A chitinase BlChiA mutant with improved specific activity and acid resistance

Abstract

The present invention discloses a chitinase BlChiA mutant with improved specific activity and acid resistance, belonging to the field of enzyme engineering. In the present invention, Bacillus subtilis is used as an expression host, the chitinase gene BlChiA derived from Bacillus licheniformis is recombinantly expressed and site-directed mutagenesis is carried out to obtain a series of dominant mutants. Mutant E469G has the highest enzyme activity under the condition of pH 4.0, is more adaptable to acidic environment compared with the wild type, and is suitable for production applications; mutant E336K / E469G / A472T improves the catalytic efficiency of chitinase BlChiA, and the specific activity is increased by 1.56 times compared with the wild type, which is beneficial to reducing its production cost; mutant D26N / E336K / E469G / A472T has enhanced tolerance to acidic conditions and has a wide pH tolerance range from pH 3.0 to 10.0, which is beneficial to industrial production and the degradation of chitin.

Description

Technical Field

[0001] The present invention relates to a chitinase BlChiA mutant with improved specific activity and acid resistance, belonging to the technical field of enzyme engineering. Background Art

[0002] Chitin, also known as chitosan, is the second most abundant natural high - molecular polysaccharide in nature after cellulose. It is widely present in the shells of crustaceans such as shrimps, crabs, insects and the exoskeletons of arthropods, and is a basic component of the fungal cell wall. Due to the stable nature of naturally occurring chitin, it is insoluble in water, dilute acids and alkalis, and only soluble in strong inorganic acids, so it is difficult to be efficiently utilized. It is estimated that the amount of chitin biosynthesized in nature every year can reach 10 10 -10 11 tons. Its degradation product, chito - oligosaccharide, has better water solubility, can be chemically modified in various ways, has a variety of physiological functions, and has excellent application value in the fields of agriculture, food, medicine, etc.

[0003] Chitinase (EC 3.2.1.14) can catalyze the hydrolysis of β - 1,4 - glycosidic bonds in N - acetylglucosamine (GlcNAc) polymers. According to the position of action on the substrate, chitinases can be divided into β - N - acetylglucosaminidase, endochitinase and exochitinase. Among them, β - N - acetylglucosaminidase can cleave the glycosidic bond from the non - reducing end to release GlcNAc monomers; endochitinase can randomly cleave glycosidic bonds in the polymer to generate oligosaccharides with different degrees of polymerization; exochitinase can cleave glycosidic bonds at the end of the polymer in units of dimers to generate chitobiose.

[0004] Since chitin has high solubility under strong acid conditions, chitinases with strong acid resistance have great advantages in the process of degrading chitin to produce chito - oligosaccharides. At present, chitinases from Bacillus licheniformis have been expressed in Bacillus subtilis, but their enzyme activities, catalytic efficiencies and acid resistances are relatively low, unable to meet the industrial needs of degrading chitin. There is an urgent need for a chitinase with high specific activity and strong acid resistance. Summary of the Invention

[0005] The present invention modifies the chitinase BlChiA derived from Bacillus licheniformis and highly expresses it in Bacillus subtilis to improve its specific activity and pH stability, which is beneficial to reducing costs and applying to industrial production.

[0006] To achieve the above object, the present invention provides a chitinase BlChiA mutant, which is obtained by site-directed mutagenesis of wild-type chitinase BlChiA. The nucleotide sequence encoding the wild-type chitinase is shown in SEQ ID NO.1, and the amino acid sequence of the wild-type chitinase is shown in SED ID NO.2. The site-directed mutagenesis is one or more of the 26th, 323rd, 336th, 469th, and 472nd positions in the amino acid sequence of chitinase BlChiA.

