002809253730000111
Site-directed mutagenesis kit or Agilent second generation QuikChange site-directed mutagenesis reagent).
The invention relates to a directional screening method of pullulanase mutants, which clones coding genes of pullulanase mutants of Bacillus longissins (B.nanoensis) and coding genes (nucleotide sequence SEQ ID NO.1) of pullulanase (amino acid sequence SEQ ID NO.2) of Bacillus longissins (B.nanoensis) into an expression vector pHY-WZX (Niu DD, Wang ZX.development of a pair of microbial expression vectors for Escherichia coli and Bacillus licheniformis. J Ind microbial Biotechnol (34: 357-362.DOI 10.1007/s 10295-0204-x.), and genetically transforms the coding genes into a Bacillus strain CCTCC NO. M208236 (the strain is preserved in China center for type culture Collection (CCTCC for short), and the recombinant pullulanase is obtained in 2008/11); after preparing enzyme solution by shaking flask fermentation and purifying, comparing and analyzing the enzyme activity of the pullulanase mutant at different temperatures and different pH values, and comparing with the original pullulanase (PulA) to directionally screen the mutant with improved enzyme activity level. The fermentation enzyme production level of pullulanase mutants was evaluated by performing the fermentation in a 30L fermentor.
The main experimental method adopted by the invention is as follows:
1. gene cloning, molecular evolution and construction of expression plasmids
Conventional Molecular Cloning procedures were performed according to the literature reference methods (Sambrook et al, Molecular Cloning: A Laboratory Manual, 1989). The bacillus longissimus pullulanase encoding gene is obtained by chemical synthesis after codon optimization and is used as a target gene in the invention; the expression vector is pHY-WZX (Wangzhenxiang, minium, Chinese invention patent ZL 200510051648; Niu & Wang.J Ind Microbiol Biotechnol, 2007). The encoding gene of the pullulanase or the mutant thereof is cloned into an expression vector pHY-WZX after PCR amplification, and a recombinant plasmid for expressing the pullulanase is obtained.
2. Extraction of plasmid DNA
The plasmid DNA was extracted using a plasmid miniprep kit from Sigma after lysing the cell wall with a certain concentration of lysozyme.
3. Gene amplification
DNA amplification was performed in 0.2mL PCR thin wall tubes. The PCR amplification conditions were: 1 × (95 ℃ C. for 5 min); 30 x (94 ℃ for 10s, 58 ℃ for 30s, 72 ℃ for 30-200 s); 1 × (72 ℃ C. for 10 min). The extension temperature and time of the PCR reaction vary depending on the amplification length. All PCR reactions were performed using Pfu DNA polymerase, unless otherwise noted.
4. Overlapping PCR
Reference is made to the literature (Krishan B R, et al, direct and cross PCR amplification to failure Tn 5. sup. pF-based sequencing of lambda phase circles. nucleic Acids Research, 1991, 22: 6177-82). The general steps are as follows: the primers (P1+ P2; P3+ P4, and P2 and P3 are reverse complementary sequences) of the fragment F1 and the fragment F2 are used for mediating the amplification of PCR to obtain a gene fragment; recovering and purifying the amplified fragments F1 and F2 by glue; the two fragments F1 and F2 obtained by purification were diluted by an appropriate ratio, mixed at a molar ratio of 1:1 as a template, and a new PCR reaction was mediated with the primers P1 (SEQ ID NO: 9) + P4 (SEQ ID NO: 10) to obtain a full-length sequence.
5. Genetic transformation of Bacillus licheniformis
The method is described in the literature (slow sensitivity, horse and horse courser, king's auspicious, influence of high osmotic pressure on the electrical conversion rate of bacteria, university of Wuxi, 2004(04): 98-100). The method mainly comprises the following steps: inoculating fresh single colony into liquid LB culture medium, culturing overnight at 37 deg.C at 200r/min, transferring 5% bacteria liquid into new LB culture medium, and culturing until OD600 is 0.75-0.90. The thalli is subjected to ice bath for 10min, and then centrifuged at 6000r/min for 10min at 4 ℃ to collect the thalli. The cells were washed repeatedly 4 times with a pre-cooled electrotransfer wash solution (0.5mol/L sorbitol, 0.5mol/L mannitol and 10% glycerol). The cell pellet was suspended in 1mL of pre-chilled electrotransfer wash solution to complete the preparation of competent cells. 1 mu L of plasmid DNA and about 100 mu L of competent cells are mixed uniformly, immediately shocked by electricity (1800v,5ms), and then added with electrotransformation tempering liquid (LB culture medium containing 0.65mol/L sorbitol and 0.45mol/L mannitol), and after being recovered at 37 ℃ and 160r/min, the corresponding resistant LB plate is coated and cultured at a proper temperature until single colonies grow out. The correct transformant is verified by methods of plasmid extraction and enzyme digestion, fermentation and enzyme production and the like.
7. Small-scale preparation of pullulanase and mutant thereof
Producing pullulanase by shake flask fermentation: 30mL of fermentation medium (0.5-1.5% of yeast extract, 1.2-3.6% of peptone, 8-20% of glucose and pH 7.0) is filled in a 250mL triangular flask, and the recombinant bacteria are inoculated and cultured for 5-7 days at 42 ℃ and 220 r/min. After fermentation, centrifuging at 3000r/min for 10min, taking supernatant, precipitating with 30-50% saturated ammonium sulfate, purifying enzyme protein with SephadexG-200 chromatographic column, and lyophilizing enzyme active component for use.
8. Fermentation preparation of pullulanase and mutant thereof
The fermentation preparation was carried out in a 30L full-automatic fermenter: the fermentation medium comprises the following components: 1-5% of maltose syrup, 0-5% of cottonseed meal, 0-4% of corn steep liquor, 0.5-5% of bean cake meal, 0.1-5% of ammonium sulfate and the balance of water, wherein the pH value is 6.0-8.0; the inoculation amount is 5-10%; in the fermentation process, the fermentation temperature is 33-45 ℃, the dissolved oxygen is controlled to be 0.1-20%, the pH is 6.0-7.8, 30-60% (w/w) of maltose syrup is fed-batch, the content of reducing sugar is maintained to be 0.1-5%, and the fermentation is carried out for 90-120 h.
9. Pullulanase enzyme activity assay
The pullulanase activity determination method is carried out by referring to the national standard GB 1886.174-2016, and the reaction is carried out in buffers with different pH values and at different temperatures. Enzyme activity is defined as: under the given reaction conditions, 1mL or 1g of product reacts per minute to produce 1 μmol of glucose, which is an enzyme activity unit expressed in U/mL or U/g.
10. Other methods of analysis
The gene and amino acid sequence alignment is carried out by adopting DNAMAN software, and the nucleotide sequence determination is carried out by adopting a Sanger method. The sequence protein content was performed according to literature procedures (Bradford. anal Chem,1976) and protein electrophoresis was performed according to literature procedures (Zhuge Kewang Zhengxiang, technical Manual of Industrial microbiology experiments, published by the light industry of China, 1994).
Example 1: selection of pullulanase mutation site
The selection of the mutant is carried out according to the following standard: 1) the optimum action temperature of the mutant is increased to 60 ℃ or above, and the enzyme activity is not reduced compared with PulA; 2) the optimum action temperature of the mutant is reduced to 50 ℃ or below, and the enzyme activity is not reduced compared with PulA; 3) the optimum action pH of the mutant is reduced to 4.5 or below, and the enzyme activity is not reduced compared with PulA; 4) the optimum action pH of the mutant is increased to 5.5 or above, and the enzyme activity is not reduced compared with PulA; 5) compared with wild pullulanase, the enzyme activity of the mutant is improved by at least 10 percent. Using one of the selection conditions as a selection criterion, 54 amino acid residues were obtained by selection, namely S99, E100, Q108, S112, A146, A235, A272, N317, T238, N322, K327, N342, A347, T355, A356, S357, G358, T385, A414, N450, K453, K460, K463, N467, K469, K476, G479, N492, N521, N523, N587, A591, K626, S630, A656, K665, G666, K669, N709, G723, T698, T730, S731, N734, G758, G769, G775, G776, N864, N870, N879, N888, G900 and G904.
