CN112831510B - Construction of recombinant bacterium for efficiently expressing chitinase and screening of high-enzyme-activity mutant - Google Patents

Abstract

Construction of a recombinant strain for efficiently expressing chitinase and screening of high-enzyme-activity mutants. The invention realizes the high-efficiency secretory expression of BchiA 1 in bacillus subtilis by means of recombinant protein technology, and solves the problem of insufficient secretion of chitinase of most microorganisms. Meanwhile, a novel chitinase high-throughput screening method is created, and screening throughput and efficiency are greatly improved. In addition, the invention relates to and discloses several high-enzyme activity chitinase mutants, namely BchchiA 1(Y10A), BchchiA 1(R301A), BchchiA 1(E327A), BchchiA 1(Y10A/R301A), BchchiA 1(Y10A/E327A), BchchiA 1(R301A/E327A) and BchchiA 1(Y10A/R301A/E327A), wherein the enzyme activities are respectively improved by 2.49 times, 0.67 times, 3.61 times, 2.39 times, 3.46 times, 4.75 times and 16.89 times compared with wild type BchchiA 1, and the activity of the BchchiA 1(Y10A/R301A/E327A) is optimal. Finally, the chitinase BchiA 1 and the optimal mutant BchiA 1(Y10A/R301A/E327A) respectively act synergistically with the monooxygenase BatLPMO10 derived from Bacillus atrophaeus, so that the hydrolysis efficiencies of the BchiA 1 and the BchiA 1(Y10A/R301A/E327A) are respectively improved by 50.00 percent and 46.71 percent.

Description

Construction of a recombinant strain highly expressing chitinase and screening of mutants with high enzymatic activity

technical field

The invention relates to the construction of a high-efficiency secreting and expressing Bacillus circus chitinase recombinant bacteria and the high-throughput screening of chitinase mutants, belonging to the technical field of genetic engineering.

Background technique

Chitin is a linear polymer linked by N-acetyl-D-glucosamine (NAG) through β-1,4 glycosidic bonds. It is the most widely distributed aminopolysaccharide in nature, especially as arthropod exoskeleton and fungi. Structural components of the cell wall. Chitin has good biocompatibility, broad-spectrum antibacterial properties, and no toxic and side effects, so it has broad application prospects in biomedical, food, environmental protection and cosmetics industries. However, the high crystallinity and poor solubility of chitin are undoubtedly a serious challenge to further enhance the application value.

Chitin oligosaccharides, with a degree of polymerization below 20, are one of the main degradation products of chitin. Due to its greatly improved solubility compared with chitin, it has outstanding performance in immune regulation, antibacterial, antitumor, and cell repair. At present, chitin oligosaccharides are mainly obtained by degrading chitin through chemical, physical and biological enzymatic methods. Among them, the biological enzymatic method is favored because of its environmental friendliness, mild reaction conditions, easily controllable degradation process, and few by-products. This method mainly relies on chitinase.

Chitinases have invaluable applications in agriculture and the environment, such as biological treatment of chitin waste, preparation of chitin oligosaccharides, use as biological control agents, and isolation of protoplasts from fungi and yeast for strain improvement Wait. Chitinases are widely distributed in eukaryotes, prokaryotes, archaea and viruses, and mainly catalyze the random cleavage of β-1,4 glycosidic bonds to generate chitin oligosaccharides with low degree of polymerization and good water solubility. However, due to the high crystallinity of chitin, the chitinase and the substrate cannot be fully contacted, resulting in low efficiency of biological enzymatic methods. Therefore, we devoted ourselves to finding strains with excellent chitinase activity. Among them, Bacillus circulans chitinase BcchiA1 has high affinity and hydrolysis activity for insoluble chitin, and has great application value.

At present, many microorganisms can secrete a certain amount of chitinase, but the problem of insufficient secretion is common. Recombinant protein technology is an important technical means to directly obtain a large amount of high-purity chitinase. Compared with other exogenous gene expression systems, microbial expression systems are widely used in protein expression technology due to their fast growth, short culture period and low cost. Commonly used are Escherichia coli, Bacillus subtilis, yeast expression systems, etc. As the most thoroughly studied and rapidly developing prokaryotic expression system, Escherichia coli has disadvantages such as non-secretory expression of most proteins, easy formation of inclusion bodies due to large intracellular aggregation, and many intracellular impurities, which lead to cumbersome and time-consuming subsequent separation and purification steps. Time. Yeast expression system is a widely used eukaryotic expression system, such as Saccharomyces cerevisiae, Pichia pastoris, etc. It is especially suitable for expressing target proteins from lower eukaryotes, but Saccharomyces cerevisiae lacks a promoter with high transcriptional strength. It is not suitable for high-level expression of exogenous proteins, and methanol, an inducer of Pichia pastoris, is easy to cause toxicity, which also makes the system have obvious defects.

