CN112831510A - Construction of a recombinant strain highly expressing chitinase and screening of mutants with high enzymatic activity - Google Patents

Abstract

Construction of a recombinant strain highly expressing chitinase and screening of mutants with high enzyme activity. The invention realizes the high-efficiency secretory expression of BcchiA1 in Bacillus subtilis by means of recombinant protein technology, and solves the problem of insufficient secretion of chitinase in most microorganisms. At the same time, a new high-throughput screening method for chitinase was created, which greatly improved the screening throughput and efficiency. In addition, the present invention relates to the disclosure of several high-enzyme activity chitinase mutants, BcchiA1(Y10A), BcchiA1(R301A), BcchiA1(E327A), BcchiA1(Y10A/R301A), BcchiA1(Y10A/E327A), BcchiA1(R301A) /E327A) and BcchiA1 (Y10A/R301A/E327A), the enzymatic activity was increased by 2.49 times, 0.67 times, 3.61 times, 2.39 times, 3.46 times, 4.75 times, and 16.89 times, respectively, compared with wild-type BcchiA1. Among them, BcchiA1 (Y10A/R301A/ E327A) has the best enzymatic activity. Finally, the synergistic effect of the chitinase BcchiA1 and its optimal mutant BcchiA1 (Y10A/R301A/E327A) and the monooxygenase BatLPMO10 from Bacillus atrophaeus, respectively, can make BcchiA1 and BcchiA1 (Y10A/R301A/E327A) The hydrolysis efficiencies of 50.00% and 46.71%, respectively.

Description

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

Technical Field

The invention relates to construction of a recombinant strain for efficiently secreting and expressing chitinase of bacillus circulans and high-throughput screening of chitinase mutants, belonging to the technical field of genetic engineering.

Background

Chitin, a linear high polymer linked by N-acetyl-D-glucosamine (NAG) through β -1,4 glycosidic bonds, is the most widely distributed aminopolysaccharide in nature, particularly as a structural component of arthropod exoskeletons and fungal cell walls. The chitin has the characteristics of good biocompatibility, broad-spectrum antibacterial property, no toxic or side effect and the like, so the chitin has wide application prospect in the biomedical, food, environmental protection and cosmetic industries. However, the high crystallinity and poor solubility of chitin are undoubtedly a serious challenge for further enhancing the application value.

Chitosan oligosaccharide, with a degree of polymerization below 20, is one of the major degradation products of chitin. The solubility is greatly improved compared with chitin, so the chitosan-chitosan composite has outstanding performances in the aspects of immunoregulation, antibiosis, tumor resistance, cell repair and the like. Currently, chitin oligosaccharides are mainly obtained by degrading chitin through three routes of chemical, physical and biological enzyme methods. The biological enzyme method is favored due to the advantages of environmental friendliness, mild reaction conditions, easy control of the degradation process, few byproducts and the like, and is mainly completed by the chitinase.

Chitinase has immeasurable application values in agriculture and environment, such as biological treatment of chitin waste, preparation of chitin oligosaccharides, application as a biological control agent, and separation of protoplasts from fungi and yeast for strain improvement. Chitinase is widely distributed in eukaryotes, prokaryotes, archaebacteria and viruses, and mainly catalyzes random breakage of beta-1, 4 glycosidic bonds to generate chitooligosaccharides with low polymerization degree and good water solubility. However, due to the high crystallinity of chitin, the chitinase cannot be sufficiently contacted with the substrate, resulting in low efficiency of the biological enzyme method. Therefore, we have focused on finding a strain excellent in chitinase activity. Among them, the Bacillus circulans chitinase BchIA 1 has high affinity and hydrolytic activity for insoluble chitin, and has great application value.

At present, many microorganisms are capable of secreting a certain amount of chitinase, but the problem of insufficient secretion is common. The recombinant protein technology is an important technical means for directly obtaining a large amount of high-purity chitinase. Compared with other exogenous gene expression systems, the microbial expression system has the characteristics of fast growth, short culture period, low cost and the like, and is widely applied to protein expression technologies, such as escherichia coli, bacillus subtilis, yeast expression systems and the like. Coli as the best-studied and rapidly-developed prokaryotic expression system has the disadvantages that most of proteins are expressed in a non-secretory mode, a large amount of intracellular aggregates are easy to form inclusion bodies, a large amount of intracellular impure proteins and the like, so that the subsequent separation and purification steps are complicated and time-consuming. The yeast expression system is used as a eukaryotic expression system with wider application, such as saccharomyces cerevisiae, pichia pastoris and the like, and is particularly suitable for expressing target proteins from lower eukaryotes, but the saccharomyces cerevisiae is not suitable for high-level expression of foreign proteins due to lack of a promoter with high transcription strength, and an inducer methanol of the pichia pastoris is easy to cause toxicity, so that the system has obvious defects.