Example 2: pullulanase combined mutant with beneficial changes of enzymatic properties and catalytic efficiency
Based on the 54 amino acid residue sites obtained in the embodiment 1, combined site-directed mutagenesis is carried out, the mutated coding gene is expressed in a bacillus licheniformis strain M208236, and after a pullulanase mutant is prepared and purified, the optimum action temperature, the optimum action pH, the specific enzyme activity and the like of the mutant are analyzed and determined, and are compared with wild pullulanase.
(1) After the pullulanase is subjected to combined mutation, the combination of the following mutation sites is beneficial to the debranching hydrolysis of starch of the pullulanase mutant at the pH value of 4.5 and/or the temperature of 60 ℃, and the enzyme activity of the pullulanase mutant at the optimum action pH value and the optimum action temperature is not lower than that of wild type PulA at the optimum action pH value and the optimum action temperature. For example:
N467G+N492A+N709R
N467G+N492A+S731C
N467G+N492A+N709R+T730S
N467G+N709R
N467G+N492A+N709R+A591S+G723S
N467G+N492A+N709R+A591S+T730S
table 1 shows the composition of some representative pullulanase mutants and their optimum pH and optimum temperature.
TABLE 1 optimum pH and optimum temperature for a portion of representative pullulanase mutants
Figure BDA0002809253730000141
Figure BDA0002809253730000151
(2) After the pullulanase is subjected to combined mutation, the combination of the following mutation sites is beneficial to the debranching hydrolysis of starch of the pullulanase mutant at the pH value of 5.5 and the temperature of 55 ℃, and the enzyme activity of the pullulanase mutant at the optimum action pH value and the optimum action temperature is not lower than that of PulA under the optimum action condition. For example:
N467G+N492A+N709R+G723S+S731C
N467G+N492A
N467G+N492A+N709R+G723S+T730S
N467G+N492A+N709R+S731C
N467G+N492A+G723S
table 2 shows the composition of some representative pullulanase mutants and their optimum pH and optimum temperature.
TABLE 2 optimum pH and optimum temperature for a portion of representative pullulanase mutants
Figure BDA0002809253730000152
(3) After the pullulanase is subjected to combined mutation, the combination of the following mutation sites is beneficial to the starch debranching hydrolysis under the lower pH (pH4.5) or lower temperature (45-50 ℃), and the enzyme activity under the respective optimal conditions is not lower than that of PulA under the optimal conditions. For example:
N467G+A591S+N709R
N467G+T730S
N467G+A591S
N467G+A591S+G723S
N467G+A591S+T730S
N467G+N492A+A591S
N467G+S731C
table 3 shows the composition of some representative pullulanase mutants and their optimum pH and optimum temperature.
TABLE 3 optimum pH and optimum temperature for a portion of representative pullulanase mutants
Figure BDA0002809253730000161
(4) After the pullulanase is subjected to combined mutation, the combination of the following mutation sites is beneficial to the improvement of the expression level of the pullulanase mutant. For example:
N467G+N709R
N467G+N492A+N709R+A591S+T730S
N467G+N492A+N709R+S731C
N467G+A591S+S731C
N467G+N492A+N709R+G723S+T730S
N467G+N492A+N709R+T730S
N467G+G723S
N467G+N492A+S731C
N467G+N492A+A591S
N467G+N492A+N709R+G723S
N467G+T730S
N467G+N492A+T730S
N467G+A591S+N709R
N467G+N492A+N709R+A591S
N467G+A591S+T730S
N467G+A591S+G723S
N467G+N492A
N467G+N492A+N709R
N467G+A591S
N467G+N492A+N709R+A591S+T730S+S731C
N467G+N492A+A591S+N709R+G723S
N467G+N492A+N709R+G723S+S731C
table 4 shows the composition of some representative pullulanase mutants and the different degrees of improvement of their expression levels in shake flask fermentation level compared with pullulanase wild type, and the enzyme activity detection is performed under the respective optimum action temperature and pH conditions.
TABLE 4 expression level changes of some representative pullulanase mutants
Figure BDA0002809253730000171
Figure BDA0002809253730000181
Example 3: fermentation preparation of pullulanase mutant
Cloning the pullulanase mutant obtained in example 2, wherein the combined mutant N467G + N492A + N709R (nucleotide sequence SEQ ID NO.3, amino acid sequence SEQ ID NO.4), the combined mutant N467G + N492A + A591S + N709R + G723S (nucleotide sequence SEQ ID NO.5, amino acid sequence SEQ ID NO.6) and the combined mutant N467G + N492A + N709R + G723S + S731C (nucleotide sequence SEQ ID NO.7, amino acid sequence SEQ ID NO.8) into an expression vector pHY-WZX, and genetically transforming into a Bacillus licheniformis strain CCTCC NO. M208236 to obtain pullulanase-producing recombinant bacteria, and the corresponding recombinant bacteria are respectively renamed as: pul3M002, Pul5M004, and Pul5M 006. Fermenting for 96h in a 30L fermentation tank (the fermentation condition is that the fermentation medium comprises 2% of malt syrup, 2% of cottonseed meal, 2% of bean cake meal, 0.5% of ammonium sulfate, the balance of water, the pH value is 7.0, the inoculation amount is 5%, the fermentation temperature is 37 ℃, the dissolved oxygen is controlled to be 15%, the pH value is 7.0, 60% (w/w) of malt syrup is fed-in and the reducing sugar content is maintained to be more than 0.1% in the fermentation process, and the highest pullulanase production levels of 3 recombinant bacteria respectively reach 2029U/mL, 4109U/mL and 4608U/mL (the enzyme activity detection condition is that the pH value is 4.5, and the temperature is 60 ℃ (national standard)). Under the same fermentation condition, the enzyme production level of wild type bacillus longissimus pullulanase (PulA) is 1106U/mL, and the expression levels of 3 pullulanase mutants are respectively improved by 0.83, 2.72 and 3.17 times.
Example 4: optimum action condition of pullulanase mutant
The pullulanase mutants obtained in example 3 were analyzed for optimal action temperature and optimal action pH.
The optimum action pH of the combined mutant N467G + N492A + N709R (nucleotide sequence 3, amino acid sequence 4) and the combined mutant N467G + N492A + A591S + N709R + G723S (nucleotide sequence 5, amino acid sequence 6) is reduced to 4.5, and the pH between 3.5 and 5.0 has high starch debranching activity (figure 1; ●,. diamond-solid); the optimum action pH of the combined mutant N467G + N492A + N709R + G723S + S731C (nucleotide sequence 7, amino acid sequence 8) is increased to 5.5, and the combined mutant has high starch debranching activity between pH5.0 and 6.0 (shown in figure 1;. tangle solidup).
The optimum action temperature of the combined mutant N467G + N492A + N709R (nucleotide sequence 3, amino acid sequence 4) is increased to 60 ℃, and the combined mutant shows higher starch debranching activity at 45-60 ℃ (figure 2; ●); compared with the wild type, the optimal action temperature of the combined mutant N467G + N492A + A591S + N709R + G723S (nucleotide sequence 5 and amino acid sequence 6) and the combined mutant N467G + N492A + N709R + G723S + S731C (nucleotide sequence 7 and amino acid sequence 8) is not obviously changed, but the temperature range for exerting the effective action is obviously widened (figure 2;. solidup-solidup).