Bacillus subtilis is non-pathogenic, does not contain exotoxin and endotoxin, and is a safe strain (GRAS) widely used in the pharmaceutical and food processing industries, especially in the production and secretion of enzymes and other active proteins. Simple and efficient. In addition, the host has the characteristics of clear genetic background, simple molecular manipulation, and short fermentation period, and has significant advantages in the field of applied microorganisms.

In order to solve the above-mentioned problems of insufficient secretion of wild-type chitinase and low activity, the present invention will further improve the enzyme activity on the basis of increasing the secretion of Bacillus circulans chitinase BcchiA1, in order to improve the meet application requirements well.

SUMMARY OF THE INVENTION

The invention firstly constructs a Bacillus subtilis recombinant bacterium that efficiently secretes Bacillus circus chitinase, and develops an efficient and concise high-throughput screening method to screen the mutation library, thereby obtaining mutations with high enzymatic activity body. In addition, monooxygenase and chitinase were used to synergize chitin-like substrates, aiming to further improve the enzymatic hydrolysis efficiency of chitinase.

The first aspect of the present invention relates to disclosing a gene encoding chitinase BcchiA1 from Bacillus circulans WL-12, characterized in that the nucleic acid sequence optimized according to the codon preference is shown in SEQ ID NO.1, and the amino acid sequence is shown in shown in SEQ ID NO.2.

The second aspect of the present invention relates to disclosing a recombinant expression plasmid pMATE-BcchiA1, characterized in that the recombinant expression plasmid is a shuttle-type plasmid of Escherichia coli and Bacillus subtilis carrying a RepB replicon, and contains the nucleic acid sequence of claim 1, and no signal peptide.

The third aspect of the present invention relates to the disclosure of three single-point mutants of chitinase, characterized in that the amino acid sequence of the mutant is mutated to Ala at position 10 of the amino acid sequence shown in SEQ ID No. The 301st Arg was mutated to Ala, or the 327th Glu was mutated to Ala, the specific activity of the enzyme was increased by 2.49 times, 0.67 times and 3.61 times, respectively, compared with the wild type of BcchiA1.

The fourth aspect of the present invention relates to the disclosure of four chitinase mutants based on single point mutation stacking, characterized in that the amino acid sequence of the mutants is mutated at Tyr position 10 of the amino acid sequence shown in SEQ ID No. 2 to Ala and 301 Arg are mutated to Ala, or 10 Tyr is mutated to Ala and 327 Glu is mutated to Ala, or 301 Arg is mutated to Ala and 327 Glu is mutated to Ala, or 10 Tyr is mutated to Ala, 301 The specific activity of the enzyme was increased by 2.39 times, 3.46 times, 4.75 times and 16.89 times, respectively, compared with the wild type of BcchiA1.

The fifth aspect of the present invention relates to the disclosure of a series of genetically engineered bacteria Bacillus subtilis 1A751, which is characterized in that it contains the recombinant expression plasmid described in any one of the first to fourth aspects of the present invention, and the recombinant bacteria is in the absence of a signal peptide. Under the action, the efficient secretory expression of BcchiA1 was successfully achieved.

The sixth aspect of the present invention relates to the disclosure of a novel chitinase directed evolution method, which includes two parts: constructing a mutation library and high-throughput screening. The establishment of the mutation library is mainly completed by error-prone PCR; the substrate used for high-throughput screening is the complex CC-RBB formed by the dye ramazol brilliant blue (RBB) and colloidal chitin (CC). Under the action of chitinase, the substrate is degraded to release free RBB, and then the absorbance value is determined. The absorbance values measured in this method range from OD ₅₇₅ to OD ₆₁₀ , and the concentration range of CC-RBB ranges from 2% to 40%. Among them, the optimal choice is OD ₅₉₅ and 2% substrate concentration.

The seventh aspect of the present invention relates to disclosing the application of the above chitinase and a series of mutants, coding genes, expression vectors and recombinant bacteria in degrading chitin substances. Among them, after the chitinase BcchiA1 and its mutant BcchiA1 (Y10A/R301A/E327A) cooperate with the monooxygenase BatLPMO10 from Bacillus atrophaeus, respectively, the hydrolysis efficiency of BcchiA1 and BcchiA1 (Y10A/R301A/E327A) can be increased. 50.00% and 46.71% respectively.

