CN110777134B - A mutant chitinase and its application - Google Patents

Abstract

The invention discloses a mutant gene SsChi18A_1 of chitinase SsChi18A, the mutation site of the gene is K186A, and its nucleotide sequence is shown in SEQ ID No.1. The invention also discloses the application of the gene in the preparation of chitinase. Experiments have shown that the activity of the mutant chitinase encoded by this gene can reach 84.2U/μmol, which is 55% higher than that of the wild-type enzyme; and like the wild-type, it can withstand high temperature of 70 °C and can withstand high temperature at pH 4-11 The mutant chitinase has the characteristics of high activity and high temperature resistance, and can be widely used in many fields such as oligosaccharide production, protoplast preparation, biological insecticides, antifungal drugs and biological pesticide production and processing. .

Description

Mutant chitinase and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to mutated chitinase and application thereof.

Background

Chitin, a poly-beta-1, 4-N-acetylglucosamine (NAG), is the second most abundant polysaccharide in nature, excluding cellulose. Chitin is mainly derived from crustacean, and is produced in water body every year up to 10¹¹Ton. The process of demineralizing and deproteinizing chitin in biomass to obtain pure chitin is usually performed with concentrated acid or alkali, but causes corrosion and environmental problems. Enzymatic degradation of chitin can produce a variety of pharmacologically active ingredients, such as N-acetylglucosamine (NAG) and chitin oligosaccharide (CHOS), and can be used as drug delivery vehicles and antioxidants, and has effects in resisting tumor, healing wound, controlling blood cholesterol, and preserving food. The chitinase has wide sources, the enzymolysis process is environment-friendly, and the application potential in industrial production is huge. At present, chitinase preparations are widely used in medicineMedicine, food, biotechnology, plant pest control and other fields.

Chitinase Chi18A (SsChi18A) from Streptomyces sp.F-3 is a good industrial enzyme. Streptomyces sp.f-3 is able to withstand high temperatures of 50 ℃ and has a relatively high chitin-degrading activity, being able to degrade chitin in the fungal cell wall.

The optimal temperature of the SsChi18A for the enzyme reaction is 70 ℃, and the protein is still stable at the temperature; the optimum pH value is 5, the enzyme still has more than 85% of activity at pH 4-6, and the enzyme is relatively stable in a buffer system with pH 4-11. In general, chitinase SsChi18A is not only thermostable, but also exhibits a wider tolerance to pH changes than fungal chitinases, indicating their potential use in industrial applications. SsChi18A enzyme activities were 54.33 and 4.81U/. mu.mol, respectively, when colloidal chitin and chitin powder were hydrolyzed by SsChi 18A; as a persistent chitinase, its hydrolysis products are mainly chitin disaccharides. The continuous chitinase plays an important role in the degradation process of natural crystalline chitin, can continuously act on molecular chains of the chitin, and reduces energy loss caused by random collision, so that the continuous chitinase has obvious advantages in the degradation process of a substrate.

In conclusion, SsChi18A is an ideal industrial enzyme, and can be used for production of chitin oligosaccharide, antifungal drugs and the like. However, in the practical application of SsChi18A, there are still problems to be solved; shortening the process flow and improving the production benefit urgently await the appearance of chitinase with higher activity. In order to expand the industrial application of SsChi18A, improving the enzymatic activity is a critical task, however, reports on SChi18A for enhancing the enzymatic activity and related mutant genes are not available at present.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a mutant chitinase and application thereof.

In one aspect, the invention provides a mutant chitinase, the amino acid sequence of which is shown in SEQ ID No. 2.

In another aspect, the present invention also provides a gene encoding the mutated chitinase; preferably, the sequence of the gene is shown as SEQ ID No. 1.

In another aspect, the present invention also provides a recombinant vector comprising a gene encoding the mutated chitinase; preferably, the recombinant vector is a recombinant expression vector; preferred are vectors of the pET series, for example, pET-15b, pET-22b, pET-28 a.

In another aspect, the present invention also provides a recombinant strain comprising the above recombinant vector, preferably, the recombinant strain is escherichia coli, e.g., escherichia coli BL 21.

In another aspect, the invention also provides the use of the mutated chitinase, the coding gene, the recombinant vector or the recombinant strain for degrading chitin-containing materials. It is understood that chitin-containing materials include materials that naturally contain chitin, or processed chitin-containing materials; in addition, chitin monomers are also a concept of chitin-containing materials.

On the other hand, the invention also provides application of the mutant chitinase, the coding gene, the recombinant vector or the recombinant strain in preparation of chitin oligosaccharide.