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
SEQUENCE LISTING
<110> Tianjin science and technology university
<120> a pullulanase mutant
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<170> PatentIn version 3.5
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<213> Bacillus longissimus (B. Nanoensis) ATCC53909
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Asp Gly Asn Thr Thr Asn Ile Val Val His Tyr Phe Arg Pro Ser Gly
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Asp Tyr Thr Asp Trp Asn Leu Trp Met Trp Pro Glu Asn Gly Asp Gly
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Ser Val Asp Ile Pro Gly Asn Pro Ser Gln Val Gly Ile Ile Val Arg
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Asn Ala Tyr Leu Asp Ala Ser Asn Gln Val Leu Val Lys Leu Ser Gln
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Thr Ala Asn Lys Asp Ile Pro Val Thr Ser Val Ser Asp Ala Asn Gln
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Val Thr Ala Val Leu Ala Gly Thr Phe Gln His Ile Phe Gly Gly Ser
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Asp Trp Ala Pro Asp Asn His Asn Thr Leu Leu Lys Lys Val Asn Ser
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Asn Leu Tyr Gln Phe Ser Gly Asn Leu Pro Glu Gly Asn Tyr Gln Tyr
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Lys Val Ala Leu Asn Asp Ser Trp Asn Asn Pro Ser Tyr Pro Ser Asp
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Asn Ile Asn Leu Thr Val Pro Ala Gly Gly Ala His Val Thr Phe Ser
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Tyr Ile Pro Ser Thr His Ala Val Tyr Asp Thr Ile Asn Asn Pro Asn
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Ala Asp Leu Gln Val Asp Ser Ser Gly Val Lys Thr Asp Leu Val Ala
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Val Thr Leu Gly Glu Asn Pro Asp Val Ser His Thr Leu Ser Ile Gln
275 280 285
Thr Glu Asp Tyr Gln Ala Gly Gln Val Ile Pro Arg Lys Val Leu Asp
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Ser Ser Gln Tyr Tyr Tyr Ser Gly Asp Asp Leu Gly Asn Thr Tyr Thr
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Lys Asn Ala Thr Thr Phe Lys Val Trp Ala Pro Thr Ser Thr Gln Val
325 330 335
Asn Val Leu Leu Tyr Asn Ser Ala Thr Gly Ala Val Thr Lys Thr Val
340 345 350
Pro Met Thr Ala Ser Gly His Gly Val Trp Glu Ala Thr Val Asn Gln
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Asp Leu Glu Asn Trp Tyr Tyr Met Tyr Glu Val Thr Gly Gln Gly Ser
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Asn Asp Pro Thr Gln Tyr Asn Trp Gly Tyr Asp Pro Arg Asn Tyr Asn
500 505 510
Val Pro Glu Gly Gln Tyr Ala Thr Asn Ala Asn Gly Thr Thr Arg Ile
515 520 525
Lys Glu Phe Lys Glu Met Val Leu Ser Leu His Gln Asp His Ile Gly
530 535 540
Val Asn Met Asp Val Val Tyr Asn His Thr Phe Ala Thr Gln Ile Ser
545 550 555 560
Asp Phe Asp Lys Ile Val Pro Glu Tyr Tyr Tyr Arg Thr Asp Asp Ala
565 570 575
Gly Asn Tyr Thr Asn Gly Ser Gly Thr Gly Asn Glu Ile Ala Ala Glu
580 585 590
Arg Pro Met Val Gln Lys Phe Ile Ile Asp Ser Leu Lys Phe Trp Val
595 600 605
Asn Glu Tyr His Val Asp Gly Phe Arg Phe Asp Leu Met Ala Leu Leu
610 615 620
Gly Lys Asp Thr Met Ser Lys Ala Ala Thr Gln Leu His Ala Ile Asp
625 630 635 640
Pro Gly Ile Ala Leu Tyr Gly Glu Pro Trp Thr Gly Gly Thr Ser Ala
645 650 655
Leu Pro Ala Asp Gln Leu Leu Thr Lys Gly Ala Gln Lys Gly Met Gly
660 665 670
Val Ala Val Phe Asn Asp Asn Leu Arg Asn Gly Leu Asp Gly Ser Val
675 680 685
Phe Asp Ser Ser Ala Gln Gly Phe Ala Thr Gly Ala Thr Gly Leu Thr
690 695 700
Asp Ala Ile Lys Asn Gly Val Glu Gly Ser Ile Asn Asp Phe Thr Ala
705 710 715 720
Ser Pro Gly Glu Thr Ile Asn Tyr Val Thr Ser His Asp Asn Tyr Thr
725 730 735
Leu Trp Asp Lys Ile Ala Gln Ser Asn Pro Asn Asp Ser Glu Ala Asp
740 745 750
Arg Ile Lys Met Asp Glu Leu Ala Gln Ala Ile Val Met Thr Ser Gln
755 760 765
Gly Ile Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg Thr Lys Gly
770 775 780
Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Val Val Asn Glu Phe Asp
785 790 795 800
Trp Ser Arg Lys Ala Gln Tyr Pro Asp Val Phe Asn Tyr Tyr Ser Gly
805 810 815
Leu Ile His Leu Arg Leu Asp His Pro Ala Phe Arg Met Thr Thr Ala
820 825 830
Asn Glu Ile Asn Ser His Leu Gln Phe Leu Asn Ser Pro Glu Asn Thr
835 840 845
Val Ala Tyr Glu Leu Ser Asp His Ala Asn Lys Asp Thr Trp Gly Asn
850 855 860
Ile Val Val Ile Tyr Asn Pro Asn Lys Thr Ala Glu Thr Ile Asn Leu
865 870 875 880
Pro Ser Gly Lys Trp Glu Ile Asn Ala Thr Ser Gly Lys Val Gly Glu
885 890 895
Ser Thr Leu Gly Gln Ala Glu Gly Ser Val Gln Val Pro Gly Ile Ser
900 905 910
Met Met Ile Leu His Gln Glu Val Ser Pro Ser Asp Gly Lys
915 920 925
<210> 3
<211> 2781
<212> DNA
<213> Artificial Synthesis
<400> 3
gacggcaaca cgacgaacat cgtcgtccat tattttcgcc cgagcggcga ctatacggac 60
tggaacctgt ggatgtggcc ggaaaacggc gacggcgccg aatatgactt taaccagccg 120
acggacagct atggcgaagt cgccagcgtc gacatcccgg gcaacccgag ccaggtcggc 180
atcatcgtcc gcaaaggcaa ctgggacgcc aaagacatcg acagcgaccg ctatatcgac 240
ctgagcaaag gccatgaaat ctggctggtc cagggcaaca gccagatctt ttatagcgaa 300
aaagacgccg aagccgccgc ccagccggcc gtcagcaacg cctatctgga cgccagcaac 360
caggtcctgg tcaaactgag ccagccgttt acgctgggcg aaggcagcag cggctttacg 420
gtccatgacg acacggccaa caaagacatc ccggtcacga gcgtcagcga cgccaaccag 480
gtcacggccg tcctggccgg cacgtttcag catatctttg gcggcagcga ctgggccccg 540
gacaaccata acacgctgct gaaaaaagtc aacagcaacc tgtatcagtt tagcggcaac 600
ctgccggaag gcaactatca gtataaagtc gccctgaacg acagctggaa caacccgagc 660
tatccgagcg acaacatcaa cctgacggtc ccggccggcg gcgcccatgt cacgtttagc 720