The beneficial effect of the present invention is to firstly utilize protein recombination technology to realize the efficient secretory expression of Bacillus circulans chitinase in Bacillus subtilis without signal peptide. Usually, the expression of most heterologous proteins needs to find a matching signal peptide, so as to realize the massive secretion of the target protein in the expression host. suitability. However, in the present invention, the chitinase BcchiA1 can be massively secreted to the outside of the cell without the aid of any signal peptide, which saves the tedious task of screening the signal peptide. Secondly, an efficient, economical and convenient high-throughput screening method for chitinase was developed, which greatly improved the screening throughput and efficiency, and provided a good technical reference for the high-throughput screening of chitinase in the future. Furthermore, a series of chitinase mutants with high enzymatic activity were screened out. The best one, BcchiA1 (Y10A/R301A/E327A), had a 16.89-fold increase in enzymatic activity compared with the wild type, which has considerable industrial application value. Finally, with the help of the synergistic effect of BatLPMO10 on BcchiA1 and its mutant BcchiA1 (Y10A/R301A/E327A), the hydrolysis efficiency of chitinase was further enhanced.

Description of drawings

Figure 1 is a graph showing the results of SDS-PAGE of chitinase BcchiA1 and its mutants secreted and expressed in Bacillus subtilis. M: protein marker; 1: extracellular sample of wild-type BcchiA1; 2: extracellular sample of mutant BcchiA1 (Y10A); 3: extracellular sample of mutant BcchiA1 (R301A); 4: mutant BcchiA1 (E327A) 5 is the extracellular sample of mutant BcchiA1 (Y10A/R301A); 6 is the extracellular sample of mutant BcchiA1 (Y10A/E327A); 7 is the extracellular sample of mutant BcchiA1 (R301A/E327A); 8 is the extracellular sample of mutant BcchiA1 (Y10A/R301A/E327A).

Figure 2 is a gel image of the SDS-PAGE results of purified chitinase and monooxygenase BatLPMO10. A is the gel image after purification of seven chitinase mutants; B is the gel image after purification of wild-type chitinase BcchiA1 and monooxygenase BatLPMO10.

Figure 3 shows the measured values of the enzyme activity of wild-type BcchiA1 and its mutants.

Figure 4 is a schematic representation of the synergy of the chitinase BcchiA1 and BatLPMO10. The abscissa is OD595, and the ordinate is the combination of different concentrations of _BatLPMO10 with a certain amount of BcchiA1.

Figure 5 is a schematic representation of the synergy of chitinase mutant BcchiA1 (Y10A/R301A/E327A) and BatLPMO10. The abscissa is OD595 and the ordinate is the combination of different concentrations of _BatLPMO10 with a certain amount of BcchiA1 (Y10A/R301A/E327A).

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

The experimental methods in the following examples, unless otherwise mentioned, are conventional methods for molecular manipulation.

The test materials used in the following examples are conventional biochemical reagents unless otherwise specified.

The quantitative tests in the following examples were all repeated three times, and the results were averaged.

Example 1 Construction of pMATE-BcchiA1 recombinant plasmid and recombinant bacteria

1. Construction of pMATE-BcchiA1 recombinant plasmid

完成片段与载体的无缝连接，转入大肠杆菌DH5α感受态细胞中，测序后完成重组质粒pMATE-BcchiA1的构建。The chitinase BcchiA1 from Bacillus circulans WL-12 has strong affinity and catalytic activity for highly insoluble chitin. Suzhou Jinweizhi Biotechnology Co., Ltd. was entrusted to synthesize the gene after optimization according to the codon preference, and a His tag was added to the C-terminal to purify the protein. Its nucleic acid sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO. 2, the gene was synthesized and cloned into the vector pUC57-Amp to obtain the recombinant plasmid pUC57-BcchiA1. Using the recombinant plasmid pUC57-BcchiA1 as a template, a primer pair consisting of primer FragmentF and primer FragmentR was used for PCR amplification to obtain a BcchiA1 fragment of about 1.9 kb. Using the pMATE plasmid as a vector template, the vector backbone was amplified from the plasmid by primers vectorF and vectorR, and a homology arm of about 20 bp was added to obtain a plasmid backbone pMATE of about 7.4 kb. The PCR product was purified by the gel recovery kit and passed through the kit.

The seamless connection between the fragment and the vector was completed, and then it was transferred into E. coli DH5α competent cells. After sequencing, the recombinant plasmid pMATE-BcchiA1 was constructed.

Table 1

2. Construction of recombinant bacteria containing pMATE-BcchiA1 recombinant plasmid

The recombinant expression plasmid pMATE-BcchiA1 in the above steps was transferred into B.subtilis 1A751, and the construction was completed after the colony was verified correctly.