In another aspect, the present invention provides a method for degrading chitin-or chitin oligosaccharide-containing material, the method comprising the step of treating chitin-or chitin oligosaccharide-containing material with the mutant chitinase, the encoding gene, the recombinant vector or the recombinant strain. It is understood that chitin-containing materials include materials that naturally contain chitin, or processed chitin-containing materials; in addition, chitin monomers are also a concept of chitin-containing materials. The chitin oligosaccharide-containing material comprises a natural chitin oligosaccharide-containing material or a processed chitin oligosaccharide-containing material; in addition, simple chitin oligosaccharides also belong to the concept of chitin oligosaccharide-containing materials.

Preferably, the oligosaccharide is a disaccharide, trisaccharide, tetrasaccharide, pentasaccharide or hexasaccharide.

Further, the temperature of the treatment is 30-80 ℃, preferably, 50-70 ℃; the pH of the treatment is 3 to 11, preferably, 4 to 7.

The invention has the beneficial effects that:

the mutant chitinase SsChi18A-K186A coded by the mutant gene SChi18A _1 has high enzyme activity, the enzyme activity is 84.2U/mu mol at 60 ℃, and is improved by 55 percent compared with the wild enzyme SChi 18A; as with the wild type, can tolerate high temperatures of 70 ℃ and is stable in a buffer system at a pH of 4-11. The mutant chitinase has larger application space in industrial and agricultural production in the actual industrial production process. The high-temperature hydrolysis reaction kettle has high hydrolysis activity and good thermal stability under the high-temperature condition, so that the biochemical reaction rate is promoted, the microbial pollution is reduced, the cost is reduced, and the benefit is improved. When the enzyme is used as industrial enzyme for medicines, foods, biotechnology, plant pest control and the like, the enzyme can keep higher activity in wider environment, and specific applications include but are not limited to:

in the process of medicine production, the mutant chitinase can be used as a microbial inhibitor to prepare an ophthalmic preparation, and can also be used as an additive of an antifungal medicament to be applied to the treatment of fungal diseases. In the food industry, the mutant chitinase can be used for producing chitin oligosaccharide, and can be used as an antimicrobial agent and an immunopotentiator to activate a host defense system; it can also be used as therapeutic agent for osteoarthritis and rheumatic arthritis and food additive for regulating human health. In the biotechnology industry, this mutant chitinase may be used in conjunction with other cell wall degrading enzymes in the preparation of fungal protoplasts. In the process of preventing and controlling plant diseases, the mutant chitinase can be used as a biocontrol agent to prevent plant pathogens and pests, and can quickly hydrolyze the cell walls of plant disease fungi, so that the cells are cracked, and the pathogenic fungi are killed.

Drawings

FIG. 1 is a graph of the results of SDS-PAGE of the expression of the SsChi18A-K186A Protein of the invention, where M is an Unstained Protein Marker (Thermo Scientific, MA, USA) and the others are mutant chitinase SChi 18A-K186A. Lane 1 is a crude enzyme solution containing the target protein in E.coli cells during heterologous expression; lane 2 is a crude enzyme solution obtained by affinity purification of a target protein containing 6 × His tag using a Ni Sepharose 6Fast Flow affinity column; lanes 5-1 and 5-2 represent the proteins obtained in the first and second elution with 5mM imidazole, respectively; the remaining 20-1 to 250-2 represent the proteins obtained in the first, second and third or fourth elution with 20mM, 40mM, 60mM, 100mM and 250mM imidazole, respectively.

FIG. 2 is a graph of the relative enzyme activities of SsChi18A-K186A and SChi18A of the present invention under optimal conditions.

FIG. 3 is a graphical representation of the results of the present invention SsChi18A-K186A and SsChi18A degrading different chitin oligosaccharides. WT is represented by the wild-type SsChi18A, and K186A is represented by the mutant protein SsChi 18A-K186A. (GlcNAc)₃、(GlcNAc)₄、(GlcNAc)₅、(GlcNAc)₆Respectively represent chitin trisaccharide, chitin tetrasaccharide, chitin pentasaccharide and chitin hexasaccharide. Wherein M represents Marker and is composed of chitin oligosaccharide with different polymerization degrees. The numbers 0, 2, 5, 10, 20 and 30 represent the spectrum of products formed by enzymatic degradation of the substrate for 0min, 2min, 5min, 10min, 20min and 30min, respectively.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific embodiments, which are illustrative of preferred embodiments of the invention and not limiting thereof, and which are capable of modifications in various equivalent embodiments, which will become apparent to those skilled in the art upon reading the foregoing disclosure. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 mutation of chitinase SsChi18A and construction of recombinant vectors

