Construction of a recombinant bacterium expressing chitinase and preparation of a high-enzymatic-activity mutant

Abstract

The invention discloses the construction of a recombinant bacterium expressing chitinase and the preparation of a high-enzymatic-activity mutant, belonging to the technical fields of genetic engineering and enzyme engineering. In the invention, the endogenous strong promoter SpoVG and the signal peptide YqxI are used to realize the secretory expression of chitinase BlChiA derived from Bacillus licheniformis in Bacillus subtilis WS9, and further site-directed mutagenesis is carried out to improve the expression level and acid tolerance of chitinase. The fermentation enzyme activity of the obtained chitinase mutant E336K / A472T reaches 2.67 U / mL. At the same time, the optimal reaction pH of the chitinase mutant is 5.0, and it has good stability in the range of pH 3.0 - pH 6.0, and the residual enzyme activity reaches more than 90%. It has the advantages of good stability and strong acid tolerance, and is suitable for obtaining oligosaccharide products with high conversion rate in industrial production.

Description

Technical Field

[0001] The present invention relates to the construction of a recombinant bacterium expressing chitinase and the preparation of a high-activity mutant, belonging to the technical fields of genetic engineering and enzyme engineering. Background Art

[0002] Chitin, also known as chitosan, chitin, chitosanamine, etc., is a linear polysaccharide formed by N-acetyl-D-glucosamine linked by β-1,4-glycosidic bonds. When the degree of deacetylation is more than 40%, it can be converted into chitosan. It is the most abundant renewable resource in the ocean and the second most abundant natural resource after cellulose. The degradation products of chitin and its analogs, amino-oligosaccharides, have excellent physiological functions and biological activities and are widely used in the fields of agriculture, medicine, and food.

[0003] Chitinase (EC 3.2.1.14) is a class of glycoside hydrolases that hydrolyze the β-1,4-glycosidic bonds of chitin to produce chito-oligosaccharides or monosaccharides. Most chitinases belong to glycoside hydrolase families 18 and 19, and a few are distributed in families 20, 23, and 48. Chitinase is widely used in the degradation of chitin. Since chitin is only soluble in strong inorganic acids, a chitinase with strong acid resistance is required during the degradation of chitin. However, natural chitinases have low expression levels, poor enzyme activity, and poor acid resistance, and generally degrade chitin through the synergistic action of multiple chitinases, which limits the production and application of chitinase. Recombinant expression to obtain a highly expressed chitinase with strong acid resistance is a good way to efficiently utilize chitin resources.

[0004] There is a possibility that the chitinase recombinantly expressed in the fungal expression system degrades the fungal cell wall, which in turn affects the growth of the host bacterium and results in a low yield of chitinase. In the prokaryotic expression system, Bacillus subtilis has the characteristics of non-pathogenicity, high safety, and a short culture period, which is also of great significance for shortening the fermentation period in industrial production. It is an ideal expression host for producing various industrial enzymes in the current food, medicine, animal husbandry and other industries. However, there are few reports on the recombinant expression of chitinase in Bacillus subtilis at present, and the enzyme activity is low and the acid resistance is poor, which limits its industrial application. Xue Jiawei et al. cloned and expressed the chitinase gene PpchiC of Pseudomonas pseudoalteromonis piscicida C923 in Escherichia coli BL21. This enzyme has a good degradation effect on chitin, but the soluble expression level is extremely low, and the recombinant protein mainly exists in the form of inclusion bodies. It shows the highest enzyme activity at pH 8.0, and when the pH is 3.5, the enzyme activity drops to 10% of the highest enzyme activity. Yin Yixu et al. heterologously expressed the chitinase gene gene02524 from Trichoderma asperellum TD3104 in Pichia pastoris, which has a certain inhibitory effect on the growth of some pathogenic bacteria. The enzyme activity of the recombinant expression of this enzyme is relatively low, and the enzyme activity is about 0.15 U / mL after 5 days of induction expression. In an environment with pH 3.0, the activity drops to 20% of the highest enzyme activity. Aiming at the problems of low expression enzyme activity and poor acid resistance of recombinant chitinase in the above-mentioned existing technologies, it is of great significance to find a chitinase with high expression enzyme activity, high acid resistance and stable properties for the efficient utilization of chitin resources. Summary of the Invention

[0005] The present invention provides a mutant of chitinase BlChiA of family 18, which is obtained by site-directed mutagenesis of chitinase BlChiA from Bacillus licheniformis. Compared with the wild-type chitinase BlChiA, the mutant has better tolerance to acidic conditions and broad pH stability, and has broad prospects in the industrial production of chitinase.