[0007] The present invention provides a series of chitinase mutants, which are mutated as follows on the basis of wild-type chitinase BlChiA:

[0008] The aspartic acid residue at the 26th position of chitinase with the amino acid sequence shown in SEQ ID NO.2 is mutated to an asparagine residue and named D26N;

[0009] The glycine residue at the 323rd position of chitinase with the amino acid sequence shown in SEQ ID NO.2 is mutated to an aspartic acid residue and named G323D;

[0010] The glutamic acid residue at the 336th position of chitinase with the amino acid sequence shown in SEQ ID NO.2 is mutated to a lysine residue and named E336K;

[0011] The glutamic acid residue at the 469th position of chitinase with the amino acid sequence shown in SEQ ID NO.2 is mutated to a glycine residue and named E469G;

[0012] The alanine residue at the 472nd position of chitinase with the amino acid sequence shown in SEQ ID NO.2 is mutated to a threonine residue and named A472T;

[0013] The glutamic acid residue at the 469th position of chitinase with the amino acid sequence shown in SEQ ID NO.2 is mutated to a glycine residue and named E469G, and the alanine residue at the 472nd position is mutated to a threonine residue and named E469G / A472T.

[0014] The aspartic acid residue at the 26th position of chitinase with the amino acid sequence shown in SEQ ID NO.2 is mutated to an asparagine residue, the glutamic acid residue at the 469th position is mutated to a glycine residue, and the alanine residue at the 472nd position is mutated to a threonine residue, and named D26N / E469G / A472T.

[0015] The glycine residue at position 323 of the chitinase with the amino acid sequence shown in SEQ ID NO.2 was mutated to an aspartic acid residue, the glutamic acid residue at position 469 was mutated to a glycine residue, and the alanine residue at position 472 was mutated to a threonine residue, and it was named G323D / E469G / A472T.

[0016] The glutamic acid residue at position 336 of the chitinase with the amino acid sequence shown in SEQ ID NO.2 was mutated to a lysine residue, the glutamic acid residue at position 469 was mutated to a glycine residue, and the alanine residue at position 472 was mutated to a threonine, and it was named E336K / E469G / A472T.

[0017] The aspartic acid residue at position 26 of the chitinase with the amino acid sequence shown in SEQ ID NO.2 was mutated to an asparagine residue, the glutamic acid residue at position 336 was mutated to a lysine residue, the glutamic acid residue at position 469 was mutated to a glycine residue, and the alanine residue at position 472 was mutated to a threonine residue, and it was named D26N / E336K / E469G / A472T.

[0018] The aspartic acid residue at position 26 of the chitinase with the amino acid sequence shown in SEQ ID NO.2 was mutated to an asparagine residue, the glycine residue at position 323 was mutated to an aspartic acid residue, the glutamic acid residue at position 336 was mutated to a lysine residue, the glutamic acid residue at position 469 was mutated to a glycine residue, and the alanine residue at position 472 was mutated to a threonine residue, and it was named D26N / G323D / E336K / E469G / A472T.

[0019] The present invention also provides a gene encoding the above chitinase mutant.

[0020] The present invention also provides a recombinant vector carrying the above gene.

[0021] In one embodiment of the present invention, the vector is a pHY300 series vector or a pHY300PLK series vector.

[0022] In one embodiment of the present invention, a promoter SpoVG and a signal peptide YqxI are connected to the recombinant vector, the nucleotide sequence of the promoter SpoVG is shown in SEQ ID NO.3, and the nucleotide sequence encoding the signal peptide YqxI is shown in SEQ ID NO.4.

[0023] The present invention also provides a microbial cell carrying the above chitinase mutant gene and the above expression vector.

[0024] In one embodiment of the present invention, the microbial cell is a recombinant prokaryotic cell or eukaryotic cell; the prokaryotic cell is a Gram-negative bacterium or Gram-positive bacterium.

[0025] In one embodiment of the present invention, the host bacterium is Bacillus subtilis WS9, and the construction method of the host bacterium Bacillus subtilis WS9 is disclosed in "Zhang K, Su L, Wu J. Enhanced extracellular pullulanase production in Bacillus subtilis using protease-deficient strains and optimal feeding. Appl Microbiol Biotechnol. 2018 Jun; 102(12):5089-5103".

[0026] In one embodiment of the present invention, the construction method of the microbial cell carrying the above chitinase mutant gene or the above expression vector is: transferring the recombinant expression vector carrying the gene encoding the mutant into the host cell by electroporation or chemical transformation.