The bacillus subtilis has no pathogenicity, does not contain exotoxin and endotoxin, is a safe strain (GRAS) widely applied to the pharmaceutical and food processing industries, has huge potential particularly in the aspects of producing and secreting active proteins such as enzyme and the like, and has simple and efficient subsequent separation and purification steps. In addition, the host has the characteristics of clear genetic background, simple molecular operation, short fermentation period and the like, and has obvious advantages in the field of applied microorganisms.

In order to solve the problems of insufficient secretion amount and low activity of wild-type chitinase, the invention further improves the enzyme activity on the basis of increasing the secretion amount of the bacillus circulans chitinase BchIA 1 so as to better meet the application requirement.

Disclosure of Invention

According to the invention, firstly, a bacillus subtilis recombinant strain capable of efficiently secreting bacillus circulans chitinase is constructed, and an efficient and simple high-throughput screening method is developed to screen a mutant library, so that a high-enzyme-activity mutant is obtained. In addition, monooxygenase and chitinase are cooperated to treat the chitin substrate, so that the enzymolysis efficiency of the chitinase is further improved.

The invention discloses a chitinase BchhiA 1 gene from Bacillus circulans WL-12, which is characterized in that the nucleic acid sequence is shown as SEQ ID NO.1 and the amino acid sequence is shown as SEQ ID NO.2 after being optimized according to codon preference.

In a second aspect, the present invention relates to the disclosure of a recombinant expression plasmid, pMATE-BcchiA1, which is characterized in that the recombinant expression plasmid is a shuttle-type plasmid of escherichia coli and bacillus subtilis carrying a RepB replicon, comprises the nucleic acid sequence of claim 1, and is free of a signal peptide.

The third aspect of the invention relates to and discloses three chitinase single-point mutants, which are characterized in that the amino acid sequence of the mutants is mutated into Ala from Tyr at the 10 th position, Ala at the 301 st position or Ala from Glu at the 327 th position of the amino acid sequence shown in SEQ ID No.2, and the specific activities of the chitinase single-point mutants are respectively improved by 2.49 times, 0.67 time and 3.61 times compared with the wild type Bchchia 1.

The fourth aspect of the invention relates to and discloses four single point mutation superposition-based chitinase mutants, which are characterized in that the amino acid sequences of the mutants are mutated into Ala from Tyr 10 and Ala from Arg 301, Ala from Tyr 10 and Glu 327, Ala from Arg 301 and Glu 327, Ala from Tyr 10 and Ala from Arg 301 and Glu 327, Ala from Arg 301 and Ala from Glu 327, respectively, and the enzyme specific activities are improved by 2.39 times, 3.46 times, 4.75 times and 16.89 times compared with the wild type BchiA 1.

The fifth aspect of the invention relates to and discloses a series of genetically engineered bacteria Bacillus subtilis 1A751, which is characterized by comprising the recombinant expression plasmid of any one of the first to fourth aspects of the invention, and the recombinant bacteria successfully realize the high-efficiency secretory expression of BchiA 1 without the action of a signal peptide.

The sixth aspect of the invention relates to and discloses a novel directed evolution method of chitinase, which comprises two parts of constructing a mutation library and high-throughput screening. The establishment of the mutation library is mainly completed by using error-prone PCR; the substrates used in high throughput screening are the staining agents Ramazol Brilliant Blue (RBB) and Colloidal Chitin (CC) formThe formed compound CC-RBB can degrade the substrate under the action of chitinase to release free RBB, and then the absorbance value is measured. The absorbance range measured in the method is OD₅₇₅-OD₆₁₀And the concentration range of CC-RBB is 2% -40%. Wherein the optimal choice is OD₅₉₅And 2% substrate concentration.