[0006] The present invention provides mutants for improving the expression level of chitinase, including any one of the following mutations:

[0007] Mutating the glutamic acid residue at position 336 of the chitinase with the amino acid sequence shown in SEQ ID NO.4 to a lysine residue, and naming it E336K;

[0008] Mutating the alanine residue at position 472 of the chitinase with the amino acid sequence shown in SEQ ID NO.4 to a threonine residue, and naming it A472T;

[0009] Mutating the glutamic acid at position 336 and the alanine at position 472 of the chitinase with the amino acid sequence shown in SEQ ID NO.4 to lysine and threonine respectively, and naming it E336K / A472T.

[0010] The present invention also provides a recombinant Bacillus subtilis expressing a chitinase BlChiA mutant. Through expression strategies such as promoter optimization, signal peptide optimization, and site-directed mutagenesis, the expressed enzyme activity of the mutant is improved, providing a basis for subsequent molecular modification to increase the expression level or catalytic efficiency of the chitinase gene BlChiA.

[0011] In one embodiment of the present invention, the control strain used is WS9-pHY300PLK-P HpaⅡ -P amyQ -SP AmyE -BlChiA, using P with the nucleotide sequence shown in SEQ ID NO.1 HpaⅡ -P amyQ as the promoter, and AmyE encoded by the nucleotide sequence shown in SEQ ID NO.2 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 Biotechnology Co., Ltd., and its nucleotide sequence is as shown in SEQ ID NO.3); the amino acid sequence of the chitinase BlChiA is as shown in SEQ ID NO.4.

[0012] In one embodiment of the present invention, the vector is a pHY300 series vector or a pHY300PLK series vector; the host bacterium is Bacillus subtilis WS9, and its 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".

[0013] In one embodiment of the present invention, the promoter of the Bacillus subtilis engineering bacterium expressing chitinase is optimized, and the optimization means that the promoter of the recombinant plasmid is the promoter P with increased transcriptional level SpoVG , and its nucleotide sequence is SEQ ID NO.5.

[0014] In one embodiment of the present invention, the signal peptide of the Bacillus subtilis engineering bacterium expressing chitinase is optimized, and the optimization means that the signal peptide of the recombinant plasmid is YqxI with increased level of secreted target protein, and the nucleotide sequence encoding the optimized signal peptide YqxI is SEQ ID NO.6.

[0015] The present invention provides a method for efficiently expressing chitinase, and this method uses the above-mentioned engineered Bacillus subtilis as the production strain.

[0016] In one embodiment of the present invention, the method is to 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 inoculation amount of 1-10%, and culture it at 30-37 °C until the OD 600 reaches 0.6-0.8, and ferment at 30-35 °C and 200-220 rpm for 36-60 h.

[0017] The present invention provides chitinase BlChiA prepared by the above method for efficiently expressing chitinase.

[0018] Beneficial effects

[0019] (1) The present invention provides a chitinase mutant E336K / A472T, whose optimal reaction pH is 5.0, and it has higher enzyme activity under acidic conditions; the mutant has better stability in the range of pH 3.0 - pH 7.0, and the residual enzyme activity reaches more than 90%. It has better tolerance to acidic conditions and broad pH stability, and has broad prospects in the industrial production of chitinase.

[0020] (2) The present invention constructs a recombinant Bacillus subtilis WS9-pHY300PLK-P SpoVG -SP YqxI -BlchiA E336K / A472T , and the chitinase activity obtained by fermenting this recombinant Bacillus subtilis is 2.67 U / mL, which has good application value in efficiently degrading chitin to obtain chitooligosaccharides. Description of the drawings

[0021] Figure 1 It is a schematic diagram of plasmid construction.