[0027] In one example of the present invention, the control strain used is WS9-pHY300PLK-P HpaⅡ -P amyQ -SP AmyE -BlChiA, with P of the nucleotide sequence shown in SEQ ID NO.5 HpaⅡ -P amyQ as the promoter, and AmyE encoded by the nucleotide sequence shown in SEQ ID NO.6 as the signal peptide, recombinantly expressing the chitinase gene BlChiA from Bacillus licheniformis (the chitinase gene with NCBI number WP_016886405 was codon-optimized and synthesized by Shanghai Jierui Bio-Engineering Co., Ltd.).

[0028] In one embodiment of the present invention, the enzyme activity of the mutant is determined by the DNS colorimetric method.

[0029] The present invention also provides a preparation method of the above chitinase mutant, and the method is: inoculating the microbial cell carrying the above gene or the above expression vector into a fermentation medium for fermentation, after the fermentation is completed, collecting the fermentation broth obtained by fermentation for centrifugation, and after the centrifugation is completed, separating and purifying the chitinase mutant from the fermentation supernatant obtained by centrifugation.

[0030] In one embodiment of the present invention, the optimal reaction pH and pH stability of mutants E469G, E336K / E469G / A472T, and D26N / E336K / A472T / E469G were characterized.

[0031] The present invention also provides the application of the above chitinase mutants, or the above genes, or the above expression vectors, or the above microbial cells, or the above preparation methods in at least one of aspects (a) to (c):

[0032] (a) Hydrolyzing chitin;

[0033] (b) Improving the catalytic efficiency of chitinase;

[0034] (c) Improving the conversion rate of chitinase to hydrolyze colloidal chitin.

[0035] Beneficial effects

[0036] (1) The present invention provides the following chitinase mutants: E469G, E336K / E469G / A472T, and D26N / E336K / E469G / A472T.

[0037] The optimal reaction pH of mutant E469G is 4.0. Compared with the wild type, its enzyme activity is higher under acidic conditions; its stability is significantly better than that of the wild type in the range of pH 3.0 - pH 10.0, and the residual enzyme activity can reach more than 85%. It has a wide pH tolerance range, and its specific activity is 1.07 times that of the wild type, which is beneficial to the production and application of this mutant.

[0038] The optimal pH of mutant E336K / E469G / A472T is 5.0. Compared with the wild type, this mutant has better acid tolerance and can maintain more than 75% of its activity in the range of pH 3.0 - pH 5.0; the stability of the mutant is better in the range of pH 3.0 - pH 9.0, and the residual enzyme activity reaches more than 85%, and its acid tolerance is significantly improved. Its specific activity is 1.56 times that of the wild type.

[0039] Mutant D26N / E336K / E469G / A472T can maintain more than 80% of its activity in the range of pH 3.0 - pH 8.0 and has a wide pH reaction range; the stability of the mutant is better in the range of pH 3.0 - pH 10.0, and the residual enzyme activity reaches more than 80%. It has a wider pH tolerance range, and its specific activity is 1.40 times that of the wild type, which is beneficial to the production and application of this mutant.

[0040] (2) The present invention provides a recombinant Bacillus subtilis expressing the above-mentioned chitinase BlChiA mutant, and the enzyme activity of the expressed mutant is increased to varying degrees compared with the wild type. Among them, the enzyme activity of mutant E469G is 1.21 times that of the wild type; the enzyme activity of mutant E336K / E469G / A472T is 1.90 times that of the wild type; the enzyme activity of D26N / E336K / E469G / A472T is the highest, which is 2.16 times that of the wild type. Description of the Drawings

[0041] Figure 1 is the pHY300PLK-P HpaⅡ-amyQ -SP AmyE -BlChiA plasmid map;

[0042] Figure 2 is the graph of the optimal reaction pH and pH stability of chitinase mutant E469G;

[0043] Figure 3 is the graph of the optimal reaction pH and pH stability of chitinase mutant E336K / E469G / A472T;

[0044] Figure 4 is the graph of the optimal reaction pH and pH stability of chitinase mutant D26N / E336K / E469G / A472T. Detailed Embodiments

[0045] The present invention discloses a chitinase mutant, a preparation method and application thereof, a DNA molecule encoding the chitinase mutant, a vector, and a host cell. It involves conventional techniques and methods in the fields of molecular biology and enzyme engineering.