Example 2 Development of a new method for directed evolution of chitinases

1. Construction of chitinase mutant library

Using the wild-type BcchiA1 nucleic acid sequence shown in SEQ ID NO.1 as a template, the target gene was amplified with primers Fragment-F and Fragment-R by TIANZ adjustable error-prone PCR kit, and random mutations were introduced into the BcchiA1 gene (1.9kb). The error-prone PCR reaction system is shown in Table 2. The error-prone PCR reaction program was: 94°C, 3 min; 94°C for 1 min, 45°C for 1 min, 72°C for 2 min, 50 cycles.

Table 2

Element Volume (μL) Error-prone PCRMix, 10x 3.0 Error-prone PCR-specific dNTPs,10 3.0 MnCl₂ for error-prone PCR 1.0 Error-prone PCR-specific dGTP 0 BcchiA1 template (5ng/μL) 3.0 Fragment-F/R(10Meach) 1.0 TaqDNA polymerase for error-prone PCR 0.5 Ultra-pure water 17.5

The PCR product that has introduced random mutations and the aforementioned pMATE vector fragment are overlapped and extended by PrimeSTAR polymerase to form a DNA polymer. Finally, the DNA polymer (POE-PCR product) was transformed into B. subtilis 1A751 strain, and the LB solid medium containing 20 μg/ml kanamycin was used for screening, that is, the construction of the mutation library was completed.

2. High-throughput screening of chitinase

The obtained mutants were selected one by one into a 96-well deep-well plate, each well containing 50 μg/ml kanamycin and 500 μl LB liquid medium, 37° C., 800 rpm shaking culture overnight, that is, as seeds for high-throughput screening. Seeds were added to 96-well deep-well plates for high-throughput screening, each well containing 500 μl of LB medium, 20 μg/ml kanamycin, 2% (w/v) CC-RBB and 1% maltose. Subsequently, it was cultured in a high-throughput orifice plate shaking culture system at 37°C and 800rpm. After culturing for 24 h, the plate was centrifuged at 4°C and 6000 rpm for 30 min. After the centrifugation was completed, the supernatant after centrifugation of the 96-well deep-well plate was transferred to a 96-well microtiter plate by means of an automatic high-throughput analysis and screening workstation, and the OD ₅₉₅ was determined.

Example 3 Further stacking of single mutation sites with high enzymatic activity-site-directed mutagenesis

1. Single-point mutants obtain two-point superimposed mutants by site-directed mutagenesis

完成片段与实施例1中的载体骨架pMATE进行无缝连接，转入大肠杆菌DH5α感受态细胞中，测序后完成重组质粒pMATE-BcchiA1(Y10A/R301A) 的构建。1. Obtainment of mutant BcchiA1 (Y10A/R301A): using single-point mutant BcchiA1 (Y10A) as a template, using the primer pairs of primers R301A-F and primers R301A-R shown in Table 3 to carry out PCR amplification to obtain BcchiA1 (Y10A/R301A) fragment of about 1.9 kb. After purification by the gel recovery kit, it was passed through the kit

The completed fragment was seamlessly connected with the vector backbone pMATE in Example 1, and transferred into E. coli DH5α competent cells. After sequencing, the construction of the recombinant plasmid pMATE-BcchiA1 (Y10A/R301A) was completed.

2. Obtainment of mutant BcchiA1 (Y10A/E327A): using single-point mutant BcchiA1 (Y10A) as a template, using the primer pairs of primers E327A-F and E327A-R shown in Table 3 to carry out PCR amplification to obtain BcchiA1 (Y10A/E327A) fragment of about 1.9 kb. See item 1 in Example 3 for the remaining steps.

3. Obtaining of mutant BcchiA1 (R301A/E327A): using single-point mutant BcchiA1 (R301A) as a template, using the primer pairs of primers E327A-F and E327A-R shown in Table 3 to carry out PCR amplification to obtain BcchiA1 (R301A/E327A) fragment of about 1.9 kb. See item 1 in Example 3 for the remaining steps.

2. Two-point mutants obtain three-point superimposed mutants by site-directed mutagenesis

Take two point mutant BcchiA1 (Y10A/R301A) as template, adopt the primer pair that primer E327A-F shown in Table 3 and primer E327A-R form to carry out PCR amplification, obtain about 1.9kb BcchiA1 (Y10A/R301A/ E327A) fragment. See item 1 in Example 3 for the remaining steps.