tatatcccga gcacgcatgc cgtctatgac acgatcaaca acccgaacgc cgacctgcag 780
gtcgacagca gcggcgtcaa aacggacctg gtcgccgtca cgctgggcga aaacccggac 840
gtcagccata cgctgagcat ccagacggaa gactatcagg ccggccaggt catcccgcgc 900
aaagtcctgg acagcagcca gtattattat agcggcgacg acctgggcaa cacgtatacg 960
aaaaacgcca cgacgtttaa agtctgggcc ccgacgagca cgcaggtcaa cgtcctgctg 1020
tataacagcg ccacgggcgc cgtcacgaaa acggtcccga tgacggccag cggccatggc 1080
gtctgggaag ccacggtcaa ccaggacctg gaaaactggt attatatgta tgaagtcacg 1140
ggccagggca gcacgcgcac ggccgtcgac ccgtatgcca cggccatcgc cccgaacggc 1200
acgcgcggca tgatcgtcga cctggccaaa acggacccgg ccggctggga aagcgacaaa 1260
catatcacgc cgaaaaacat cgaagacgaa gtcatctatg aaatggacgt ccgcgacttt 1320
agcatcgaca gcaacagcgg catgaaaaac aaaggcaaat atctggccct gacggaaaaa 1380
ggcacgaaag gcccggacgg cgtcaaaacg ggcgtcgaca gcctgaaaca gctgggcatc 1440
acgcatgtcc agctgcagcc ggtctttgcc tttgccagcg tcaacgaaaa cgacccgacg 1500
cagtataact ggggctatga cccgcgcaac tataacgtcc cggaaggcca gtatgccacg 1560
aacgccaacg gcacgacgcg catcaaagaa tttaaagaaa tggtcctgag cctgcatcag 1620
gaccatatcg gcgtcaacat ggacgtcgtc tataaccata cgtttgccac gcagatcagc 1680
gactttgaca aaatcgtccc ggaatattat tatcgcacgg acgacgccgg caactatacg 1740
aacggcagcg gcacgggcaa cgaaatcgcc gccgaacgcc cgatggtcca gaaatttatc 1800
atcgacagcc tgaaattttg ggtcaacgaa tatcatgtcg acggctttcg ctttgacctg 1860
atggccctgc tgggcaaaga cacgatgagc aaagccgcca cgcagctgca tgccatcgac 1920
ccgggcatcg ccctgtatgg cgaaccgtgg acgggcggca cgagcgccct gccggccgac 1980
cagctgctga cgaaaggcgc ccagaaaggc atgggcgtcg ccgtctttaa cgacaacctg 2040
cgcaacggcc tggacggcag cgtctttgac agcagcgccc agggctttgc cacgggcgcc 2100
acgggcctga cggacgccat caaacgcggc gtcgaaggca gcatcaacga ctttacggcc 2160
agcccgggcg aaacgatcaa ctatgtcacg agccatgaca actatacgct gtgggacaaa 2220
atcgcccaga gcaacccgaa cgacagcgaa gccgaccgca tcaaaatgga cgaactggcc 2280
caggccatcg tcatgacgag ccagggcatc ccgtttatgc agggcggcga agaaatgctg 2340
cgcacgaaag gcggcaacga caacagctat aacgccggcg acgtcgtcaa cgaatttgac 2400
tggagccgca aagcccagta tccggacgtc tttaactatt atagcggcct gatccatctg 2460
cgcctggacc atccggcctt tcgcatgacg acggccaacg aaatcaacag ccatctgcag 2520
tttctgaaca gcccggaaaa cacggtcgcc tatgaactga gcgaccatgc caacaaagac 2580
acgtggggca acatcgtcgt catctataac ccgaacaaaa cggccgaaac gatcaacctg 2640
ccgagcggca aatgggaaat caacgccacg agcggcaaag tcggcgaaag cacgctgggc 2700
caggccgaag gcagcgtcca ggtcccgggc atcagcatga tgatcctgca tcaggaagtc 2760
agcccgagcg acggcaaata a 2781
<210> 4
<211> 926
<212> PRT
<213> Artificial Synthesis
<400> 4
Asp Gly Asn Thr Thr Asn Ile Val Val His Tyr Phe Arg Pro Ser Gly
1 5 10 15
Asp Tyr Thr Asp Trp Asn Leu Trp Met Trp Pro Glu Asn Gly Asp Gly
20 25 30
Ala Glu Tyr Asp Phe Asn Gln Pro Thr Asp Ser Tyr Gly Glu Val Ala
35 40 45
Ser Val Asp Ile Pro Gly Asn Pro Ser Gln Val Gly Ile Ile Val Arg
50 55 60
Lys Gly Asn Trp Asp Ala Lys Asp Ile Asp Ser Asp Arg Tyr Ile Asp
65 70 75 80
Leu Ser Lys Gly His Glu Ile Trp Leu Val Gln Gly Asn Ser Gln Ile
85 90 95
Phe Tyr Ser Glu Lys Asp Ala Glu Ala Ala Ala Gln Pro Ala Val Ser
100 105 110
Asn Ala Tyr Leu Asp Ala Ser Asn Gln Val Leu Val Lys Leu Ser Gln
115 120 125
Pro Phe Thr Leu Gly Glu Gly Ser Ser Gly Phe Thr Val His Asp Asp
130 135 140
Thr Ala Asn Lys Asp Ile Pro Val Thr Ser Val Ser Asp Ala Asn Gln
145 150 155 160
Val Thr Ala Val Leu Ala Gly Thr Phe Gln His Ile Phe Gly Gly Ser
165 170 175
Asp Trp Ala Pro Asp Asn His Asn Thr Leu Leu Lys Lys Val Asn Ser
180 185 190
Asn Leu Tyr Gln Phe Ser Gly Asn Leu Pro Glu Gly Asn Tyr Gln Tyr
195 200 205
Lys Val Ala Leu Asn Asp Ser Trp Asn Asn Pro Ser Tyr Pro Ser Asp
210 215 220
Asn Ile Asn Leu Thr Val Pro Ala Gly Gly Ala His Val Thr Phe Ser
225 230 235 240
Tyr Ile Pro Ser Thr His Ala Val Tyr Asp Thr Ile Asn Asn Pro Asn
245 250 255
Ala Asp Leu Gln Val Asp Ser Ser Gly Val Lys Thr Asp Leu Val Ala
260 265 270
Val Thr Leu Gly Glu Asn Pro Asp Val Ser His Thr Leu Ser Ile Gln
275 280 285
Thr Glu Asp Tyr Gln Ala Gly Gln Val Ile Pro Arg Lys Val Leu Asp
290 295 300
Ser Ser Gln Tyr Tyr Tyr Ser Gly Asp Asp Leu Gly Asn Thr Tyr Thr
305 310 315 320
Lys Asn Ala Thr Thr Phe Lys Val Trp Ala Pro Thr Ser Thr Gln Val
325 330 335
Asn Val Leu Leu Tyr Asn Ser Ala Thr Gly Ala Val Thr Lys Thr Val
340 345 350
Pro Met Thr Ala Ser Gly His Gly Val Trp Glu Ala Thr Val Asn Gln
355 360 365
Asp Leu Glu Asn Trp Tyr Tyr Met Tyr Glu Val Thr Gly Gln Gly Ser
370 375 380
Thr Arg Thr Ala Val Asp Pro Tyr Ala Thr Ala Ile Ala Pro Asn Gly
385 390 395 400
Thr Arg Gly Met Ile Val Asp Leu Ala Lys Thr Asp Pro Ala Gly Trp
405 410 415
Glu Ser Asp Lys His Ile Thr Pro Lys Asn Ile Glu Asp Glu Val Ile
420 425 430
Tyr Glu Met Asp Val Arg Asp Phe Ser Ile Asp Ser Asn Ser Gly Met
435 440 445
Lys Asn Lys Gly Lys Tyr Leu Ala Leu Thr Glu Lys Gly Thr Lys Gly
450 455 460
Pro Asp Gly Val Lys Thr Gly Val Asp Ser Leu Lys Gln Leu Gly Ile
465 470 475 480
Thr His Val Gln Leu Gln Pro Val Phe Ala Phe Ala Ser Val Asn Glu
485 490 495
Asn Asp Pro Thr Gln Tyr Asn Trp Gly Tyr Asp Pro Arg Asn Tyr Asn
500 505 510
Val Pro Glu Gly Gln Tyr Ala Thr Asn Ala Asn Gly Thr Thr Arg Ile
515 520 525
Lys Glu Phe Lys Glu Met Val Leu Ser Leu His Gln Asp His Ile Gly
530 535 540
Val Asn Met Asp Val Val Tyr Asn His Thr Phe Ala Thr Gln Ile Ser
545 550 555 560
Asp Phe Asp Lys Ile Val Pro Glu Tyr Tyr Tyr Arg Thr Asp Asp Ala
565 570 575
Gly Asn Tyr Thr Asn Gly Ser Gly Thr Gly Asn Glu Ile Ala Ala Glu
580 585 590
Arg Pro Met Val Gln Lys Phe Ile Ile Asp Ser Leu Lys Phe Trp Val
595 600 605
Asn Glu Tyr His Val Asp Gly Phe Arg Phe Asp Leu Met Ala Leu Leu
610 615 620
Gly Lys Asp Thr Met Ser Lys Ala Ala Thr Gln Leu His Ala Ile Asp