[0046] The experimental materials and reagents involved in the following examples are as follows:

[0047] 1. Strains and Vectors

[0048] The Bacillus subtilis WS9, Bacillus subtilis WB168, and plasmid pHY300PLK used in the present invention are all commercial hosts or vectors.

[0049] Bacillus subtilis WS9 is based on Bacillus subtilis WS5 (which has been disclosed in Patent CN106754466, deposit number CCTCC M 2016536), and nprB, bpr, mpr, and epr have been further knocked out. The specific construction method is disclosed in "Zhang K, Su L, Wu J. Enhanced extracellular pullulanase production in Bacillus subtilis using protease - deficient strains and optimal feeding. Appl Microbiol Biotechnol. 2018 Jun;102(12):5089 - 5103".

[0050] 2. Enzymes and Kits

[0051] The 2×Phanta Max Master Mix high - fidelity DNA polymerase and the seamless cloning kit ClonExpress II One Step Cloning Kit were purchased from Novoprotein Scientific Inc., the protein concentration kit was purchased from Beyotime Biotechnology Co., Ltd., DpnⅠ was purchased from NEB Inc., the restriction endonucleases were purchased from Takara Company, and the plasmid extraction, gel recovery, and purification kits were purchased from Tiangen Biochemical Technology Co., Ltd.

[0052] The media involved in the following examples are as follows:

[0053] LB liquid medium: Tryptone 10 g·L -1 , Yeast extract 5 g·L -1 , Sodium chloride 10 g·L -1 ,

[0054] LB solid medium: Tryptone 10 g·L -1 , Yeast extract 5 g·L -1 , Sodium chloride 10 g·L -1 , Agar powder 20 g·L -1

[0055] TB fermentation medium: Tryptone 12 g·L -1 , Yeast extract 24 g·L -1 , Glycerol 5 g·L -1 , KH 2 PO 4 2.31 g·L -1 , K 2 HPO 4 ·3H 2 O 16.43 g·L -1 .

[0056] The detection methods involved in the following examples are as follows:

[0057] Enzyme activity assay method:

[0058] The DNS method was used to determine the total reducing sugar content. Using 1% (w / v) colloidal chitin substrate, 250 μL of colloidal chitin and 150 μL of citrate phosphate buffer (50 mmol·L -1 ) were preheated at the reaction temperature for 10 min, 100 μL of appropriately diluted fermentation supernatant / pure enzyme solution was added and mixed evenly, reacted in a water bath at 60 °C for 1 h, 2 mL of DNS was added and mixed evenly to terminate the reaction, boiled in boiling water for 10 min, and immediately cooled to room temperature; adding an equal amount of inactivated enzyme solution as a blank control. Centrifuged at 12000 r·min -1 for 5 min, and the absorbance of the supernatant was measured at 540 nm. Enzyme activity definition: The amount of enzyme required to release 1 μmol of GlcNAc per minute at 60 °C is defined as one enzyme activity unit (1 U).

[0059] Protein concentration assay method:

[0060] The protein standard curve was prepared and the protein concentration of the pure enzyme was determined according to the instructions of the protein concentration kit. The protein concentration of each sample was calculated based on the absorbance at A595 nm of the microplate reader and the standard curve.

[0061] Specific activity assay method:

[0062] The specific activity was calculated according to the following formula:

[0063] Specific activity (U / mg) = Enzyme activity (U / mL) / Protein content (mg / mL)

[0064] The primer sequences involved in the following examples are as follows:

[0065] Table 1 Primer sequences

[0066]

[0067]

[0068] Example 1: Expression of wild-type chitinase

[0069] 1. Construction of chitinase-expressing engineering strain

[0070] The chitinase gene BlChiA from Bacillus licheniformis with NCBI accession number WP_016886405 was codon-optimized and synthesized by Shanghai Genry Biological Engineering Co., Ltd. Its nucleotide sequence is shown in SEQ ID NO.1. Using the nucleotide sequence shown in SEQID NO.5 as the PHpaⅡ -P amyQ is a promoter, AmyE encoded by the nucleotide sequence shown in SEQ ID NO.6 is a signal peptide, and strain WS9-pHY300PLK-P that recombinantly expresses the chitinase gene BlChiA HpaⅡ-amyQ -SP AmyE -BlChiA is used as the starting strain.