[0022] Figure 2 It is the SDS-PAGE analysis of chitinase recombinant engineering bacteria containing different promoters; where M: Marker; P1: P hag ; P2: P tufA ; P3: P43; P4: P SpoVG ; P5: P HpaⅡ -P amyQ ; P6: P fusA ; P7: P HpaⅡ -P amyQ’ ; P8: P amyE . a: Fermentation supernatant; b: Broken supernatant; c: Broken precipitate.

[0023] Figure 3 SDS-PAGE analysis of recombinant engineering bacteria of chitinase containing different signal peptides; where M: Marker; SP1: AmyE; SP2: YqxI; SP3: PhrC. a: Fermentation supernatant; b: Broken supernatant; c: Broken precipitate.

[0024] Figure 4 Graphs of the optimal reaction temperature, thermal stability, optimal reaction pH, and pH stability of chitinase mutant E336K / A472T. Specific implementation manners

[0025] For the biological experimental methods not specifically described in the following examples, they are all carried out according to the specific methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook, or according to the product instructions of the kits.

[0026] The experimental materials and reagents involved in the following examples are as follows:

[0027] 1. Strains and vectors

[0028] The Bacillus subtilis WB168, E. coli JM109, and plasmid pHY300PLK used in the present invention are all commercial hosts or vectors.

[0029] Bacillus subtilis WS9 is based on Bacillus subtilis WS5 (which has been publicly disclosed in patent CN106754466, deposit number CCTCC M 2016536), and nprB, bpr, mpr, and epr are further knocked out. The specific construction method is publicly 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".

[0030] 2. Enzymes and kits

[0031] 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., DpnⅠ was purchased from NEB Inc., the restriction endonucleases and the B. subtilis Secretory Protein Expression System kit were purchased from Takara Inc., and the plasmid extraction, gel extraction and purification kits were purchased from Tiangen Biochemical Technology Co., Ltd., and the protein concentration kit was purchased from Beyotime Biotechnology Co., Ltd.

[0032] The culture media involved in the following examples are as follows:

[0033] LB liquid medium: Tryptone 10 g·L -1 , Yeast extract 5 g·L -1 , Sodium chloride 10 g·L -1 ,

[0034] 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

[0035] 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 .

[0036] Flask fermentation and acquisition of crude enzyme solution:

[0037] The seed solution was cultured in LB liquid medium containing 100 μg / mL tetracycline, and after culturing at 37 °C and 200 rpm for 10 h, the seed solution was transferred to TB fermentation medium containing 100 μg / mL tetracycline at an inoculation amount of 5%, and cultured at 37 °C and 200 rpm until the OD 600 reached 0.6 - 0.8 and then transferred to 33 °C and 200 rpm for 48 h to obtain the fermentation broth. The fermentation broth was centrifuged at 4 °C and 8000 rpm for 10 min to remove the bacteria, and the centrifuged supernatant was the crude chitinase enzyme solution containing different mutants.

[0038] The detection methods involved in the following examples are as follows:

[0039] Enzyme activity assay method:

[0040] The total reducing sugar content was determined by the 3,5-dinitrosalicylic acid (DNS) method. 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. Centrifuge at 12000 r·min -1 for 5 min, take the supernatant and measure the absorbance 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).

[0041] Protein concentration assay method:

[0042] The protein standard curve was made 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.

[0043] Specific activity calculation method:

[0044] Specific activity (U / mg) = Enzyme activity (U / mL) / Protein concentration (mg / mL)

[0045] The chitinase purification method involved in the following examples is as follows:

[0046] (1) While stirring, the crude enzyme solution was slowly added with ammonium sulfate at a concentration of 26% by mass relative to the enzyme solution, stirred until the ammonium sulfate was dissolved, and allowed to stand at 4 °C for 8 - 10 h to precipitate proteins, obtaining a mixture.

[0047] (2) The mixture obtained in step (1) was centrifuged (8000 rpm, 10 min) to collect the precipitate, and then redissolved with the minimum volume of pH 6.5 20 mM KH 2 PO 4 -Na 2 HPO 4 buffer solution (solution A), and after redissolution, the solid matter was removed by centrifugation again, and the supernatant was collected to obtain a redissolved solution; the redissolved solution was dialyzed in buffer solution A at 4 °C for 24 h, and after dialysis, centrifuged at 12000 rpm / min at 4 °C for 5 min, and the supernatant was collected.