(1) Obtaining chitinase SsChi18A gene from NCBI database, connecting SChi18A gene with plasmid pET28a to obtain pET28 a-SChi 18A plasmid, wherein the nucleotide sequence of pET28 a-SChi 18A plasmid is shown as SEQ ID No. 3. Designing mutation primers by taking the recombinant plasmids as templates, and carrying out site-directed mutation, wherein the primer sequences are as follows:

K186A-sense：TCGGACGGAGGCGCACTCGACGCGG；

K186A-antisense：GAGTGCGCCTCCGTCCGAGGCGTCC；

the PCR amplification reaction system is as follows:

the reaction procedure was as follows:

preheating at 94 deg.C for 5 min; denaturation, at 94 ℃ for 30 s; annealing at 55-65 ℃ for 30 s; 30 cycles; extension, at 72 ℃ for 2 min; re-extension at 72 deg.C for 10 min; 4 ℃ and forever.

(2) 3 μ L of the PCR product was verified by agarose gel electrophoresis, leaving a reaction product with a clear band. The wild-type plasmid in the product was digested with DpnI restriction enzyme to ensure that all transformants were mutated in the next step. The digestion system is as shown in the following table, the system is fully mixed and reacts in water bath at 37 ℃ for 15 min;

(3) purifying the digested PCR product, wherein the experimental steps refer to GV-High-Efficiency DNA Fragments Purification Kit of Changsheng biotechnology Limited liability company of Beijing ancient cooking;

(4) transformation of E.coli DH 5. alpha. competent cells with the mutant plasmid:

add 10. mu.L of the purified above mutant plasmid into 50. mu.L of E.coli DH 5. alpha. competent cells, ice-bath for 30 min; carrying out heat shock for 90s at 42 ℃; ice-bath for 2min, adding 1mL liquid LB, and culturing in a shaker at 37 deg.C for 1-1.5 h; centrifuge at 8000rpm for 2min, discard the supernatant (leave a little bottom liquid). The remaining solution was spread on LB plate containing 50. mu.g/mL kanamycin and cultured by inversion overnight at 37 ℃; the next day, a single clone was picked, inoculated into 5mL of LB medium containing 50. mu.g/mL kanamycin, and cultured overnight at 37 ℃ at 200 rpm;

(5) extracting plasmids, wherein the experimental steps refer to GV-Plasmid DNA Mini Extraction Kit of Changsheng biotechnology Limited company of Beijing ancient cooking;

(6) taking 3 mu L of plasmid to carry out 1% agarose gel electrophoresis detection, and leaving the plasmid with clear bands;

(7) 10 μ L of plasmid solution was sequenced in sterile EP tubes. And comparing the sequencing result with the original sequence to confirm whether the site-directed mutagenesis is successful. The plasmid with the correct sequencing is a recombinant vector pET28a-SsChi18A-K186A of a mutant gene SsChi18A _1 containing chitinase SChi18A, and the nucleotide sequence of the plasmid is shown as SEQ ID No. 4.

Example 2 construction of recombinant engineered bacterium containing the mutant Gene SsChi18A _1

In the embodiment, the recombinant engineering bacteria containing the mutant gene SsChi18A _1 are constructed, and the specific steps are as follows: adding 2 μ L of the above mutation plasmid pET28a-SsChi18A-K186A with correct sequencing into 50 μ L of Escherichia coli BL-21 competent cells, and ice-cooling for 30 min; carrying out heat shock for 90s at 42 ℃; ice-bath for 2min, adding 1mL liquid LB, and culturing in a shaker at 37 deg.C for 1-1.5 h; centrifuge at 8000rpm for 2min, discard the supernatant (leave a little bottom liquid). The remaining solution was spread on LB plate containing 50. mu.g/mL kanamycin, spread evenly to dryness, and cultured by inversion overnight at 37 ℃; the next day, the single clone is selected and inoculated in 5mL LB culture medium containing antibiotics, and cultured overnight at 37 ℃ and 200rpm, and the recombinant engineering bacterium containing the mutant gene SsChi18A _1 is obtained.