[0071] Using the ClonExpress II One Step Cloning Kit, the AmyE nucleotide fragment shown in SEQ ID NO.6 was ligated after the p15A ori fragment of the pHY300PLK vector, and pHY300PLK-SP was obtained by seamless ligation AmyE vector.

[0072] The chitinase gene BlChiA shown in SEQ ID NO.1 was ligated between the NcoⅠ and HindⅢ sites of pHY300PLK-SP AmyE and transformed into competent E.coli JM109 cells and cultured overnight. Screening was carried out on the resistance plate, positive transformants were picked to extract plasmids and sequenced, and the plasmids in the positive recombinant bacteria with correct sequencing were extracted to obtain the vector pHY300PLK-SP AmyE -BlChiA.

[0073] Using the vector pHY300PLK-SP AmyE -BlChiA as a template, the linearized pHY300PLK-SP AmyE -BlChiA vector fragment was obtained by PCR amplification using primers F0 and R0 (see Table 1), and the amplification product was recovered after agarose gel electrophoresis.

[0074] On this basis, using the Bacillus subtilis 168 genome as a template, primers P SpoVG -F, P SpoVG -R were used to amplify the promoter P SpoVG , and the ClonExpress II One Step Cloning Kit was used to seamlessly ligate the promoter fragment and the linear vector pHY300PLK-SP AmyE -BlChiA to obtain the recombinant plasmid pHY300PLK-P SpoVG -SP AmyE -BlChiA; primers SP YqxI -F, SP YqxI -R were used to amplify the signal peptide YqxI to replace pHY300PLK-P SpoVG -SP AmyE- The signal peptide AmyE on the -BlChiA vector. The recombinant plasmid with correct sequencing was transformed into the expression host strain WS9, and the constructed Bacillus subtilis recombinant strain was named WS9-pHY300PLK-P SpoVG -SP YqxI -BlChiA.

[0075] 2. Expression of chitinase

[0076] The above-mentioned Bacillus subtilis recombinant strain WS9-pHY300PLK-P SpoVG -SP YqxI -BlChiA was cultured in LB liquid medium containing 100 μg / mL tetracycline to obtain a seed solution. After culturing at 37°C and 200 rpm for 8 - 10 h, the seed solution was inoculated into TB fermentation medium containing 100 μg / mL tetracycline at an inoculation amount of 5% (v / v). It was cultured at 37°C and 200 rpm until OD 600 reached 0.6 - 0.8 and then transferred to 33°C and 200 rpm for 48 h to obtain a fermentation broth. The fermentation broth was centrifuged at 4°C and 8000 rpm for 10 min to remove the cells, and the centrifuged supernatant was collected to obtain a crude enzyme solution, which was the crude enzyme solution of the wild enzyme.

[0077] Example 2: Construction and expression of chitinase mutants

[0078] (1) Preparation of chitinase single mutants

[0079] Primers introducing corresponding mutations were designed and synthesized respectively to perform site-directed mutagenesis on the chitinase gene. Taking the construction process of the mutant with the 26th aspartic acid residue mutated to asparagine residue as an example:

[0080] Using the method of site-directed mutagenesis, with the recombinant plasmid pHY300PLK-P SpoVG -SP YqxI -BlChiA as the template, primers F1 and R1 were designed to mutate the 26th aspartic acid residue to asparagine residue. The above plasmid was amplified by PCR, and DpnⅠ was added to fully digest the template under the condition of 37°C water bath. An appropriate amount of the product was transformed into B.subtilis SCK6 competent cells, and positive transformants were picked to extract plasmids and sequence.

[0081] Table 2 PCR reaction system

[0082]

[0083] PCR conditions: Pre-denaturation at 98°C for 5 min; denaturation at 98°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 kb / min, 25 cycles, extension at 72°C for 10 min, and incubation at 4°C.

[0084] The correctly sequenced mutant plasmid as described above was transformed into the host bacterium Bacillus subtilis WS9 and expressed to obtain the mutant D26N.