[0048] (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, use 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 to elute the miscellaneous proteins; Second, use 2 column volumes of solution B (25 mM Tris-HCl buffer, 500 mM NaCl, 300 mM imidazole) to elute BlChiA and collect it. Then, use a 30 KDa ultrafiltration tube to remove imidazole from the collected enzyme solution and replace the buffer with 50 mM phosphate buffer to obtain the purified chitinase.

[0049] The primers used in the following examples are shown in the following table:

[0050] Table 1 Primer sequences

[0051]

[0052]

[0053] Example 1: Construction of recombinant Bacillus subtilis containing different promoters

[0054] 1. Vector pHY300PLK-SP AmyE - Construction of BlChiA

[0055] Design primers F0 and R0, use the signal peptide AmyE carried by Bacillus subtilis amylase amyE (Gene ID: 938356) as a template for PCR amplification of the nucleotide fragment and recover it (as shown in SEQ ID NO.2). Use the ClonExpress II OneStep Cloning Kit to ligate the AmyE fragment after the p15A ori fragment of the pHY300PLK vector to obtain the pHY300PLK-SP AmyE vector.

[0056] The chitinase gene BlChiA from Bacillus licheniformis was codon-optimized and synthesized by Shanghai Jierui Biotechnology Co., Ltd., and the nucleotide sequence is as shown in SEQ ID NO.3. It was ligated between the NcoⅠ and HindⅢ sites of pHY300PLK-SP AmyE and transformed into E. coli JM109 competent cells and cultured overnight. Screen on the resistance plate, pick positive transformants to extract plasmids and sequence them. Extract the plasmid from the positive recombinant bacteria with correct sequencing to obtain the vector pHY300PLK-SP AmyE -BlChiA.

[0057] 2. Construction of vectors with different promoters

[0058] The promoters used in this invention are derived as follows: Using the Bacillus subtilis 168 genome as a template, primers were designed according to the nucleotide sequences of the promoters (Table 1), and the promoter fragments P hag , P tufA , P 43 , P SpoVG , P fusA , P amyE were obtained by PCR amplification using the corresponding primers. P HpaⅡ -P amyQ , P HpaⅡ -P amyQ’ were constructed and stored in our laboratory previously, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.7 respectively.

[0059] Taking the construction process of the vector pHY300PLK-P hag -SP AmyE -BlChiA as an example, 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 F1 and R1 (see Table 1). The amplification product was recovered after agarose gel electrophoresis. Using the Bacillus subtilis 168 genome as a template, the promoter fragment P hag -F and P hag -R were used to amplify the promoter P hag fragment. The ClonExpress II One Step Cloning Kit was used to seamlessly ligate the promoter fragment and the plasmid backbone, and the ligation product pHY300PLK-P hag -SP AmyE -BlChiA was transformed into E. coli JM109 competent cells. Screening was carried out on the resistance plate, and positive transformants were picked to extract plasmids and sequence.

[0060] Using the same method as above, vectors pHY300PLK-P tufA -SP AmyE -BlChiA, pHY300PLK-P 43 -SP AmyE -BlChiA, pHY300PLK-P SpoVG -SP AmyE -BlChiA, pHY300PLK-P HpaⅡ -P amyQ -SP AmyE-BlChiA, pHY300PLK-P fusA -SP AmyE -BlChiA, pHY300PLK-P HpaⅡ -P amyQ’ -SP AmyE -BlChiA and pHY300PLK-P amyE -SP AmyE -BlChiA.

[0061] The plasmids with correct sequencing mentioned above were separately transformed into the host bacterium Bacillus subtilis WS9, and the obtained strains were named WS9-pHY300PLK-P hag -SP AmyE -BlChiA, WS9-pHY300PLK-P tufA -SP AmyE -BlChiA, WS9-pHY300PLK-P 43 -SP AmyE -BlChiA, WS9-pHY300PLK-P SpoVG -SP AmyE -BlChiA, WS9-pHY300PLK-P HpaⅡ -P amyQ -SP AmyE -BlChiA, WS9-pHY300PLK-P fusA -SP AmyE -BlChiA, WS9-pHY300PLK-P HpaⅡ -P amyQ’ -SP AmyE -BlChiA and WS9-pHY300PLK-P amyE -SP AmyE -BlChiA.