In summary, the mutants BcchiA1(Y10A/R301A), BcchiA1(Y10A/E327A), BcchiA1(R301A/E327A) and BcchiA1(Y10A/R301A/E327A) were obtained in sequence, that is, the amino acid sequences of the mutants are listed in SEQ ID No.2 The amino acid sequence shown has a mutation of Tyr 10 to Ala and Arg 301 to Ala, or Tyr 10 to Ala and Glu 327 to Ala, or Arg 301 to Ala and Glu 327 Mutation to Ala, or mutation of Tyr at position 10 to Ala, Arg at position 301 to Ala, and mutation of Glu at position 327 to Ala. After sequencing and verification, the recombinant plasmid was constructed successfully. The recombinant expression plasmids containing the chitinase mutants obtained in the above steps were transferred into B.subtilis1A751 respectively, and the construction was completed after the colony was verified correctly.

table 3

primer name Sequence (5'-3') use Y10A-R TCGGATAtgcGCCCACGATCTTATATGAATCTGCCA Tyr at position 10 is mutated to Ala Y10A-F catataAGATCGTGGGCgcatatccgagctgggcagcatac Tyr at position 10 is mutated to Ala R301A-R CCATCCCAGCCtgcGCCATAGAACGGCACTCC Arg at position 301 is mutated to Ala R301A-F TTCTATGGCgcaGGCTGGGATGGCTGC Arg at position 301 is mutated to Ala E327A-R CTGCCCGCtgcCCATGTGCCCACTGAGCT Glu at position 327 was mutated to Ala E327A-F AGTGGGCACATGGgcaGCGGGCAGCTTTGACTTTT Glu at position 327 was mutated to Ala

Example 4 Evaluation of wild-type chitinase and its mutants

1. Shake flask fermentation of B.subtilis 1A751 strain containing BcchiA1 wild-type or mutant recombinant plasmid

Pick a single colony and activate the seeds in 5ml LB liquid medium containing 20μg/ml kanamycin, shake at 37°C, 200rpm for 14-16h, and then transfer it into 2×SR fermentation medium according to the inoculum of 1%. Medium composition (g/L): yeast powder 50, peptone 30, K ₂ HPO ₄ 6. In addition, 50 μg/ml kanamycin and 3% maltose were added to the fermentation medium. After 48 h of fermentation, the supernatant was collected by centrifugation at 14000g for 10 min at 4°C, which was the extracellular fraction sample. Take 80 μl of extracellular supernatant sample and mix it with 20 μl of loading buffer, then bath in boiling water for 20 min. Subsequently, SDS-PAGE was performed using NuPAGE 10% Bis-Tris protein precast gel, and the results are shown in Figure 1.

2. Purification of chitinase

The purification process of chitinase was carried out at 4°C. The 48-h fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken and filtered through a 0.22 μm filter. Binding with Ni NTA Beads 6FF for 2 hours was loaded onto the column (1.5 x 8 cm). Impurities and weakly bound proteins were eluted with a gradient of buffer (50 mm Tris-HCl, 500 mm NaCl, 50-300 mm imidazole), followed by buffer (50 mm Tris-HCl, 500 mm imidazole, and 500 mm NaCl) to elute the protein of interest. The concentration of purified protein was determined by BCA protein quantification kit, and the purity of chitinase was checked by SDS-PAGE. The results are shown in Figure 2.

3. Determination of chitinase activity

The chitinase enzyme activity was determined by spectrophotometry, and one unit of chitinase enzyme activity was defined as: the amount of enzyme required to increase the absorbance by 0.01 per hour under the reaction conditions of 50 °C was one activity unit (U).

1. Preparation of colloidal chitin: Weigh 10g of chitin, add 100ml of 85% phosphoric acid, react at 4°C for 24h, add water and wash repeatedly until neutral. The white colloidal substance formed at this time maintains a humidity of 90–95%;

2. Combination of colloidal chitin and dye RBB: fully mix 25 g of the above colloidal chitin with 100 ml of 0.84% RBB solution, the obtained suspension is bathed in boiling water for 60 min, filtered to remove the filtrate, and the dyed colloidal substance is suspended Fix the color in 25ml of 1.5% sodium dichromate and 1.5% potassium sodium tartrate solution, take a boiling water bath for 10min, filter, and finally obtain a blue colloidal substrate CC-RBB;

3. Determination of enzyme activity: Take 1ml of purified BcchiA1, add 1ml of 40% (w/v) CC-RBB colloid solution, incubate at 50°C for 1h, centrifuge at 12000rpm for 5min, measure OD ₅₉₅ , inactivate in boiling water bath for 10min as a negative control ;

4. Determination of protein concentration: Measure with BCA protein quantification kit.

Finally, the 48h fermentation broth supernatant of wild-type and mutant chitinase was used as crude enzyme solution to purify the target protein, and then the enzyme activity was determined. The results are shown in Figure 3.