[0085] The following five mutants were successfully obtained by the above method:

[0086] A mutant in which the aspartic acid residue at position 26 of chitinase was mutated to an asparagine residue (named D26N); a mutant in which the glycine residue at position 323 of chitinase was mutated to an aspartic acid residue (named G323D) was constructed using primers F2 and R2; a mutant in which the glutamic acid residue at position 336 of chitinase was mutated to a lysine residue (named E336K) was constructed using primers F3 and R3; a mutant in which the glutamic acid residue at position 469 of chitinase was mutated to a glycine residue (named E469G) was constructed using primers F4 and R4; a mutant in which the alanine residue at position 472 of chitinase was mutated to threonine (named A472T) was constructed using primers F5 and R5.

[0087] (2) Preparation of chitinase combinatorial mutants

[0088] Using the site-directed mutagenesis method, with the plasmid pHY300PLK-P SpoVG -SP YqxI -BlChiA A472T carrying the mutant A472T as a template, primers F4 and R4 were used to mutate the glutamic acid residue at position 469 to a glycine residue. PCR amplification, transformation of B. subtilis SCK6 competent cells, plasmid extraction and sequencing were carried out by the same method as above. The correctly sequenced mutant plasmid was transformed into the host bacterium Bacillus subtilis WS9 and expressed to obtain the mutant E469G / A472T.

[0089] Using the site-directed mutagenesis method, with the plasmid pHY300PLK-P SpoVG -SP YqxI -BlChiA E469G / A472T carrying the mutant E469G / A472T as a template, primers F1 and R1 were used to mutate the aspartic acid residue at position 26 to an asparagine residue. PCR amplification, transformation of B. subtilis SCK6 competent cells, plasmid extraction and sequencing were carried out by the same method as above. The correctly sequenced mutant plasmid was transformed into the host bacterium Bacillus subtilis WS9 and expressed to obtain the mutant D26N / E469G / A472T.

[0090] Using the site-directed mutagenesis method, with the plasmid pHY300PLK-P carrying the mutant E469G / A472TSpoVG -SP YqxI -BlChiA E469G / A472T Using the plasmid carrying the mutant E469G / A472T, pHY300PLK-P

[0091] as a template, primers F2 and R2 were used to mutate the glycine residue at position 323 to an aspartic acid residue. PCR amplification, transformation of competent B. subtilis SCK6 cells, plasmid extraction and sequencing were carried out using the same method as above. The correctly sequenced mutant plasmid was transformed into the host strain Bacillus subtilis WS9 and expressed to obtain the mutant G323D / E469G / A472T. SpoVG -SP YqxI -BlChiA E469G / A472T Using the plasmid carrying the mutant E469G / A472T, pHY300PLK-P

[0092] as a template, primers F3 and R3 were used to mutate the glutamic acid residue at position 336 to a lysine residue. PCR amplification, transformation of competent B. subtilis SCK6 cells, plasmid extraction and sequencing were carried out using the same method as above. The correctly sequenced mutant plasmid was transformed into the host strain Bacillus subtilis WS9 and expressed to obtain the mutant E336K / E469G / A472T. SpoVG -SP YqxI -BlChiA E336K / E469G / A472T Using the plasmid carrying the mutant E336K / E469G / A472T, pHY300PLK-P

[0093] as a template, primers F1 and R1 were used to mutate the aspartic acid residue at position 26 to an asparagine residue. PCR amplification, transformation of competent B. subtilis SCK6 cells, plasmid extraction and sequencing were carried out using the same method as above. The correctly sequenced mutant plasmid was transformed into the host strain Bacillus subtilis WS9 and expressed to obtain the mutant D26N / E336K / E469G / A472T. SpoVG -SP YqxI -BlChiA D26N / E336K / E469G / A472TUsing [template], the glycine residue at the 323rd position was mutated to an aspartic acid residue using primers F2 and R2. PCR amplification, transformation of competent B. subtilis SCK6 cells, plasmid extraction, and sequencing were performed using the same method as above. The correctly sequenced mutant plasmid was transformed into the host bacterium Bacillus subtilis WS9 and expressed to obtain the mutant D26N / G323D / E336K / E469G / A472T.

[0094] (3) Expression of mutant enzyme

[0095] The expression process of the mutant was as described in Example 1.