625 630 635 640
Pro Gly Ile Ala Leu Tyr Gly Glu Pro Trp Thr Gly Gly Thr Ser Ala
645 650 655
Leu Pro Ala Asp Gln Leu Leu Thr Lys Gly Ala Gln Lys Gly Met Gly
660 665 670
Val Ala Val Phe Asn Asp Asn Leu Arg Asn Gly Leu Asp Gly Ser Val
675 680 685
Phe Asp Ser Ser Ala Gln Gly Phe Ala Thr Gly Ala Thr Gly Leu Thr
690 695 700
Asp Ala Ile Lys Arg Gly Val Glu Gly Ser Ile Asn Asp Phe Thr Ala
705 710 715 720
Ser Pro Gly Glu Thr Ile Asn Tyr Val Thr Ser His Asp Asn Tyr Thr
725 730 735
Leu Trp Asp Lys Ile Ala Gln Ser Asn Pro Asn Asp Ser Glu Ala Asp
740 745 750
Arg Ile Lys Met Asp Glu Leu Ala Gln Ala Ile Val Met Thr Ser Gln
755 760 765
Gly Ile Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg Thr Lys Gly
770 775 780
Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Val Val Asn Glu Phe Asp
785 790 795 800
Trp Ser Arg Lys Ala Gln Tyr Pro Asp Val Phe Asn Tyr Tyr Ser Gly
805 810 815
Leu Ile His Leu Arg Leu Asp His Pro Ala Phe Arg Met Thr Thr Ala
820 825 830
Asn Glu Ile Asn Ser His Leu Gln Phe Leu Asn Ser Pro Glu Asn Thr
835 840 845
Val Ala Tyr Glu Leu Ser Asp His Ala Asn Lys Asp Thr Trp Gly Asn
850 855 860
Ile Val Val Ile Tyr Asn Pro Asn Lys Thr Ala Glu Thr Ile Asn Leu
865 870 875 880
Pro Ser Gly Lys Trp Glu Ile Asn Ala Thr Ser Gly Lys Val Gly Glu
885 890 895
Ser Thr Leu Gly Gln Ala Glu Gly Ser Val Gln Val Pro Gly Ile Ser
900 905 910
Met Met Ile Leu His Gln Glu Val Ser Pro Ser Asp Gly Lys
915 920 925
<210> 5
<211> 2781
<212> DNA
<213> Artificial Synthesis
<400> 5
gacggcaaca cgacgaacat cgtcgtccat tattttcgcc cgagcggcga ctatacggac 60
tggaacctgt ggatgtggcc ggaaaacggc gacggcgccg aatatgactt taaccagccg 120
acggacagct atggcgaagt cgccagcgtc gacatcccgg gcaacccgag ccaggtcggc 180
atcatcgtcc gcaaaggcaa ctgggacgcc aaagacatcg acagcgaccg ctatatcgac 240
ctgagcaaag gccatgaaat ctggctggtc cagggcaaca gccagatctt ttatagcgaa 300
aaagacgccg aagccgccgc ccagccggcc gtcagcaacg cctatctgga cgccagcaac 360
caggtcctgg tcaaactgag ccagccgttt acgctgggcg aaggcagcag cggctttacg 420
gtccatgacg acacggccaa caaagacatc ccggtcacga gcgtcagcga cgccaaccag 480
gtcacggccg tcctggccgg cacgtttcag catatctttg gcggcagcga ctgggccccg 540
gacaaccata acacgctgct gaaaaaagtc aacagcaacc tgtatcagtt tagcggcaac 600
ctgccggaag gcaactatca gtataaagtc gccctgaacg acagctggaa caacccgagc 660
tatccgagcg acaacatcaa cctgacggtc ccggccggcg gcgcccatgt cacgtttagc 720
tatatcccga gcacgcatgc cgtctatgac acgatcaaca acccgaacgc cgacctgcag 780
gtcgacagca gcggcgtcaa aacggacctg gtcgccgtca cgctgggcga aaacccggac 840
gtcagccata cgctgagcat ccagacggaa gactatcagg ccggccaggt catcccgcgc 900
aaagtcctgg acagcagcca gtattattat agcggcgacg acctgggcaa cacgtatacg 960
aaaaacgcca cgacgtttaa agtctgggcc ccgacgagca cgcaggtcaa cgtcctgctg 1020
tataacagcg ccacgggcgc cgtcacgaaa acggtcccga tgacggccag cggccatggc 1080
gtctgggaag ccacggtcaa ccaggacctg gaaaactggt attatatgta tgaagtcacg 1140
ggccagggca gcacgcgcac ggccgtcgac ccgtatgcca cggccatcgc cccgaacggc 1200
acgcgcggca tgatcgtcga cctggccaaa acggacccgg ccggctggga aagcgacaaa 1260
catatcacgc cgaaaaacat cgaagacgaa gtcatctatg aaatggacgt ccgcgacttt 1320
agcatcgaca gcaacagcgg catgaaaaac aaaggcaaat atctggccct gacggaaaaa 1380
ggcacgaaag gcccggacgg cgtcaaaacg ggcgtcgaca gcctgaaaca gctgggcatc 1440
acgcatgtcc agctgcagcc ggtctttgcc tttgccagcg tcaacgaaaa cgacccgacg 1500
cagtataact ggggctatga cccgcgcaac tataacgtcc cggaaggcca gtatgccacg 1560
aacgccaacg gcacgacgcg catcaaagaa tttaaagaaa tggtcctgag cctgcatcag 1620
gaccatatcg gcgtcaacat ggacgtcgtc tataaccata cgtttgccac gcagatcagc 1680
gactttgaca aaatcgtccc ggaatattat tatcgcacgg acgacgccgg caactatacg 1740
aacggcagcg gcacgggcaa cgaaatcgcc agcgaacgcc cgatggtcca gaaatttatc 1800
atcgacagcc tgaaattttg ggtcaacgaa tatcatgtcg acggctttcg ctttgacctg 1860
atggccctgc tgggcaaaga cacgatgagc aaagccgcca cgcagctgca tgccatcgac 1920
ccgggcatcg ccctgtatgg cgaaccgtgg acgggcggca cgagcgccct gccggccgac 1980
cagctgctga cgaaaggcgc ccagaaaggc atgggcgtcg ccgtctttaa cgacaacctg 2040
cgcaacggcc tggacggcag cgtctttgac agcagcgccc agggctttgc cacgggcgcc 2100
acgggcctga cggacgccat caaacgcggc gtcgaaggca gcatcaacga ctttacggcc 2160
agcccgagcg aaacgatcaa ctatgtcacg agccatgaca actatacgct gtgggacaaa 2220
atcgcccaga gcaacccgaa cgacagcgaa gccgaccgca tcaaaatgga cgaactggcc 2280
caggccatcg tcatgacgag ccagggcatc ccgtttatgc agggcggcga agaaatgctg 2340
cgcacgaaag gcggcaacga caacagctat aacgccggcg acgtcgtcaa cgaatttgac 2400
tggagccgca aagcccagta tccggacgtc tttaactatt atagcggcct gatccatctg 2460
cgcctggacc atccggcctt tcgcatgacg acggccaacg aaatcaacag ccatctgcag 2520
tttctgaaca gcccggaaaa cacggtcgcc tatgaactga gcgaccatgc caacaaagac 2580
acgtggggca acatcgtcgt catctataac ccgaacaaaa cggccgaaac gatcaacctg 2640
ccgagcggca aatgggaaat caacgccacg agcggcaaag tcggcgaaag cacgctgggc 2700
caggccgaag gcagcgtcca ggtcccgggc atcagcatga tgatcctgca tcaggaagtc 2760
agcccgagcg acggcaaata a 2781
<210> 6
<211> 926
<212> PRT
<213> Artificial Synthesis
<400> 6
Asp Gly Asn Thr Thr Asn Ile Val Val His Tyr Phe Arg Pro Ser Gly
1 5 10 15
Asp Tyr Thr Asp Trp Asn Leu Trp Met Trp Pro Glu Asn Gly Asp Gly
20 25 30
Ala Glu Tyr Asp Phe Asn Gln Pro Thr Asp Ser Tyr Gly Glu Val Ala
35 40 45
Ser Val Asp Ile Pro Gly Asn Pro Ser Gln Val Gly Ile Ile Val Arg
50 55 60
Lys Gly Asn Trp Asp Ala Lys Asp Ile Asp Ser Asp Arg Tyr Ile Asp
65 70 75 80
Leu Ser Lys Gly His Glu Ile Trp Leu Val Gln Gly Asn Ser Gln Ile
85 90 95
Phe Tyr Ser Glu Lys Asp Ala Glu Ala Ala Ala Gln Pro Ala Val Ser
100 105 110
Asn Ala Tyr Leu Asp Ala Ser Asn Gln Val Leu Val Lys Leu Ser Gln
115 120 125
Pro Phe Thr Leu Gly Glu Gly Ser Ser Gly Phe Thr Val His Asp Asp