Comprehensively, the specific activity of wild-type chitinase BcchiA1 was 56.16±0.92U/mg; the specific activities of single-point mutant strains BcchiA1(Y10A), BcchiA1(R301A) and BcchiA1(E327A) were higher than those of wild-type BcchiA1, respectively. 2.49-fold, 0.67-fold, and 3.61-fold; two-point stacking mutant strains BcchiA1 (Y10A/R301A), BcchiA1 (Y10A/E327A) and BcchiA1 (R301A/E327A) had 2.39-fold, 3.46-fold higher enzymatic activities than wild-type BcchiA1, respectively The enzyme activity of the three-point superimposed mutant strain BcchiA1 (Y10A/R301A/E327A) was the highest, which was 1004.83±0.87U/mg, which was 16.89 times higher than that of the wild type.

Example 5 Synergistic effect of chitinase and monooxygenase BatLPMO10

Mix 500 μl of different concentrations of monooxygenase BatLPMO10 (0, 9, 18, 36, 72, 144, 288 nmol) with 1000 μl of 40% CC-RBB solution, add 1 mM ascorbic acid, and place the 7 groups in a shaker at 50 °C. 200rpm reaction for 1h. Subsequently, 500 μl of 9 nmol purified BcchiA1 was added to each of the 7 groups, and the reaction was incubated for 1 h according to the conditions of the enzyme activity assay described above. Finally, the reaction mixture was centrifuged at 12,000 rpm for 5 min and the _OD595 was determined. All reactions were repeated three times.

Taking the chitinase mutant BcchiA1 (Y10A/R301A/E327A) with the most obvious increase in enzyme activity as the research object, the above steps were repeated.

In order to determine the synergistic effect of BatLPMO10 on chitinase, the chitin substrate was pretreated by adding different concentrations of BatLPMO10 under the premise of keeping the amount of BcchiA1 and BcchiA1 (Y10A/R301A/E327A) unchanged. The results showed that the addition amount of BatLPMO10 in the two groups was significantly positively correlated with OD ₅₉₅ , indicating that BatLPMO10 had a significant promoting effect on substrate pretreatment, so that BcchiA1 and BcchiA1 (Y10A/R301A/E327A) could be more sufficient with chitin. Compared with the experimental group using chitinase alone, the highest OD ₅₉₅ can be increased by 50.00% and 46.71%, respectively. The results are shown in Figures 4 and 5.

sequence listing

<110> Tianjin Industrial Biotechnology Research, Chinese Academy of Sciences

<120> Construction of a recombinant strain highly expressing chitinase and screening of mutants with high enzyme activity