[0096] Example 3: Analysis of wild-type chitinase and mutant enzyme activities

[0097] The enzyme activities of the wild-type chitinase and mutants in Example 1 were measured respectively. The enzyme activities of the mutants were improved to varying degrees. Among them, the enzyme activity of the mutant E336K / E469G / A472T was 1.89 times that of the wild-type and 3.69 times that of the unoptimized starting strain; the enzyme activity of D26N / E336K / E469G / A472T was the highest, which was 2.10 times that of the wild-type and 4.11 times that of the unoptimized starting strain.

[0098] Table 3 Enzyme activities of wild-type and mutant chitinases

[0099]

[0100] Example 4: Determination of chitinase specific activity

[0101] 1. Purification of chitinase

[0102] Purified wild-type chitinase and mutants were obtained by the following method:

[0103] (1) While stirring, the crude enzyme solution of wild-type chitinase obtained by fermentation in Example 1 was slowly added to ammonium sulfate with a concentration of 26% (mass fraction relative to the enzyme solution). Stir until the ammonium sulfate was dissolved, and the protein was precipitated by standing at 4 °C for 8 - 10 h to obtain a mixture.

[0104] (2) The mixture obtained in step (1) was centrifuged (8000 rpm, 10 min) to collect the precipitate, and then the minimum volume of 20 mM KH 2 PO 4 -Na 2 HPO 4Resuspend in buffer (Solution A). After resuspension, centrifuge again to remove solids, collect the supernatant to obtain the resuspended solution. Dialyze the resuspended solution in buffer A at 4°C for 24 h. After dialysis, centrifuge at 12,000 rpm / min at 4°C for 5 min and collect the supernatant.

[0105] (3) Filter the supernatant obtained in step (2) through a 0.22-μm organic membrane to prepare a sample for loading. Purify BlChiA using a Ni-NTA column. The protein elution steps are as follows: First step, elute the miscellaneous proteins with 2 column volumes of Solution A (25 mM Tris-HCl buffer, 500 mM NaCl, pH 7.4) and a gradient of Solution A containing 15, 30, and 45 mM imidazole; Second step, elute BlChiA with 2 column volumes of Solution B (25 mM Tris-HCl buffer, 500 mM NaCl, 300 mM imidazole) and collect. Then, remove the imidazole from the collected enzyme solution using a 30-KDa ultrafiltration tube and replace the buffer with 50 mM phosphate buffer to obtain the purified chitinase.

[0106] 2. Determination of the protein concentration of the pure enzyme

[0107] Prepare a protein standard curve and determine the protein concentration of the pure enzyme according to the instructions of the protein concentration kit. Calculate the protein concentration based on the absorbance of each sample at A595 nm on the microplate reader and the standard curve.

[0108] 3. Determination of specific activity

[0109] Determine the enzyme activity of the pure enzyme solution according to the aforementioned method to calculate the specific activities of the wild type and mutants E336K / E469G / A472T.

[0110] Specific activity calculation formula: Specific activity (U / mg) = Enzyme activity (U / mL) / Protein concentration (mg / mL)

[0111] As shown in Table 4, the specific activity of mutant E469G is 1.07 times that of the wild type, the specific activity of mutant E336K / E469G / A472T is 1.56 times that of the wild type, and the specific activity of mutant D26N / E336K / E469G / A472T is 1.40 times that of the wild type.

[0112] Table 4 Specific activities of wild type and mutants of chitinase

[0113]

[0114]

[0115] Example 5: Characterization of the enzymatic properties of chitinase mutant E469G

[0116] The optimal reaction pH and pH stability of the recombinant enzyme were determined using the DNS method.

[0117] The measurement range of the optimal reaction pH and pH stability was pH 3.0 - 10.0. The pH stability was measured after the enzyme solution was incubated at 4°C for 24 h.

[0118] When determining the optimal reaction pH of the recombinant enzyme, the highest enzyme activity measured was defined as 100%, and the ratio of the enzyme activity measured under other conditions to the highest enzyme activity was defined as the relative enzyme activity.