The seventh aspect of the invention relates to and discloses the chitinase, a series of mutants, coding genes, expression vectors and recombinant bacteria, and application of the chitinase, the mutants, the coding genes, the expression vectors and the recombinant bacteria in degrading chitin substances. Wherein, the chitinase BchiA 1 and the mutant BchiA 1(Y10A/R301A/E327A) thereof respectively cooperate with the monooxygenase BatLPMO10 derived from Bacillus atrophaeus to respectively improve the hydrolysis efficiency of BchiA 1 and BchiA 1(Y10A/R301A/E327A) by 50.00 percent and 46.71 percent respectively.

The invention has the beneficial effects that firstly, the protein recombination technology is utilized to realize the high-efficiency secretory expression of the chitinase of the bacillus circulans in the bacillus subtilis under the condition of no signal peptide. Usually, most of heterologous proteins need to be expressed by finding a signal peptide matched with the heterologous proteins so as to realize the massive secretion of the target protein in an expression host, but a signal peptide matched with all the target proteins cannot be found so far, namely the universality is lacked. However, the chitinase BchIA 1 can be secreted to the outside of the cell in a large amount without any signal peptide, so that the heavy task of screening the signal peptide is saved. Secondly, an efficient, economic and convenient chitinase high-throughput screening method is developed, so that the screening throughput and efficiency are greatly improved, and good technical reference is provided for the subsequent chitinase high-throughput screening. Moreover, a series of chitinase mutants with high enzyme activity are screened, and the optimum BchIA 1(Y10A/R301A/E327A) enzyme activity is improved by 16.89 times compared with the wild type chitinase, so that the chitinase has considerable industrial application value. Finally, the efficiency of chitinase hydrolysis was further enhanced by virtue of the synergistic effect of BatLPMO10 on BchhiA 1 and its mutant BchhiA 1 (Y10A/R301A/E327A).

Drawings

FIG. 1 is a SDS-PAGE result of secretory expression of chitinase BchIA 1 and its mutant in Bacillus subtilis. M: protein marker; 1: an extracellular sample that is wild-type BcchiA 1; 2 is an extracellular sample of mutant BcchiA1 (Y10A); 3 is an extracellular sample of mutant BcchiA1 (R301A); 4 is an extracellular sample of mutant BcchiA1 (E327A); 5 is an extracellular sample of mutant BchiA 1 (Y10A/R301A); 6 is an extracellular sample of mutant BchiA 1 (Y10A/E327A); 7 is an extracellular sample of mutant BchiA 1 (R301A/E327A); the sample 8 is an extracellular sample of mutant BchiA 1 (Y10A/R301A/E327A).

FIG. 2 is a SDS-PAGE pectin binding pattern of purified chitinase and the monooxygenase BatLPMO 10. A is a glue picture of seven purified chitinase mutants; b is a gel diagram of the purified wild-type chitinase BchiA 1 and the monooxygenase BatLPMO 10.

FIG. 3 shows the measured values of the enzyme activities of the wild type BchiA 1 and its mutants.

FIG. 4 is a schematic representation of the synergy of chitinase BchhiA 1 and BatLPMO 10. The abscissa is OD₅₉₅The ordinate is the combination of different concentrations of BatLPMO10 with a certain amount of BcchiA 1.

FIG. 5 is a schematic representation of the synergy of chitinase mutant BchhiA 1(Y10A/R301A/E327A) and BatLPMO 10. The abscissa is OD₅₉₅The ordinate is the combination of different concentrations of BatLPMO10 with a certain amount of Bchia 1 (Y10A/R301A/E327A).

Detailed Description

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

The experimental procedures in the following examples, unless otherwise mentioned, are conventional procedures for molecular manipulations.

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

The quantitative tests in the following examples, all set up in triplicate, and the results averaged.

EXAMPLE 1 construction of pMATE-Bcchia1 recombinant plasmid and recombinant bacterium

Firstly, constructing pMATE-Bcchia1 recombinant plasmid

The chitinase BchhiA 1 from Bacillus circulans WL-12 has strong affinity and catalytic activity for highly insoluble chitin.The gene is artificially synthesized by Suzhou Jinwei Zhi Biotechnology Limited company after being entrusted to optimization according to codon preference, a His label is added at the C end for purifying protein, the nucleic acid sequence of the His label is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, and the gene is cloned to a vector pUC57-Amp after being synthesized, so that a recombinant plasmid pUC 57-BchhiA 1 is obtained. The recombinant plasmid pUC 57-BchiA 1 was used as a template, and a primer pair consisting of the primer FragmentF and the primer FragmentR was used for PCR amplification to obtain a BchiA 1 fragment of about 1.9 kb. The vector backbone was amplified from the plasmid by primers vectorF and vectorR using the pMATE plasmid as a vector template and homology arms of about 20bp were added to give an approximately 7.4kb plasmid backbone pMATE. Purifying PCR products by using a gel recovery kit, and passing the PCR products through the kit