Example 3: recombinant expression of mutant Gene SsChi18A _1

Fermenting, expressing and purifying the mutant chitinase SsChi18A-K186A encoded by the mutant gene SChi18A _1, and specifically comprising the following steps:

(1) heterologous expression of recombinant protein:

the recombinant engineering bacteria containing the mutant gene SsChi18A _1 obtained in example 2 are taken and cultured in 5mL LB culture medium (containing 50 ug/mL kanamycin) at the temperature of 37 ℃ and 200rpm overnight;

transferring the overnight-cultured bacterial liquid into a 1L triangular flask (containing 50 ug/mL kanamycin) filled with 300mL of LB medium, and culturing at 37 ℃ for about 3h until OD600 is 0.6-0.8; adding IPTG with final concentration of 0.5mM, and performing induced culture at 20 ℃ for 20 h; centrifuging at 8000rpm and 4 deg.C for 10min to obtain thallus precipitate;

with NaH at pH 8.0₂PO₄Resuspending the bacteria in NaCl buffer solution, and placing the bacteria solution in a 100mL centrifuge tube; placing the centrifuge tube ON ice, ultrasonically breaking cells, setting the time to be 9s ON and 10s OFF for 90 times; centrifuging at 11000rpm at 4 ℃ for 30 min; the supernatant was filtered through a 0.22 μm filter head and the filtrate was combined with column packing and prepared for affinity purification.

(2) And (3) purifying the recombinant protein:

the target protein containing 6 XHis tag was affinity purified using a GE Healthcare Ni Sepharose 6Fast Flow affinity column. Mixing the crude enzyme solution obtained in the last step with a filler, and rotating and combining for 2h at 4 ℃ to ensure that nickel on the filler is fully combined with His labels on the proteins;

with NaH at pH 8.0₂PO₄Preparing imidazole mother liquor with the concentration of 1M by NaCl buffer solution, then diluting to 5mM, 20mM, 60mM, 100mM and 250mM respectively, eluting protein by imidazole with the concentration, and collecting the protein into 10mL EP tubes respectively;

regenerating the nickel column after eluting the protein, and adding corresponding solutions into the nickel column according to the following sequence during regeneration: 50mM EDTA → distilled water → 1M NaCl → distilled water → 0.1M nickel sulfate → distilled water → 70% ethanol → 20% ethanol for storage;

the collected enzyme solution was subjected to SDS-PAGE: mixing the sample with SDS buffer at ratio of 4:1 (16 μ L sample, 4 μ L buffer), 10 μ L Marker, and treating at 105 deg.C for 10 min; when the sample is spotted, 12 mu L of sample spots and 5 mu L of Marker spots are spotted. Electrophoresis was performed with 80V and the voltage was adjusted to 180V when the protein sample was in a straight line. Dyeing with Coomassie brilliant blue R-250 dye solution for 30min after electrophoresis, decolorizing with decolorizing solution for about 2h, and scanning with scanner for observation;

finding out several tubes with purer target protein bands, adding NaH with pH of 5.0₂PO₄-citric acid buffer, ultrafiltration at 4900rpm at 4 ℃ until the pH of the filtered buffer is 5.0;

the enzyme solution after ultrafiltration was filtered through a sterile 0.22 μm filter head into a sterilized 10mL centrifuge tube and stored at 4 ℃. If long-term storage is required, the temperature is kept at-80 ℃.

(3) Content determination of recombinant protein:

diluting the protein of interest to an appropriate concentration (measured without exceeding the range of the standard curve); the control group was added with 0.1mL of NaH at pH 5.0₂PO₄Citrate buffer, 0.1mL of diluted solution of the protein of interest is added to the experimental group. Each tube was then mixed with 1mL of Coomassie brilliant blue staining solution and shaken well. Standing for 10min, and measuring OD 595; each group of three replicates was assayed three times for nine replicates; protein amount and protein concentration were calculated from the standard curve.

As shown in FIG. 1, SDS-PAGE showed that the band of mutant chitinase SsChi18A-K186A was a standard protein of about 45kDa, similar in size to that expected. The results show that the target protein SsChi18A-K186A is successfully obtained by the invention.

The amino acid sequence of SsChi18A-K186A is shown as SEQ ID No.2, and the coding gene sequence is shown as SEQ ID No. 1.

A recombinant vector pET28a-SsChi18A containing the wild-type gene SsChi18A, the nucleotide sequence of which is shown in SEQ ID No.3, was prepared in the same manner as described above, and expression and purification of the protein were carried out.