<130> 2019

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1998

<212> DNA

<213> Bacillus circulans

<400> 1

atggcagatt catataagat cgtgggctat tatccgagct gggcagcata cggccgcaac 60

tataacgtgg cagatatcga tcctacgaag gtgacgcaca tcaactacgc gtttgcggat 120

atctgctgga atggaatcca cggaaatccg gatccttctg gtcctaaccc cgttacatgg 180

acgtgccaga atgaaaagag ccagacgatc aacgtgccga acggcacgat cgtgcttggc 240

gatccgtgga ttgatactgg taaaacgttc gctggtgaca cgtgggatca gccgatcgcg 300

ggcaatatca accaacttaa caaacttaaa caaacaaacc cgaatcttaa aacgatcatc 360

tcagtgggcg gctggacgtg gagcaacaga ttcagcgatg ttgcagcgac ggcggcaaca 420

agagaagtgt tcgcgaacag cgcggtggat tttttacgca agtataactt tgatggcgtg 480

gatttagact gggaatatcc ggttagcgga ggactggacg gcaatagcaa acgcccggaa 540

gacaaacaaa actatacact gctgcttagc aagatccgcg agaagctgga tgctgctggt 600

gcggttgatg gcaaaaagta tctgcttaca attgcgagcg gcgcaagcgc aacatacgca 660

gcgaacacgg agcttgcaaa gattgcggcg atcgtggatt ggatcaacat catgacatat 720

gatttcaacg gcgcgtggca gaaaatcagc gcgcacaacg cacctcttaa ctatgatccc 780

gctgcgagcg cagcgggagt tcccgatgca aacacgttca acgtggcggc tggtgcgcaa 840

ggtcatcttg atgctggtgt gccggcggcg aaacttgttc tgggagtgcc gttctatggc 900

agaggctggg atggctgcgc acaagcggga aacggccagt accaaacatg cactggtggc 960

agctcagtgg gcacatggga ggcgggcagc tttgactttt atgatcttga agcaaattat 1020

atcaataaaa acggatacac acgctattgg aacgatacgg cgaaggttcc gtatttatac 1080

aacgcatcaa acaagagatt tatcagctat gacgatgcgg aatcagtggg ctataagaca 1140

gcgtacatta aaagcaaggg tttaggcgga gcgatgtttt gggaacttag cggcgaccgc 1200

aacaaaacac tgcagaataa gctgaaagcg gatcttccga cgggaggcac agtgcctccg 1260

gttgatacaa cggcgccttc agttccggga aatgcacgca gcacgggcgt gacagcgaat 1320

agcgtgacgc tggcgtggaa cgcaagcaca gacaatgttg gcgtgacggg ctacaacgtg 1380

tataatggcg cgaatttagc aacaagcgtt actggtacga cagcgacaat ctctggttta 1440

acggcgggaa cgtcatacac attcacgatt aaagcgaaag acgcagcggg caatttaagc 1500

gcggcatcaa atgcggtgac ggtgtcaaca acggcgcaac cgggcggaga tacgcaagca 1560

ccgacggcgc cgacaaatct ggcgagcaca gcacagacga caagcagcat tacgctgagc 1620

tggacagcaa gcacggataa tgtgggcgtt acgggctacg atgtgtacaa cggaacagcg 1680

cttgcgacaa cggtgactgg tacaacggcg acaatcagcg gactggcggc ggatacatca 1740

tatacgttca cggtgaaagc gaaggatgcg gcgggcaatg tgtcagcggc aagcaatgcg 1800

gtgagcgtga aaacagcagc ggaaacgaca aacccgggcg tgagcgcgtg gcaagttaat 1860

acggcgtata cagcgggcca gctggttacg tacaacggaa agacgtacaa atgtttacaa 1920

ccgcatacgt ctttagcggg atgggaaccg tcaaacgtgc cggcgctgtg gcagcttcaa 1980

catcaccatc atcatcat 1998

<210> 2

<211> 666

<212> PRT

<213> Bacillus circulans

<400> 2

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

1 5 10 15

Tyr Gly Arg Asn Tyr Asn Val Ala Asp Ile Asp Pro Thr Lys Val Thr

20 25 30

His Ile Asn Tyr Ala Phe Ala Asp Ile Cys Trp Asn Gly Ile His Gly

35 40 45

Asn Pro Asp Pro Ser Gly Pro Asn Pro Val Thr Trp Thr Cys Gln Asn

50 55 60

Glu Lys Ser Gln Thr Ile Asn Val Pro Asn Gly Thr Ile Val Leu Gly

65 70 75 80

Asp Pro Trp Ile Asp Thr Gly Lys Thr Phe Ala Gly Asp Thr Trp Asp

85 90 95

Gln Pro Ile Ala Gly Asn Ile Asn Gln Leu Asn Lys Leu Lys Gln Thr

100 105 110

Asn Pro Asn Leu Lys Thr Ile Ile Ser Val Gly Gly Trp Thr Trp Ser

115 120 125

Asn Arg Phe Ser Asp Val Ala Ala Thr Ala Ala Thr Arg Glu Val Phe

130 135 140

Ala Asn Ser Ala Val Asp Phe Leu Arg Lys Tyr Asn Phe Asp Gly Val

145 150 155 160

Asp Leu Asp Trp Glu Tyr Pro Val Ser Gly Gly Leu Asp Gly Asn Ser

165 170 175

Lys Arg Pro Glu Asp Lys Gln Asn Tyr Thr Leu Leu Leu Ser Lys Ile

180 185 190

Arg Glu Lys Leu Asp Ala Ala Gly Ala Val Asp Gly Lys Lys Tyr Leu

195 200 205

Leu Thr Ile Ala Ser Gly Ala Ser Ala Thr Tyr Ala Ala Asn Thr Glu

210 215 220

Leu Ala Lys Ile Ala Ala Ile Val Asp Trp Ile Asn Ile Met Thr Tyr

225 230 235 240

Asp Phe Asn Gly Ala Trp Gln Lys Ile Ser Ala His Asn Ala Pro Leu

245 250 255

Asn Tyr Asp Pro Ala Ala Ser Ala Ala Gly Val Pro Asp Ala Asn Thr

260 265 270

Phe Asn Val Ala Ala Gly Ala Gln Gly His Leu Asp Ala Gly Val Pro

275 280 285

Ala Ala Lys Leu Val Leu Gly Val Pro Phe Tyr Gly Arg Gly Trp Asp