130 135 140
Thr Ala Asn Lys Asp Ile Pro Val Thr Ser Val Ser Asp Ala Asn Gln
145 150 155 160
Val Thr Ala Val Leu Ala Gly Thr Phe Gln His Ile Phe Gly Gly Ser
165 170 175
Asp Trp Ala Pro Asp Asn His Asn Thr Leu Leu Lys Lys Val Asn Ser
180 185 190
Asn Leu Tyr Gln Phe Ser Gly Asn Leu Pro Glu Gly Asn Tyr Gln Tyr
195 200 205
Lys Val Ala Leu Asn Asp Ser Trp Asn Asn Pro Ser Tyr Pro Ser Asp
210 215 220
Asn Ile Asn Leu Thr Val Pro Ala Gly Gly Ala His Val Thr Phe Ser
225 230 235 240
Tyr Ile Pro Ser Thr His Ala Val Tyr Asp Thr Ile Asn Asn Pro Asn
245 250 255
Ala Asp Leu Gln Val Asp Ser Ser Gly Val Lys Thr Asp Leu Val Ala
260 265 270
Val Thr Leu Gly Glu Asn Pro Asp Val Ser His Thr Leu Ser Ile Gln
275 280 285
Thr Glu Asp Tyr Gln Ala Gly Gln Val Ile Pro Arg Lys Val Leu Asp
290 295 300
Ser Ser Gln Tyr Tyr Tyr Ser Gly Asp Asp Leu Gly Asn Thr Tyr Thr
305 310 315 320
Lys Asn Ala Thr Thr Phe Lys Val Trp Ala Pro Thr Ser Thr Gln Val
325 330 335
Asn Val Leu Leu Tyr Asn Ser Ala Thr Gly Ala Val Thr Lys Thr Val
340 345 350
Pro Met Thr Ala Ser Gly His Gly Val Trp Glu Ala Thr Val Asn Gln
355 360 365
Asp Leu Glu Asn Trp Tyr Tyr Met Tyr Glu Val Thr Gly Gln Gly Ser
370 375 380
Thr Arg Thr Ala Val Asp Pro Tyr Ala Thr Ala Ile Ala Pro Asn Gly
385 390 395 400
Thr Arg Gly Met Ile Val Asp Leu Ala Lys Thr Asp Pro Ala Gly Trp
405 410 415
Glu Ser Asp Lys His Ile Thr Pro Lys Asn Ile Glu Asp Glu Val Ile
420 425 430
Tyr Glu Met Asp Val Arg Asp Phe Ser Ile Asp Ser Asn Ser Gly Met
435 440 445
Lys Asn Lys Gly Lys Tyr Leu Ala Leu Thr Glu Lys Gly Thr Lys Gly
450 455 460
Pro Asp Gly Val Lys Thr Gly Val Asp Ser Leu Lys Gln Leu Gly Ile
465 470 475 480
Thr His Val Gln Leu Gln Pro Val Phe Ala Phe Ala Ser Val Asn Glu
485 490 495
Asn Asp Pro Thr Gln Tyr Asn Trp Gly Tyr Asp Pro Arg Asn Tyr Asn
500 505 510
Val Pro Glu Gly Gln Tyr Ala Thr Asn Ala Asn Gly Thr Thr Arg Ile
515 520 525
Lys Glu Phe Lys Glu Met Val Leu Ser Leu His Gln Asp His Ile Gly
530 535 540
Val Asn Met Asp Val Val Tyr Asn His Thr Phe Ala Thr Gln Ile Ser
545 550 555 560
Asp Phe Asp Lys Ile Val Pro Glu Tyr Tyr Tyr Arg Thr Asp Asp Ala
565 570 575
Gly Asn Tyr Thr Asn Gly Ser Gly Thr Gly Asn Glu Ile Ala Ser Glu
580 585 590
Arg Pro Met Val Gln Lys Phe Ile Ile Asp Ser Leu Lys Phe Trp Val
595 600 605
Asn Glu Tyr His Val Asp Gly Phe Arg Phe Asp Leu Met Ala Leu Leu
610 615 620
Gly Lys Asp Thr Met Ser Lys Ala Ala Thr Gln Leu His Ala Ile Asp
625 630 635 640
Pro Gly Ile Ala Leu Tyr Gly Glu Pro Trp Thr Gly Gly Thr Ser Ala
645 650 655
Leu Pro Ala Asp Gln Leu Leu Thr Lys Gly Ala Gln Lys Gly Met Gly
660 665 670
Val Ala Val Phe Asn Asp Asn Leu Arg Asn Gly Leu Asp Gly Ser Val
675 680 685
Phe Asp Ser Ser Ala Gln Gly Phe Ala Thr Gly Ala Thr Gly Leu Thr
690 695 700
Asp Ala Ile Lys Arg Gly Val Glu Gly Ser Ile Asn Asp Phe Thr Ala
705 710 715 720
Ser Pro Ser Glu Thr Ile Asn Tyr Val Thr Ser His Asp Asn Tyr Thr
725 730 735
Leu Trp Asp Lys Ile Ala Gln Ser Asn Pro Asn Asp Ser Glu Ala Asp
740 745 750
Arg Ile Lys Met Asp Glu Leu Ala Gln Ala Ile Val Met Thr Ser Gln
755 760 765
Gly Ile Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg Thr Lys Gly
770 775 780
Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Val Val Asn Glu Phe Asp
785 790 795 800
Trp Ser Arg Lys Ala Gln Tyr Pro Asp Val Phe Asn Tyr Tyr Ser Gly
805 810 815
Leu Ile His Leu Arg Leu Asp His Pro Ala Phe Arg Met Thr Thr Ala
820 825 830
Asn Glu Ile Asn Ser His Leu Gln Phe Leu Asn Ser Pro Glu Asn Thr
835 840 845
Val Ala Tyr Glu Leu Ser Asp His Ala Asn Lys Asp Thr Trp Gly Asn
850 855 860
Ile Val Val Ile Tyr Asn Pro Asn Lys Thr Ala Glu Thr Ile Asn Leu
865 870 875 880
Pro Ser Gly Lys Trp Glu Ile Asn Ala Thr Ser Gly Lys Val Gly Glu
885 890 895
Ser Thr Leu Gly Gln Ala Glu Gly Ser Val Gln Val Pro Gly Ile Ser
900 905 910
Met Met Ile Leu His Gln Glu Val Ser Pro Ser Asp Gly Lys
915 920 925
<210> 7
<211> 2781
<212> DNA
<213> Artificial Synthesis
<400> 7
gacggcaaca cgacgaacat cgtcgtccat tattttcgcc cgagcggcga ctatacggac 60
tggaacctgt ggatgtggcc ggaaaacggc gacggcgccg aatatgactt taaccagccg 120
acggacagct atggcgaagt cgccagcgtc gacatcccgg gcaacccgag ccaggtcggc 180
atcatcgtcc gcaaaggcaa ctgggacgcc aaagacatcg acagcgaccg ctatatcgac 240
ctgagcaaag gccatgaaat ctggctggtc cagggcaaca gccagatctt ttatagcgaa 300
aaagacgccg aagccgccgc ccagccggcc gtcagcaacg cctatctgga cgccagcaac 360
caggtcctgg tcaaactgag ccagccgttt acgctgggcg aaggcagcag cggctttacg 420
gtccatgacg acacggccaa caaagacatc ccggtcacga gcgtcagcga cgccaaccag 480
gtcacggccg tcctggccgg cacgtttcag catatctttg gcggcagcga ctgggccccg 540
gacaaccata acacgctgct gaaaaaagtc aacagcaacc tgtatcagtt tagcggcaac 600
ctgccggaag gcaactatca gtataaagtc gccctgaacg acagctggaa caacccgagc 660
tatccgagcg acaacatcaa cctgacggtc ccggccggcg gcgcccatgt cacgtttagc 720
tatatcccga gcacgcatgc cgtctatgac acgatcaaca acccgaacgc cgacctgcag 780
gtcgacagca gcggcgtcaa aacggacctg gtcgccgtca cgctgggcga aaacccggac 840
gtcagccata cgctgagcat ccagacggaa gactatcagg ccggccaggt catcccgcgc 900
aaagtcctgg acagcagcca gtattattat agcggcgacg acctgggcaa cacgtatacg 960
aaaaacgcca cgacgtttaa agtctgggcc ccgacgagca cgcaggtcaa cgtcctgctg 1020
tataacagcg ccacgggcgc cgtcacgaaa acggtcccga tgacggccag cggccatggc 1080
gtctgggaag ccacggtcaa ccaggacctg gaaaactggt attatatgta tgaagtcacg 1140
ggccagggca gcacgcgcac ggccgtcgac ccgtatgcca cggccatcgc cccgaacggc 1200
acgcgcggca tgatcgtcga cctggccaaa acggacccgg ccggctggga aagcgacaaa 1260
catatcacgc cgaaaaacat cgaagacgaa gtcatctatg aaatggacgt ccgcgacttt 1320
agcatcgaca gcaacagcgg catgaaaaac aaaggcaaat atctggccct gacggaaaaa 1380