And (3) completing the seamless connection of the fragment and the vector, transferring the fragment into escherichia coli DH5 alpha competent cells, and completing the construction of a recombinant plasmid pMATE-Bcchia1 after sequencing.

TABLE 1

Secondly, constructing recombinant bacteria containing pMATE-Bcchia1 recombinant plasmid

And (3) transferring the recombinant expression plasmid pMATE-BchchiA 1 obtained in the step into B.subtilis 1A751, and completing construction after the colony is verified to be correct.

Example 2 development of a novel method for directed evolution of chitinase

Construction of chitinase mutant library

The wild type BchiA 1 nucleic acid sequence shown in SEQ ID NO.1 is used as a template, a TIANZ adjustable error-prone PCR kit is used, primers Fragment-F and Fragment-R are used for amplifying a target gene, and random mutation is introduced into a BchiA 1 gene (1.9 kb). The error-prone PCR reaction system is shown in Table 2. The error-prone PCR reaction procedure was: 94 ℃ for 3 min; 94 ℃ for 1min, 45 ℃ for 1min, 72 ℃ for 2min, 50 cycles.

TABLE 2

Composition (I)	Volume (μ L)
		Error prone PCRMix, 10 ×	3.0
Error-prone PCR dedicated dNTP,10	3.0
		Error-prone special MnCl for PCR2	1.0
Error-prone PCR special dGTP	0
		Bchchia 1 template (5 ng/. mu.L)	3.0
Fragment-F/R(10Meach)	1.0
		Error-prone PCR special TaqDNA polymerase	0.5
Ultrapure water	17.5

PCR products into which random mutations had been introduced were extended with the pMATE vector fragment described above by PrimeSTAR polymerase to achieve overlap of the two templates to form DNA multimers. Finally, DNA multimers (POE-PCR products) were transformed into B.subtilis 1A751 strain and screened on LB solid medium containing 20. mu.g/ml kanamycin, thereby completing the construction of a mutant library.

High throughput screening of chitinase II

The obtained mutants were individually selected to 96-well deep-well plates containing 50. mu.g/ml kanamycin and 500. mu.l LB liquid medium in each well, and were cultured overnight at 37 ℃ with shaking at 800rpm, and then used as seeds for high-throughput screening. Seeds were added to a 96-well deep-well plate corresponding to the high-throughput screen containing 500. mu.l of LB medium, 20. mu.g/ml kanamycin, 2% (w/v) CC-RBB and 1% maltose per well. Subsequently, the cells were cultured in a high-throughput well plate shaking culture system at 37 ℃ and 800 rpm. After 24h incubation, the plates were centrifuged at 6000rpm for 30min at 4 ℃. After the centrifugation is finished, transferring the centrifuged supernatant of the 96-hole deep-hole plate to a 96-hole enzyme label plate by means of a full-automatic high-throughput analysis and screening workstation, and determining OD₅₉₅。

Example 3 further site-directed mutagenesis of high enzyme activity Single mutant sites

One, the single-point mutant obtains two-point superposition mutant by site-directed mutagenesis

1. Obtaining of mutant BchchiA 1 (Y10A/R301A): using single-site mutant BchiA 1(Y10A) as a template, PCR amplification was carried out using a primer set composed of primers R301A-F and R301A-R shown in Table 3, to obtain a BchiA 1(Y10A/R301A) fragment of about 1.9 kb. Purifying with gel recovery kit, and passing through kit

The fragment was seamlessly ligated to the vector backbone pMATE of example 1, transformed into E.coli DH 5. alpha. competent cells, and sequenced to complete the construction of the recombinant plasmid pMATE-Bcchia1 (Y10A/R301A).

2. Obtaining of mutant BchchiA 1 (Y10A/E327A): using single-site mutant BchiA 1(Y10A) as a template, PCR amplification was carried out using a primer set composed of primers E327A-F and E327A-R shown in Table 3, to obtain a BchiA 1(Y10A/E327A) fragment of about 1.9 kb. See example 3, item 1 for the rest of the procedure.