290 295 300

Gly Cys Ala Gln Ala Gly Asn Gly Gln Tyr Gln Thr Cys Thr Gly Gly

305 310 315 320

Ser Ser Val Gly Thr Trp Glu Ala Gly Ser Phe Asp Phe Tyr Asp Leu

325 330 335

Glu Ala Asn Tyr Ile Asn Lys Asn Gly Tyr Thr Arg Tyr Trp Asn Asp

340 345 350

Thr Ala Lys Val Pro Tyr Leu Tyr Asn Ala Ser Asn Lys Arg Phe Ile

355 360 365

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

370 375 380

Ser Lys Gly Leu Gly Gly Ala Met Phe Trp Glu Leu Ser Gly Asp Arg

385 390 395 400

Asn Lys Thr Leu Gln Asn Lys Leu Lys Ala Asp Leu Pro Thr Gly Gly

405 410 415

Thr Val Pro Pro Val Asp Thr Thr Ala Pro Ser Val Pro Gly Asn Ala

420 425 430

Arg Ser Thr Gly Val Thr Ala Asn Ser Val Thr Leu Ala Trp Asn Ala

435 440 445

Ser Thr Asp Asn Val Gly Val Thr Gly Tyr Asn Val Tyr Asn Gly Ala

450 455 460

Asn Leu Ala Thr Ser Val Thr Gly Thr Thr Ala Thr Ile Ser Gly Leu

465 470 475 480

Thr Ala Gly Thr Ser Tyr Thr Phe Thr Ile Lys Ala Lys Asp Ala Ala

485 490 495

Gly Asn Leu Ser Ala Ala Ser Asn Ala Val Thr Val Ser Thr Thr Ala

500 505 510

Gln Pro Gly Gly Asp Thr Gln Ala Pro Thr Ala Pro Thr Asn Leu Ala

515 520 525

Ser Thr Ala Gln Thr Thr Ser Ser Ile Thr Leu Ser Trp Thr Ala Ser

530 535 540

Thr Asp Asn Val Gly Val Thr Gly Tyr Asp Val Tyr Asn Gly Thr Ala

545 550 555 560

Leu Ala Thr Thr Val Thr Gly Thr Thr Ala Thr Ile Ser Gly Leu Ala

565 570 575

Ala Asp Thr Ser Tyr Thr Phe Thr Val Lys Ala Lys Asp Ala Ala Gly

580 585 590

Asn Val Ser Ala Ala Ser Asn Ala Val Ser Val Lys Thr Ala Ala Glu

595 600 605

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

610 615 620

Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu Gln

625 630 635 640

Pro His Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro Ala Leu

645 650 655

Trp Gln Leu Gln His His His His His His

660 665

Claims (12)

1. A chitinase mutant, characterized in that the nucleic acid sequence of the chitinase mutant is gag mutated to gca at the 979-981 th position of the nucleic acid sequence shown in SEQ ID No. 1.

2. The chitinase mutant according to claim 1, wherein the nucleic acid sequence of the chitinase mutant has a mutation at the 28-30 th position of the nucleic acid sequence shown in SEQ ID No.1, such as the tat mutation is gca and the gag at the 979-981 th position, or the aga at the 901-903 th position is gca and the gag at the 979-981 th position is gca.

3. The chitinase mutant according to claim 2, wherein the nucleic acid sequence of the chitinase mutant has a mutation at the 28-30 th position of the nucleic acid sequence shown in SEQ ID No.1, such as a mutation at the 28-30 th position of gca, a mutation at the 901-903 th position of aga to gca and a mutation at the 979-981 th position of gag to gca.

4. A recombinant expression plasmid comprising an amino acid sequence or a nucleic acid sequence according to any one of claims 1 to 3.

5. The recombinant expression plasmid of claim 4, wherein the recombinant expression plasmid carries a RepB replicon.

6. The recombinant expression plasmid of claim 5, wherein the recombinant expression plasmid is pMATE.

7. The recombinant expression plasmid of claim 6, wherein the recombinant expression plasmid pMATE is free of a signal peptide.

8. A genetically engineered bacterium comprising the recombinant expression plasmid of claim 4.

9. The genetically engineered bacterium of claim 8, wherein the genetically engineered bacterium is Bacillus subtilis 1A 751.

10. Use of the chitinase mutant according to any one of claims 1 to 3 or the recombinant expression plasmid according to any one of claims 4 to 7 or the genetically engineered bacterium according to any one of claims 8 to 9 for digesting chitin-like substances.

11. Use of a mutant chitinase according to any one of claims 1 to 3 in combination with a monooxygenase for degrading chitin-like substances.

12. Use of a chitinase mutant according to claim 3 in combination with the monooxygenase BatLPMO10 from Bacillus atrophaeus in the hydrolysis of colloidal chitin.

Images