ggcacgaaag gcccggacgg cgtcaaaacg ggcgtcgaca gcctgaaaca gctgggcatc 1440
acgcatgtcc agctgcagcc ggtctttgcc tttgccagcg tcaacgaaaa cgacccgacg 1500
cagtataact ggggctatga cccgcgcaac tataacgtcc cggaaggcca gtatgccacg 1560
aacgccaacg gcacgacgcg catcaaagaa tttaaagaaa tggtcctgag cctgcatcag 1620
gaccatatcg gcgtcaacat ggacgtcgtc tataaccata cgtttgccac gcagatcagc 1680
gactttgaca aaatcgtccc ggaatattat tatcgcacgg acgacgccgg caactatacg 1740
aacggcagcg gcacgggcaa cgaaatcgcc gccgaacgcc cgatggtcca gaaatttatc 1800
atcgacagcc tgaaattttg ggtcaacgaa tatcatgtcg acggctttcg ctttgacctg 1860
atggccctgc tgggcaaaga cacgatgagc aaagccgcca cgcagctgca tgccatcgac 1920
ccgggcatcg ccctgtatgg cgaaccgtgg acgggcggca cgagcgccct gccggccgac 1980
cagctgctga cgaaaggcgc ccagaaaggc atgggcgtcg ccgtctttaa cgacaacctg 2040
cgcaacggcc tggacggcag cgtctttgac agcagcgccc agggctttgc cacgggcgcc 2100
acgggcctga cggacgccat caaacgcggc gtcgaaggca gcatcaacga ctttacggcc 2160
agcccgagcg aaacgatcaa ctatgtcacg tgccatgaca actatacgct gtgggacaaa 2220
atcgcccaga gcaacccgaa cgacagcgaa gccgaccgca tcaaaatgga cgaactggcc 2280
caggccatcg tcatgacgag ccagggcatc ccgtttatgc agggcggcga agaaatgctg 2340
cgcacgaaag gcggcaacga caacagctat aacgccggcg acgtcgtcaa cgaatttgac 2400
tggagccgca aagcccagta tccggacgtc tttaactatt atagcggcct gatccatctg 2460
cgcctggacc atccggcctt tcgcatgacg acggccaacg aaatcaacag ccatctgcag 2520
tttctgaaca gcccggaaaa cacggtcgcc tatgaactga gcgaccatgc caacaaagac 2580
acgtggggca acatcgtcgt catctataac ccgaacaaaa cggccgaaac gatcaacctg 2640
ccgagcggca aatgggaaat caacgccacg agcggcaaag tcggcgaaag cacgctgggc 2700
caggccgaag gcagcgtcca ggtcccgggc atcagcatga tgatcctgca tcaggaagtc 2760
agcccgagcg acggcaaata a 2781
<210> 8
<211> 926
<212> PRT
<213> Artificial Synthesis
<400> 8
Asp Gly Asn Thr Thr Asn Ile Val Val His Tyr Phe Arg Pro Ser Gly
1 5 10 15
Asp Tyr Thr Asp Trp Asn Leu Trp Met Trp Pro Glu Asn Gly Asp Gly
20 25 30
Ala Glu Tyr Asp Phe Asn Gln Pro Thr Asp Ser Tyr Gly Glu Val Ala
35 40 45
Ser Val Asp Ile Pro Gly Asn Pro Ser Gln Val Gly Ile Ile Val Arg
50 55 60
Lys Gly Asn Trp Asp Ala Lys Asp Ile Asp Ser Asp Arg Tyr Ile Asp
65 70 75 80
Leu Ser Lys Gly His Glu Ile Trp Leu Val Gln Gly Asn Ser Gln Ile
85 90 95
Phe Tyr Ser Glu Lys Asp Ala Glu Ala Ala Ala Gln Pro Ala Val Ser
100 105 110
Asn Ala Tyr Leu Asp Ala Ser Asn Gln Val Leu Val Lys Leu Ser Gln
115 120 125
Pro Phe Thr Leu Gly Glu Gly Ser Ser Gly Phe Thr Val His Asp Asp
130 135 140
Thr Ala Asn Lys Asp Ile Pro Val Thr Ser Val Ser Asp Ala Asn Gln
145 150 155 160
Val Thr Ala Val Leu Ala Gly Thr Phe Gln His Ile Phe Gly Gly Ser
165 170 175
Asp Trp Ala Pro Asp Asn His Asn Thr Leu Leu Lys Lys Val Asn Ser
180 185 190
Asn Leu Tyr Gln Phe Ser Gly Asn Leu Pro Glu Gly Asn Tyr Gln Tyr
195 200 205
Lys Val Ala Leu Asn Asp Ser Trp Asn Asn Pro Ser Tyr Pro Ser Asp
210 215 220
Asn Ile Asn Leu Thr Val Pro Ala Gly Gly Ala His Val Thr Phe Ser
225 230 235 240
Tyr Ile Pro Ser Thr His Ala Val Tyr Asp Thr Ile Asn Asn Pro Asn
245 250 255
Ala Asp Leu Gln Val Asp Ser Ser Gly Val Lys Thr Asp Leu Val Ala
260 265 270
Val Thr Leu Gly Glu Asn Pro Asp Val Ser His Thr Leu Ser Ile Gln
275 280 285
Thr Glu Asp Tyr Gln Ala Gly Gln Val Ile Pro Arg Lys Val Leu Asp
290 295 300
Ser Ser Gln Tyr Tyr Tyr Ser Gly Asp Asp Leu Gly Asn Thr Tyr Thr
305 310 315 320
Lys Asn Ala Thr Thr Phe Lys Val Trp Ala Pro Thr Ser Thr Gln Val
325 330 335
Asn Val Leu Leu Tyr Asn Ser Ala Thr Gly Ala Val Thr Lys Thr Val
340 345 350
Pro Met Thr Ala Ser Gly His Gly Val Trp Glu Ala Thr Val Asn Gln
355 360 365
Asp Leu Glu Asn Trp Tyr Tyr Met Tyr Glu Val Thr Gly Gln Gly Ser
370 375 380
Thr Arg Thr Ala Val Asp Pro Tyr Ala Thr Ala Ile Ala Pro Asn Gly
385 390 395 400
Thr Arg Gly Met Ile Val Asp Leu Ala Lys Thr Asp Pro Ala Gly Trp
405 410 415
Glu Ser Asp Lys His Ile Thr Pro Lys Asn Ile Glu Asp Glu Val Ile
420 425 430
Tyr Glu Met Asp Val Arg Asp Phe Ser Ile Asp Ser Asn Ser Gly Met
435 440 445
Lys Asn Lys Gly Lys Tyr Leu Ala Leu Thr Glu Lys Gly Thr Lys Gly
450 455 460
Pro Asp Gly Val Lys Thr Gly Val Asp Ser Leu Lys Gln Leu Gly Ile
465 470 475 480
Thr His Val Gln Leu Gln Pro Val Phe Ala Phe Ala Ser Val Asn Glu
485 490 495
Asn Asp Pro Thr Gln Tyr Asn Trp Gly Tyr Asp Pro Arg Asn Tyr Asn
500 505 510
Val Pro Glu Gly Gln Tyr Ala Thr Asn Ala Asn Gly Thr Thr Arg Ile
515 520 525
Lys Glu Phe Lys Glu Met Val Leu Ser Leu His Gln Asp His Ile Gly
530 535 540
Val Asn Met Asp Val Val Tyr Asn His Thr Phe Ala Thr Gln Ile Ser
545 550 555 560
Asp Phe Asp Lys Ile Val Pro Glu Tyr Tyr Tyr Arg Thr Asp Asp Ala
565 570 575
Gly Asn Tyr Thr Asn Gly Ser Gly Thr Gly Asn Glu Ile Ala Ala Glu
580 585 590
Arg Pro Met Val Gln Lys Phe Ile Ile Asp Ser Leu Lys Phe Trp Val
595 600 605
Asn Glu Tyr His Val Asp Gly Phe Arg Phe Asp Leu Met Ala Leu Leu
610 615 620
Gly Lys Asp Thr Met Ser Lys Ala Ala Thr Gln Leu His Ala Ile Asp
625 630 635 640
Pro Gly Ile Ala Leu Tyr Gly Glu Pro Trp Thr Gly Gly Thr Ser Ala
645 650 655
Leu Pro Ala Asp Gln Leu Leu Thr Lys Gly Ala Gln Lys Gly Met Gly
660 665 670
Val Ala Val Phe Asn Asp Asn Leu Arg Asn Gly Leu Asp Gly Ser Val
675 680 685
Phe Asp Ser Ser Ala Gln Gly Phe Ala Thr Gly Ala Thr Gly Leu Thr
690 695 700
Asp Ala Ile Lys Arg Gly Val Glu Gly Ser Ile Asn Asp Phe Thr Ala
705 710 715 720
Ser Pro Ser Glu Thr Ile Asn Tyr Val Thr Cys His Asp Asn Tyr Thr
725 730 735
Leu Trp Asp Lys Ile Ala Gln Ser Asn Pro Asn Asp Ser Glu Ala Asp
740 745 750
Arg Ile Lys Met Asp Glu Leu Ala Gln Ala Ile Val Met Thr Ser Gln
755 760 765
Gly Ile Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg Thr Lys Gly
770 775 780
Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Val Val Asn Glu Phe Asp