3. Obtaining of mutant BchchiA 1 (R301A/E327A): using single-site mutant BchiA 1(R301A) as a template, PCR amplification was carried out using a primer pair consisting of primer E327A-F and primer E327A-R shown in Table 3, to obtain a BchiA 1(R301A/E327A) fragment of about 1.9 kb. See example 3, item 1 for the rest of the procedure.

Secondly, obtaining three-point superposition mutant from two-point mutant by site-directed mutagenesis

Using a two-site mutant BchhiA 1(Y10A/R301A) as a template, PCR amplification was performed using a primer pair consisting of a primer E327A-F and a primer E327A-R shown in Table 3, to obtain a BchhiA 1(Y10A/R301A/E327A) fragment of about 1.9 kb. See example 3, item 1 for the rest of the procedure.

In conclusion, mutants BchchiA 1(Y10A/R301A), BchchiA 1(Y10A/E327A), BchchiA 1(R301A/E327A) and BchchiA 1(Y10A/R301A/E327A) are obtained in sequence, namely the amino acid sequence of the mutants is mutated into Ala at the position 10 Tyr and Ala at the position 301 Arg or Ala at the position 10 Tyr and Ala at the position 327 Glu, or Ala at the position 301 and Ala at the position 327 Arg or Ala at the position 10 Tyr and Ala at the position 301 Arg and Ala at the position 301 and Glu at the position 327 of Ala in the amino acid sequence shown in SEQ ID No. 2. After sequencing verification, the recombinant plasmid is successfully constructed. And (3) respectively transferring the recombinant expression plasmids containing the chitinase mutant obtained in the step into B.subtilis 1A751, and completing construction after the colony verification is correct.

TABLE 3

Primer name	Sequence (5 '-3')	Use of
			Y10A-R	TCGGATAtgcGCCCACGATCTTATATGAATCTGCCA	Mutation of Tyr 10 to Ala
Y10A-F	catataAGATCGTGGGCgcatatccgagctgggcagcatac	Mutation of Tyr 10 to Ala
			R301A-R	CCATCCCAGCCtgcGCCATAGAACGGCACTCC	Mutation of Arg at position 301 to Ala
R301A-F	TTCTATGGCgcaGGCTGGGATGGCTGC	Mutation of Arg at position 301 to Ala
			E327A-R	CTGCCCGCtgcCCATGTGCCCACTGAGCT	Mutation of Glu 327 to Ala
E327A-F	AGTGGGCACATGGgcaGCGGGCAGCTTTGACTTTT	Mutation of Glu 327 to Ala

Example 4 evaluation of wild-type chitinase and its mutants

First, shake flask fermentation of B.subtilis 1A751 strain containing Bchchia 1 wild type or mutant recombinant plasmid

Single colonies were picked and seeds were activated in 5ml LB liquid medium containing 20. mu.g/ml kanamycin, 37 ℃ with shaking at 200rpmCulturing for 14-16h, inoculating into 2 × SR fermentation medium according to the inoculum size of 1%, and culturing medium components (g/L): 50 parts of yeast powder, 30 parts of peptone and K parts₂HPO₄6. Furthermore, 50. mu.g/ml kanamycin and 3% maltose were added to the fermentation medium. After fermentation for 48h, centrifuging at 14000g for 10min at 4 ℃ and collecting the supernatant, namely the extracellular component sample. 80 μ l of extracellular supernatant sample was mixed with 20 μ l of loading buffer and then subjected to boiling water bath for 20 min. Subsequently, SDS-PAGE detection was performed using NuPAGE 10% Bis-Tris protein pre-gel, and the results are shown in FIG. 1.

Secondly, purifying chitinase

The chitinase purification process is completed at 4 ℃. Centrifuging the 48h fermentation liquid at 10000rpm for 10min, taking the supernatant, and filtering with a 0.22 mu m filter membrane. Bind with Ni NTA Beads 6FF for 2 hours before loading on the column (1.5X 8 cm). The impurities and weakly bound proteins were eluted with a gradient of buffer (50mm Tris-HCl, 500mm NaCl,50-300mm imidazole) followed by elution of the protein of interest with buffer (50mm Tris-HCl, 500mm imidazole and 500mm NaCl). The BCA protein quantification kit measures the purified protein concentration and the purity of the chitinase is checked using SDS-PAGE, and the results are shown in FIG. 2.