Example 4: comparison of the enzymatic Properties of the mutant chitinase SsChi18A-K186A with the wild-type enzyme SChi18A

(1) The mutant chitinase SsChi18A-K186A and the wild-type enzyme SChi18A have enzyme activities under optimal conditions:

diluting mutant chitinase SsChi18A-K186A and wild type enzyme SChi18A to 0.01 mu mol/mL; to a control tube was added 100. mu.L of NaH at pH 5.0₂PO₄Citric acid buffer, 100. mu.L of diluted enzyme solution was added to the test group tubes, and 100. mu.L of colloidal chitin (dissolved in 50mM NaH at pH 5.0) was added to each tube₂PO₄-citric acid buffer), reacting at 60 ℃ for 10 min;

adding 300 μ L DNS into each tube, and boiling in water bath for 10 min; rapidly cooling, shaking, centrifuging, collecting supernatant, and determining OD 550; three replicates of each enzyme were run; calculating the reducing sugar amount according to a standard curve, and calculating the specific enzyme activity according to a formula; the enzyme activities of the two proteins were compared.

The optimal enzyme activities of mutant chitinase SsChi18A-K186A and wild-type chitinase SChi18A are shown in FIG. 2. The enzyme activity of the mutant xylanase SsChi18A-K186A is 1.55 times that of the wild xylanase SChi18A, which proves that the mutant chitinase has stronger enzyme activity on chitosan and has potential application value.

(2) Determination of oligosaccharide product profiles at 60 ℃ of mutant xylanase SsChi18A-K186A and wild-type enzyme SChi 18A:

diluting mutant chitinase SsChi18A-K186A and wild type enzyme SChi18A to 0.01 nmol/mL; the experimental tube was filled with 40. mu.L of diluted enzyme solution, and 60. mu.L of 1mg/mL chitin oligosaccharide solution (dissolved in 50mM Na pH 5.0)₂HPO₄-citric acid buffer), reacting for 30min at 60 ℃; sampling at 0, 2, 5, 10, 20 and 30 min;

detecting a product spectrum by adopting a method of FACE electrophoresis: firstly, a supersaturated 7-amino-1, 3-naphthalene disulfonate (ANDS) solution dissolved in 15% acetic acid is mixed with a sample in equal volume (5 mu L of each solution) and reacted for 1 hour in a dark place; an equal volume of NaCNBH was then added₃Adding the solution into the mixture, and incubating overnight at 42 ℃; adding 15 mu L of 50% sucrose solution into the mixed solution; and the labeled product was used for electrophoresis at a ratio of 7. mu.L/well; the images were scanned using a ChemiDocTM MP imaging system and the image files were stored in TIFF format.

The spectrum of oligosaccharide products from mutant chitinase SsChi18A-K186A treated at different times with wild-type chitinase SChi18A at 60 ℃ is shown in FIG. 3. The mutant chitinase SsChi18A-K186A can finish the degradation of chitin pentasaccharide within 10min and finish the degradation of chitin hexasaccharide within 20 min; while the wild-type chitinase SsChi18A can not degrade the oligosaccharide within 30 min. The result proves that the mutant chitinase also has stronger enzyme activity on chitin oligosaccharide, and has potential industrial application value.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong university