785 790 795 800
Trp Ser Arg Lys Ala Gln Tyr Pro Asp Val Phe Asn Tyr Tyr Ser Gly
805 810 815
Leu Ile His Leu Arg Leu Asp His Pro Ala Phe Arg Met Thr Thr Ala
820 825 830
Asn Glu Ile Asn Ser His Leu Gln Phe Leu Asn Ser Pro Glu Asn Thr
835 840 845
Val Ala Tyr Glu Leu Ser Asp His Ala Asn Lys Asp Thr Trp Gly Asn
850 855 860
Ile Val Val Ile Tyr Asn Pro Asn Lys Thr Ala Glu Thr Ile Asn Leu
865 870 875 880
Pro Ser Gly Lys Trp Glu Ile Asn Ala Thr Ser Gly Lys Val Gly Glu
885 890 895
Ser Thr Leu Gly Gln Ala Glu Gly Ser Val Gln Val Pro Gly Ile Ser
900 905 910
Met Met Ile Leu His Gln Glu Val Ser Pro Ser Asp Gly Lys
915 920 925
<210> 9
<211> 28
<212> DNA
<213> Artificial sequence
<400> 9
ataggatccg acggcaacac gacgaaca 28
<210> 10
<211> 23
<212> DNA
<213> Artificial sequence
<400> 10
gacttcctga tgcaggatca ttc 23

Claims (11)

1. A pullulanase mutant, which is obtained by carrying out any one of the following mutations on the basis of the amino acid sequence of a wild type pullulanase shown in SEQ ID No. 2:
n467+ N709, N467+ N492+ N709, N492+ S630+ N709+ N734, N492+ N734, N467+ N492+ S731, N467+ N492+ N709+ T730, N467+ N492+ N709+ a591+ G723 or N467+ N492+ N709+ a591+ T730;
n467+ N492, N467+ N492+ G723, N467+ N492+ N709+ S731, N467+ N492+ N709+ G723+ T730, E100+ a317+ N450+ N523+ T730, E100+ K460, E100+ a317+ N450+ K463+ N523+ N864, or E100+ a317+ N450+ N523+ G758+ N846;
n467+ A591+ N709, N467+ T730, N467+ A591, N467+ T731, N492+ S630+ N734, N467+ A591+ G723, N467+ A591+ T730, E100+ K460+ N734+ N870, or N467G + N492+ A591;
n467+ G723, N467+ N492+ A591, N467+ N492+ T730, N467+ A591+ S731, N467+ N492+ N709+ G723, N467+ N492+ N709+ A591+ T730+ S731, A317+ N467+ G476+ N709, A317+ N467+ S630+ S731+ G900, or E100+ K460+ A591+ N734+ G758.
2. The pullulanase mutant according to claim 1, wherein the mutant is obtained by carrying out any one of the following mutations based on the amino acid sequence of the wild-type pullulanase represented by SEQ ID No. 2:
N467G + N709R, N467G + N492A + N709R, N467G + N492A + S731C, N467G + N492A + N709R + T730S, N467G + N492A + N709R + A591S + G723S or N467G + N492A + N709R + A591S + T730S.
3. The pullulanase mutant according to claim 1, wherein the mutant is obtained by carrying out any one of the following mutations based on the amino acid sequence of the wild-type pullulanase represented by SEQ ID No. 2:
N467G + N492A, N467G + N492A + G723S, N467G + N492A + N709R + S731C, N467G + N492A + N709R + G723S + S731C or N467G + N492A + N709R + G723S + T730S.
4. The pullulanase mutant according to claim 1, wherein the mutant is obtained by carrying out any one of the following mutations based on the amino acid sequence of the wild-type pullulanase represented by SEQ ID No. 2:
N467G + A591S + N709R, N467G + T730S, N467G + A591S, N467G + A591S + G723S, N467G + A591S + T730S, N467G + N492A + A591S or N467G + S731C.
5. The pullulanase mutant according to claim 1, wherein the mutant is obtained by carrying out any one of the following mutations based on the amino acid sequence of the wild-type pullulanase represented by SEQ ID No. 2:
n467+ N492, N467+ A591, N467+ N709, N467+ G723, N467+ T730, N467+ N492+ A591, N467+ N492+ N709, N467+ N492+ T730, N467+ N492+ S731, N467+ A591+ N709, N467+ A591+ G723, N467+ A591+ T730, N467+ A591+ S731, N467+ N492+ N709+ A591, N467+ N492+ G723, N467+ N492+ N709+ T730, N467+ N492+ S731, N467+ N591 + A591+ N709+ G723, N467+ N492+ G492 + K591 + G723, N467+ N709+ N591 + N492+ S730, N467+ N492+ N591 + S730, N467+ N591 + S492 + K591 + N730, N467+ N591 + S730, N467+ S730 or N467+ S492 + K591 + S730.
6. A gene encoding the mutant of any one of claims 2 to 5.
7. A recombinant vector or a recombinant strain comprising the coding gene of claim 6.
8. The recombinant vector or recombinant strain of claim 7, wherein the recombinant vector uses expression vectors including, but not limited to, pHY-WZX, pHY300plk, pUB110, pE194, pHT1469 plasmids; the host cells adopted by the recombinant strain include but are not limited to escherichia coli, bacillus subtilis, bacillus licheniformis, bacillus pumilus, bacillus megaterium, saccharomyces cerevisiae, pichia pastoris, aspergillus niger, aspergillus oryzae or trichoderma reesei.
9. Use of a mutant according to any one of claims 1 to 5 in starch hydrolysis.
10. Use of the recombinant vector or the recombinant strain according to claim 7 for producing pullulanase.
11. The use according to claim 10, wherein the pullulanase mutant is produced by fermentation by:
inoculating the strain into a fermentation tank according to the inoculation amount of 5% -10%; in the fermentation process, the fermentation temperature is 33-45 ℃, the dissolved oxygen is controlled to be 0.1-20%, the pH value is 6.0-7.8, 30-60% of maltose syrup is fed-batch, and the content of reducing sugar is maintained to be 0.1-5%; the fermentation lasts for 90-120h, the sampling analysis is carried out at regular time in the fermentation process, and the fermentation end point is controlled to be that the enzyme activity increase value is less than 5-25U/h within 4 hours of fermentation.
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CN109321552A (en) * 2018-10-11 2019-02-12 山东隆科特酶制剂有限公司 A kind of novel Pullulanase and its gene, engineering bacteria and preparation method
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WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

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