Determination of chitinase Activity

The activity of the chitinase is measured by spectrophotometry, and 1 unit of the chitinase is defined as: the amount of enzyme required for an increase in absorbance of 0.01 per hour under the reaction conditions of 50 ℃ was one activity unit (U).

1. Preparing colloidal chitin: 10g chitin is weighed, 100ml 85% phosphoric acid is added, reaction is carried out for 24h at 4 ℃, and water is added for repeated washing until the chitin is neutral. The white colloidal substance formed at this time maintains a humidity of 90-95%;

2. binding of colloidal chitin to stain RBB: fully mixing 25g of the colloidal chitin with 100ml of 0.84% RBB solution, carrying out boiling water bath on the obtained suspension for 60min, filtering, removing filtrate, suspending the dyed colloidal substance in 25ml of 1.5% sodium dichromate and 1.5% potassium sodium tartrate solution for fixation, carrying out boiling water bath for 10min, and filtering to finally obtain a blue colloidal substrate CC-RBB;

3. and (3) measuring enzyme activity: 1ml of purified Bchia 1 was taken and 1ml of 40% (w)V) CC-RBB colloidal solution, incubating at 50 deg.C for 1h, centrifuging at 12000rpm for 5min, and determining OD₅₉₅Inactivating in boiling water bath for 10min as negative control;

4. protein concentration determination: the assay was performed with BCA protein quantification kit.

And finally, taking supernatant of 48h fermentation liquor of the wild type chitinase and the mutant as crude enzyme liquid, purifying target protein, and further determining enzyme activity, wherein the result is shown in figure 3.

Comprehensively, the enzyme specific activity of the wild type chitinase BchiA 1 is 56.16 +/-0.92U/mg; the enzyme specific activities of the single-point mutant strains BchchiA 1(Y10A), BchchiA 1(R301A) and BchchiA 1(E327A) are respectively improved by 2.49 times, 0.67 time and 3.61 times compared with that of the wild type BchchiA 1; the enzyme activities of two-point superimposed mutant strains BchchiA 1(Y10A/R301A), BchchiA 1(Y10A/E327A) and BchchiA 1(R301A/E327A) are respectively improved by 2.39 times, 3.46 times and 4.75 times compared with that of wild type BchchiA 1; the enzyme activity of the three-point superposition mutant strain BchiA 1(Y10A/R301A/E327A) is the highest and is 1004.83 +/-0.87U/mg, which is 16.89 times higher than that of the wild type.

Example 5 synergistic Effect of chitinase and the monooxygenase BatLPMO10

Mu.l of different concentrations of the monooxygenase BatLPMO10(0,9,18,36,72,144,288nmol) were mixed with 1000. mu.l of 40% CC-RBB solution, 1mM ascorbic acid was added, and 7 groups were reacted for 1h at 200rpm in a shaker at 50 ℃. Subsequently, 500. mu.l of 9nmol of purified BchhiA 1 was added to each of the 7 groups, and the reaction was incubated for 1 hour under the enzyme activity measurement conditions described above. Finally, the reaction mixture was centrifuged at 12,000rpm for 5min, and the OD was measured₅₉₅. All reactions were repeated three times.

The chitinase mutant BchhiA 1(Y10A/R301A/E327A) with the most obvious activity improvement is taken as a research object, and the steps are repeated.

To determine the synergistic effect of BatLPMO10 on chitinase, the chitin substrate was pretreated by adding BatLPMO10 at different concentrations while keeping the addition amounts of BchIA 1 and BchIA 1(Y10A/R301A/E327A) constant. The results show that the addition amounts and OD of the two groups of BatLPMO10₅₉₅Shows obvious positive correlation, which shows that the BatLPMO10 has obvious promotion effect on the pretreatment of the substrate, so thatBoth BchhiA 1 and BchhiA 1(Y10A/R301A/E327A) reacted more fully with chitin, up to an OD of₅₉₅The results are shown in fig. 4 and 5, which are respectively increased by 50.00% and 46.71% compared with the experimental group using chitinase alone.

Sequence listing

<110> research on Tianjin Industrial biotechnology of Chinese academy of sciences

<120> construction of recombinant bacterium for efficiently expressing chitinase and screening of high-enzyme-activity mutant

<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 (10)

1. A chitinase encoding Bacillus circulans characterized in that the nucleic acid sequence optimized according to codon preference is shown in SEQ ID NO. 1.