<120> a mutant chitinase and use thereof

<130> 111

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1098

<212> DNA

<213> Artificial Sequence

<220>

<223> mutant

<400> 1

ggttacttca ccgagtgggg cgtctacggc cgcgactacc acgtcaagaa cctggtgacc 60

tccggctcgg ccgccaagat cacccacatc aactacgggt tcggcaacgt caagaacggc 120

cagtgcgccg tcgacgacac cttcgccgcc tacgagaagg cctacaccgc cgaccagtcc 180

gtcagcggca ccgccgacac ctgggaccag ccgctgcgcg gcaacttcaa ccagctgcgc 240

cagctgaagg cgaagtaccc gcacatcaag gtgctgtact ccttcggcgg ctggacctac 300

tccggcggct tcgcccaggc cgcgcagaac ccggccgcgt tcgccaggtc ctgcaagcag 360

gtcgtggagg acccgcgctg ggcggatgtc ttcgacggca tcgacatcga ctgggagtac 420

ccgaacgcct gcggtctgac ctgcgactcc agcggcccgg cggccttcag gaacctcgtg 480

caggccctgc gcgccgagtt cggcgaggac tacctggtca ccgccgcgat caccgcggac 540

gcctcggacg gaggcgcact cgacgcggcc gactacggcg gcgccgccca gtacctcgac 600

tggtacaacg tgatgacgta cgactacttc ggcgcctggg acaggaccgg ccccaccgcc 660

ccccactcgc cgctgacctc ctacaacggc atcccgaagc agggcttcga ctcggcctcc 720

gccatcgcca agctcagggc caagggcgtt ccggcgcaca agctgctgct cggcatcggc 780

ttctacggcc gcggctggac cggcgtgacc cagtccgccc cgggcggcac cgccaccggt 840

ccggcgcccg gcacctacga ggcgggcgtc gaggactaca aggtcctgaa gaactcctgc 900

ccggccaccg gcaccatcgc cggcacggcc tacgcccact gcggcaccaa ctggtggtcc 960

tacgacaccc cggccaccat cggcaccaag atgaactggg tcaagtccca gggcctgggc 1020

ggtgccttct tctgggagtt cagcggcgac accagcaacg gtgagctggt cagcgccatc 1080

aacagcggcc tgtcgtaa 1098

<210> 2

<211> 365

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant

<400> 2

Gly Tyr Phe Thr Glu Trp Gly Val Tyr Gly Arg Asp Tyr His Val Lys

1 5 10 15

Asn Leu Val Thr Ser Gly Ser Ala Ala Lys Ile Thr His Ile Asn Tyr

20 25 30

Gly Phe Gly Asn Val Lys Asn Gly Gln Cys Ala Val Asp Asp Thr Phe

35 40 45

Ala Ala Tyr Glu Lys Ala Tyr Thr Ala Asp Gln Ser Val Ser Gly Thr

50 55 60

Ala Asp Thr Trp Asp Gln Pro Leu Arg Gly Asn Phe Asn Gln Leu Arg

65 70 75 80

Gln Leu Lys Ala Lys Tyr Pro His Ile Lys Val Leu Tyr Ser Phe Gly

85 90 95

Gly Trp Thr Tyr Ser Gly Gly Phe Ala Gln Ala Ala Gln Asn Pro Ala

100 105 110

Ala Phe Ala Arg Ser Cys Lys Gln Val Val Glu Asp Pro Arg Trp Ala

115 120 125

Asp Val Phe Asp Gly Ile Asp Ile Asp Trp Glu Tyr Pro Asn Ala Cys

130 135 140

Gly Leu Thr Cys Asp Ser Ser Gly Pro Ala Ala Phe Arg Asn Leu Val

145 150 155 160

Gln Ala Leu Arg Ala Glu Phe Gly Glu Asp Tyr Leu Val Thr Ala Ala

165 170 175

Ile Thr Ala Asp Ala Ser Asp Gly Gly Ala Leu Asp Ala Ala Asp Tyr

180 185 190

Gly Gly Ala Ala Gln Tyr Leu Asp Trp Tyr Asn Val Met Thr Tyr Asp

195 200 205

Tyr Phe Gly Ala Trp Asp Arg Thr Gly Pro Thr Ala Pro His Ser Pro

210 215 220

Leu Thr Ser Tyr Asn Gly Ile Pro Lys Gln Gly Phe Asp Ser Ala Ser

225 230 235 240

Ala Ile Ala Lys Leu Arg Ala Lys Gly Val Pro Ala His Lys Leu Leu

245 250 255

Leu Gly Ile Gly Phe Tyr Gly Arg Gly Trp Thr Gly Val Thr Gln Ser

260 265 270

Ala Pro Gly Gly Thr Ala Thr Gly Pro Ala Pro Gly Thr Tyr Glu Ala

275 280 285

Gly Val Glu Asp Tyr Lys Val Leu Lys Asn Ser Cys Pro Ala Thr Gly

290 295 300

Thr Ile Ala Gly Thr Ala Tyr Ala His Cys Gly Thr Asn Trp Trp Ser

305 310 315 320

Tyr Asp Thr Pro Ala Thr Ile Gly Thr Lys Met Asn Trp Val Lys Ser

325 330 335

Gln Gly Leu Gly Gly Ala Phe Phe Trp Glu Phe Ser Gly Asp Thr Ser

340 345 350

Asn Gly Glu Leu Val Ser Ala Ile Asn Ser Gly Leu Ser

355 360 365

<210> 3

<211> 6428

<212> DNA

<213> Artificial Sequence

<220>

<223> mutant

<400> 3

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 180

cgacggagct cgaattctta cgacaggccg ctgttgatgg cgctgaccag ctcaccgttg 240

ctggtgtcgc cgctgaactc ccagaagaag gcaccgccca ggccctggga cttgacccag 300

ttcatcttgg tgccgatggt ggccggggtg tcgtaggacc accagttggt gccgcagtgg 360

gcgtaggccg tgccggcgat ggtgccggtg gccgggcagg agttcttcag gaccttgtag 420

tcctcgacgc ccgcctcgta ggtgccgggc gccggaccgg tggcggtgcc gcccggggcg 480

gactgggtca cgccggtcca gccgcggccg tagaagccga tgccgagcag cagcttgtgc 540

gccggaacgc ccttggccct gagcttggcg atggcggagg ccgagtcgaa gccctgcttc 600

gggatgccgt tgtaggaggt cagcggcgag tggggggcgg tggggccggt cctgtcccag 660

gcgccgaagt agtcgtacgt catcacgttg taccagtcga ggtactgggc ggcgccgccg 720

tagtcggccg cgtcgagctt gcctccgtcc gaggcgtccg cggtgatcgc ggcggtgacc 780

aggtagtcct cgccgaactc ggcgcgcagg gcctgcacga ggttcctgaa ggccgccggg 840

ccgctggagt cgcaggtcag accgcaggcg ttcgggtact cccagtcgat gtcgatgccg 900

tcgaagacat ccgcccagcg cgggtcctcc acgacctgct tgcaggacct ggcgaacgcg 960

gccgggttct gcgcggcctg ggcgaagccg ccggagtagg tccagccgcc gaaggagtac 1020

agcaccttga tgtgcgggta cttcgccttc agctggcgca gctggttgaa gttgccgcgc 1080

agcggctggt cccaggtgtc ggcggtgccg ctgacggact ggtcggcggt gtaggccttc 1140

tcgtaggcgg cgaaggtgtc gtcgacggcg cactggccgt tcttgacgtt gccgaacccg 1200

tagttgatgt gggtgatctt ggcggccgag ccggaggtca ccaggttctt gacgtggtag 1260

tcgcggccgt agacgcccca ctcggtgaag taacccatat ggctgccgcg cggcaccagg 1320