[0119] When determining the pH stability of the recombinant enzyme, the enzyme activity of the untreated enzyme solution was defined as 100%, and the ratio of the enzyme activity of the treated enzyme solution to the enzyme activity of the untreated enzyme solution was defined as the residual enzyme activity.

[0120] The optimal reaction pH of the mutant E469G was 4.0. Compared with the wild type, it had higher enzyme activity under acidic conditions. Its stability was significantly better than that of the wild type in the range of pH 3.0 - pH 10.0, and the residual enzyme activity could reach more than 85%, with a wide pH tolerance range, which was beneficial to the production and application of this mutant.

[0121] Example 6: Characterization of the enzymatic properties of chitinase mutant E336K / E469G / A472T

[0122] The enzymatic properties of chitinase mutant E336K / E469G / A472T were characterized using the same method as in Example 5. The optimal pH of chitinase mutant E336K / E469G / A472T was 5.0. Compared with the wild type, this mutant had better acid tolerance and could maintain more than 75% of its activity in the range of pH 3.0 - pH 5.0. The mutant had better stability in the range of pH 3.0 - pH 9.0, and the residual enzyme activity reached more than 85%, with a significantly improved acid tolerance.

[0123] Example 7: Characterization of the enzymatic properties of chitinase mutant D26N / E336K / E469G / A472T

[0124] The enzymatic properties of chitinase mutant D26N / E336K / E469G / A472T were characterized using the same method as in Example 5. Chitinase mutant D26N / E336K / E469G / A472T could maintain more than 80% of its activity in the range of pH 3.0 - pH 8.0, with a wide pH reaction range. The mutant had better stability in the range of pH 3.0 - pH 9.0, and the residual enzyme activity reached more than 85%, with a wider pH tolerance range, which was beneficial to the production and application of this mutant.

[0125] Comparative Example 1:

[0126] For the mutants constructed as in Example 2, enzymatic property characterization was carried out in the same method. The results showed that the optimal pH of mutant E469G / A472T was 5.0, and the residual enzyme activity was over 85% in the range of pH 4.0 - pH 7.0; the optimal pH of mutant D26N / E469G / A472T was 5.0, and the residual enzyme activity was over 85% in the range of pH 4.0 - pH 7.0; the optimal pH of mutant G323D / E469G / A472T was 6.0, and the residual enzyme activity was over 85% in the range of pH 4.0 - pH 8.0; the optimal pH of mutant D26N / G323D / E336K / E469G / A472T was 6.0, and the residual enzyme activity was over 85% in the range of pH 3.0 - pH 8.0.

[0127] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A chitinase mutant, characterized in that the glutamic acid residue at position 336 of the amino acid sequence shown in SEQ ID NO.2 is mutated to a lysine residue, the glutamic acid residue at position 469 is mutated to a glycine residue, and the alanine residue at position 472 is mutated to a threonine residue.

2. A gene encoding the mutant according to claim 1.

3. A recombinant vector carrying the gene according to claim 2, characterized in that using the pHY300PLK series vector as the starting vector, containing the promoter SpoVG of the nucleotide sequence shown in SEQ ID NO.3 and the signal peptide YqxI encoded by the nucleotide sequence shown in SEQ ID NO.

4.

4. A recombinant microbial cell expressing the chitinase according to claim 1, characterized in that the recombinant microbial cell is a recombinant prokaryotic cell or a recombinant eukaryotic cell.

5. The recombinant microbial cell according to claim 4, characterized in that The recombinant microbial cell is recombinant Bacillus subtilis, using Bacillus subtilis WS9, SCK6 or RIK1285 as the host and carrying the recombinant vector as described in claim 3.

6. A method for producing a chitinase mutant, characterized in that fermentation is carried out using the recombinant Bacillus subtilis according to claim 5.

7. The method according to claim 6, characterized in that Transfer the recombinant Bacillus subtilis cultured under the conditions of 30-38°C and 180-220 rpm for 6-12 h into the fermentation medium at an inoculum amount of 1-10%, and culture at 30-37°C until the OD 600 is 0.6-0.8, and ferment at 30-35°C and 200-220 rpm for 36-60 h.

8. Use of the mutant according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, the recombinant microbial cell according to claim 4 or 5, or the method according to claim 6 or 7 in the degradation of chitin.

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