2. The chitinase according to claim 1, characterized in that the amino acid sequence is as shown in SEQ ID No. 2.

3. The chitinase mutant is characterized in that the amino acid sequence of the mutant is mutated into Ala from Tyr at the 10 th position of the amino acid sequence shown in SEQ ID No.2, or mutated into Ala from Arg at the 301 th position, or mutated into Ala from Glu at the 327 th position;

in another preferred embodiment, the amino acid sequence of the mutant is mutated from Tyr at position 10 to Ala and Arg at position 301 to Ala, or from Tyr at position 10 to Ala and Glu at position 327 to Ala, or from Arg at position 301 to Ala and Glu at position 327 to Ala, in the amino acid sequence shown in SEQ ID No. 2;

in another preferred embodiment, the amino acid sequence of the mutant is mutated from Tyr to Ala at position 10, Arg to Ala at position 301 and Glu to Ala at position 327 of the amino acid sequence shown in SEQ ID No. 2.

4. The chitinase mutant according to claim 3, characterized in that the mutant has a nucleic acid sequence in which the tat mutation at position 28-30 of the nucleic acid sequence shown in SEQ ID No.1 is gca, or the aga mutation at position 901-903 is gca, or the gag mutation at position 979-981 is gca;

in another preferred embodiment, the tat mutation at positions 28-30 of the nucleic acid sequence shown in SEQ ID No.1 of the mutant is gca and the aga at positions 901-903 is gca, or the tat mutation at positions 28-30 is gca and the gag at positions 979-981 is gca, or the aga at positions 901-903 is gca and the gag at positions 979-981 is gca;

in another preferred embodiment, the tat mutation at position 28-30 of the nucleic acid sequence shown in SEQ ID No.1 of the mutant nucleic acid sequence is gca, the aga mutation at position 901-903 is gca and the gag mutation at position 979-981 is gca.

5. A recombinant expression plasmid comprising the nucleic acid sequence or amino acid sequence of any one of claims 1 to 4.

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

preferably, the plasmid is pMATE;

more preferably, said plasmid pMATE is free of a signal peptide.

7. A genetically engineered bacterium comprising the recombinant expression plasmid of claim 5;

preferably, the genetically engineered bacterium is Bacillus subtilis 1A 751.

8. An efficient and rapid directional evolution method of chitinase comprises the construction of a mutation library and high-throughput screening:

1) constructing a mutation library: the method comprises the steps of taking a nucleic acid sequence SEQ ID NO.1 of BchhiA 1 as a template, introducing mutation through an adjustable error-prone PCR kit, connecting the mutation with a plasmid vector skeleton, and finally transferring the mutation into bacillus subtilis;

2) high-throughput screening: selecting a substrate as a compound CC-RBB formed by a coloring agent Ramazol Brilliant Blue (RBB) and Colloidal Chitin (CC), selecting monoclonals in the mutation library one by one into a 96-hole deep-hole plate containing a seed culture medium, carrying out shake culture overnight, then transferring into a high-throughput screening culture medium containing the substrate CC-RBB, carrying out culture overnight, centrifuging, taking a supernatant, and measuring OD.

Preferably, the concentration of the substrate CC-RBB is 2% -40%, and the measured light absorption value range OD₅₇₅-OD₆₁₀；

More preferably, the substrate CC-RBB concentration is 20%, and the absorbance range OD is determined₅₉₅。

9. The directed evolution method of claim 8, wherein the seed culture medium for high throughput screening is: 500. mu.l of LB liquid medium containing 50. mu.g/ml kanamycin; the screening culture medium is as follows: 500. mu.l of LB medium containing 20. mu.g/ml kanamycin, 2% (w/v) CC-RBB and 1% maltose.

10. The use of chitinase according to any one of claims 1 to 4 or recombinant expression plasmid according to any one of claims 5 to 6 or genetically engineered bacteria according to claim 7 for enzymatic hydrolysis of chitin-like substances;

preferably, the chitinase and monooxygenase act synergistically in degrading chitin-like substances;

more preferably, the chitinase interacts synergistically with the monooxygenase BatLPMO10 from Bacillus atrophaeus at the presence of hydrocolloid chitin.

Images