ccgctgctgt gatgatgatg atgatggctg ctgcccatgg tatatctcct tcttaaagtt 1380

aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg 1440

tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc 1500

accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat 1560

cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc 1620

gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg 1680

ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag 1740

cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg 1800

cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc 1860

agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt 1920

ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa 1980

ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag 2040

tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact 2100

gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc 2160

ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga 2220

ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt 2280

ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg 2340

cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag 2400

ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat 2460

tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat 2520

gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc 2580

gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt 2640

agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa 2700

acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg 2760

ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct 2820

ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 2880

acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc 2940

tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct 3000

ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca 3060

tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc 3120

gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg 3180

ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg 3240

ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag 3300

gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa 3360

gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc 3420

gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct 3480

gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct 3540

ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac 3600

cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc 3660

tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat 3720

cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat 3780

taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat 3840

cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg 3900

gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg 3960

ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag 4020

ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga 4080

gcagattgta ctgagagtgc accatatatg cggtgtgaaa taccgcacag atgcgtaagg 4140

agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 4200

gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 4260

tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 4320

aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 4380

aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 4440

ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 4500

tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 4560

agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 4620

gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 4680

tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 4740

acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 4800

tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 4860

caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 4920

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 4980

aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa 5040

tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa 5100

ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc 5160

aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca 5220

tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac 5280

ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt 5340

actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc 5400

aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt 5460

ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat 5520

gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga 5580

acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca 5640

tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga 5700

tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct 5760

cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc 5820

tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca 5880

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 5940

ttccccgaaa agtgccacct gaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa 6000

atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 6060

aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 6120

tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 6180

cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa 6240

atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 6300

cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 6360

tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc 6420

attcgcca 6428

<210> 4

<211> 6428

<212> DNA

<213> Artificial Sequence

<220>

<223> mutant

<400> 4

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 180

cgacggagct cgaattctta cgacaggccg ctgttgatgg cgctgaccag ctcaccgttg 240

ctggtgtcgc cgctgaactc ccagaagaag gcaccgccca ggccctggga cttgacccag 300

ttcatcttgg tgccgatggt ggccggggtg tcgtaggacc accagttggt gccgcagtgg 360

gcgtaggccg tgccggcgat ggtgccggtg gccgggcagg agttcttcag gaccttgtag 420

tcctcgacgc ccgcctcgta ggtgccgggc gccggaccgg tggcggtgcc gcccggggcg 480

gactgggtca cgccggtcca gccgcggccg tagaagccga tgccgagcag cagcttgtgc 540

gccggaacgc ccttggccct gagcttggcg atggcggagg ccgagtcgaa gccctgcttc 600

gggatgccgt tgtaggaggt cagcggcgag tggggggcgg tggggccggt cctgtcccag 660

gcgccgaagt agtcgtacgt catcacgttg taccagtcga ggtactgggc ggcgccgccg 720

tagtcggccg cgtcgagtgc gcctccgtcc gaggcgtccg cggtgatcgc ggcggtgacc 780

aggtagtcct cgccgaactc ggcgcgcagg gcctgcacga ggttcctgaa ggccgccggg 840

ccgctggagt cgcaggtcag accgcaggcg ttcgggtact cccagtcgat gtcgatgccg 900

tcgaagacat ccgcccagcg cgggtcctcc acgacctgct tgcaggacct ggcgaacgcg 960

gccgggttct gcgcggcctg ggcgaagccg ccggagtagg tccagccgcc gaaggagtac 1020

agcaccttga tgtgcgggta cttcgccttc agctggcgca gctggttgaa gttgccgcgc 1080

agcggctggt cccaggtgtc ggcggtgccg ctgacggact ggtcggcggt gtaggccttc 1140

tcgtaggcgg cgaaggtgtc gtcgacggcg cactggccgt tcttgacgtt gccgaacccg 1200

tagttgatgt gggtgatctt ggcggccgag ccggaggtca ccaggttctt gacgtggtag 1260

tcgcggccgt agacgcccca ctcggtgaag taacccatat ggctgccgcg cggcaccagg 1320

ccgctgctgt gatgatgatg atgatggctg ctgcccatgg tatatctcct tcttaaagtt 1380

aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg 1440

tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc 1500

accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat 1560

cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc 1620

gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg 1680

ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag 1740

cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg 1800

cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc 1860

agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt 1920

ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa 1980

ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag 2040

tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact 2100

gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc 2160

ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga 2220

ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt 2280

ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg 2340

cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag 2400

ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat 2460

tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat 2520

gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc 2580

gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt 2640

agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa 2700

acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg 2760

ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct 2820

ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 2880

acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc 2940

tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct 3000

ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca 3060

tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc 3120

gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg 3180

ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg 3240

ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag 3300

gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa 3360

gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc 3420

gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct 3480

gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct 3540

ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac 3600

cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc 3660

tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat 3720

cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat 3780

taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat 3840

cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg 3900

gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg 3960

ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag 4020

ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga 4080

gcagattgta ctgagagtgc accatatatg cggtgtgaaa taccgcacag atgcgtaagg 4140

agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 4200

gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 4260

tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 4320

aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 4380

aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 4440

ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 4500

tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 4560

agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 4620

gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 4680

tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 4740

acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 4800

tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 4860

caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 4920

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 4980

aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa 5040

tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa 5100

ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc 5160

aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca 5220

tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac 5280

ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt 5340

actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc 5400

aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt 5460

ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat 5520

gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga 5580

acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca 5640

tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga 5700

tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct 5760

cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc 5820

tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca 5880

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 5940

ttccccgaaa agtgccacct gaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa 6000

atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 6060

aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 6120

tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 6180

cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa 6240

atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 6300

cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 6360

tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc 6420

attcgcca 6428

Claims (16)

1. A mutant chitinase, the amino acid sequence of the mutant chitinase is shown in SEQ ID No.2. 2. A gene encoding the mutated chitinase of claim 1. 3. The gene according to claim 2, wherein the sequence of the gene is shown in SEQ ID No.1. 4. A recombinant vector containing the gene of claim 2 or 3. 5. The recombinant vector according to claim 4, wherein the recombinant vector is a recombinant expression vector. 6 . The recombinant vector according to claim 4 , wherein the backbone vector of the recombinant vector is derived from pET-15b, pET-22b or pET-28a. 7 . 7. A recombinant strain comprising the recombinant vector of any one of claims 4-6. 8. The recombinant strain according to claim 7, wherein the recombinant strain is Escherichia coli. 9. The recombinant strain according to claim 8, wherein the Escherichia coli is Escherichia coli BL21. 10. The mutated chitinase of claim 1, the gene of any of claims 2-3, the recombinant vector of any of claims 4-6, or the gene of any of claims 7-9 Use of recombinant strains in degrading chitin-containing materials. 11. The mutated chitinase of claim 1, the gene of any of claims 2-3, the recombinant vector of any of claims 4-6, or the gene of any of claims 7-9 Application of recombinant strains in the preparation of chitin oligosaccharides. 12. A method for degrading chitin-containing or chitin-oligosaccharide-containing materials, the method comprising utilizing the mutant chitinase of claim 1, the gene of any one of claims 2-3 . The step of processing the material containing chitin or chitin oligosaccharide by the recombinant vector described in any one of claims 4-6 or the recombinant strain described in any one of claims 7-9. 13. The method according to claim 12, wherein the temperature of the treatment is 30°C-80°C. 14. The method according to claim 13, wherein the temperature of the treatment is 50°C-70°C. 15. The method of claim 12, wherein the pH of the treatment is 3-11. 16. The method of claim 15, wherein the pH of the treatment is 4-7.

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