[0062] Table 2 Enzyme activity in the supernatant of the fermentation broth of chitinase engineering bacteria carrying different promoters

[0063]

[0064] The above-obtained engineering bacteria were separately subjected to shake-flask fermentation, and the enzyme activity of the crude enzyme solution in the supernatant of the fermentation broth was measured. Among them, the engineering bacterium WS9-pHY300PLK-P HpaⅡ -P amyQ -SP AmyE -BlChiA is the control strain of the present invention. The enzyme activity detection results: the control strain WS9-pHY300PLK-P HpaⅡ -P amyQ -SP AmyE-The enzyme activity of the engineered strain of -BlChiA chitinase is 0.72 U / mL; WS9-pHY300PLK-P SpoVG -SP AmyE -The enzyme activity of the engineered strain of -BlChiA chitinase is 0.96 U / mL, which is 1.33 times that of the former one.

[0065] Example 2: Construction of recombinant Bacillus subtilis containing different signal peptides

[0066] According to the sequence information of signal peptides YqxI and PhrC in NCBI, the corresponding primers SP YqxI -F, SP YqxI -R and SP PhrC -F, SP PhrC -R were designed and synthesized, and PCR amplification was carried out using pHY300PLK-P SpoVG -SP AmyE -BlChiA as the template to obtain plasmids containing the corresponding signal peptides. The amplification products were fully digested with DpnⅠ to remove the template, and then an appropriate amount of the digested products was taken to transform E. coli JM109 competent cells. Positive transformants were screened on resistance plates, and the plasmids were extracted and sequenced. After correct sequencing, they were respectively transformed into the host strain Bacillus subtilis WS9, and the obtained strains were named WS9-pHY300PLK-P SpoVG -SP YqxI -BlChiA and WS9-pHY300PLK-P SpoVG -SP PhrC -BlChiA. The above strains were respectively subjected to shake flask fermentation and the enzyme activity of the crude enzyme solution was measured.

[0067] Enzyme activity detection results: WS9-pHY300PLK-P SpoVG -SP AmyE -The enzyme activity of the engineered strain of -BlChiA chitinase is 0.96 U / mL. WS9-pHY300PLK-P SpoVG -SP PhrC -The enzyme activity of the engineered strain of -BlChiA chitinase is 1.20 U / mL; WS9-pHY300PLK-P SpoVG -SP YqxI -The enzyme activity of the engineered strain of -BlChiA chitinase is 1.41 U / mL.

[0068] The recombinant engineered strain WS9-pHY300PLK-P SpoVG -SP YqxI -The chitinase activity produced by -BlChiA is that of WS9-pHY300PLK-P before signal peptide optimization SpoVG -SP AmyE-1.47 times the enzyme activity produced by BlChiA; it is the control strain WS9-pHY300PLK-P HpaⅡ -P amyQ -SP AmyE -1.96 times the enzyme activity produced by BlChiA.

[0069] Example 3: Construction of a chitinase mutant with increased expression level

[0070] Using the recombinant plasmid pHY300PLK-P SpoVG -SP YqxI -BlChiA as a template, primers F2 and R2 (Table 1) were designed to mutate the glutamate residue at position 336 of chitinase to a lysine residue. The resulting plasmid was transformed into the host bacterium Bacillus subtilis WS9, and the resulting strain was named WS9-pHY300PLK-P SpoVG -SP YqxI -BlChiA E336K . Using the recombinant plasmid pHY300PLK-P SpoVG -SP YqxI -BlChiA as a template, primers F3 and R3 (Table 1) were designed to mutate the alanine residue at position 472 to a threonine residue. The resulting plasmid was transformed into the host bacterium Bacillus subtilis WS9, and the resulting strain was named WS9-pHY300PLK-P SpoVG -SP YqxI -BlChiA A472T . Using the plasmid pHY300PLK-P SpoVG -SP YqxI -BlChiA carrying the mutant E336K as a template, primers F3 and R3 (Table 1) were used to mutate the alanine residue at position 472 to a threonine residue. The resulting plasmid was transformed into the host bacterium Bacillus subtilis WS9, and the resulting strain was named WS9-pHY300PLK-P E336K -SP SpoVG -SP YqxI -BlChiA E336K / A472T .

[0071] Taking the construction process of the combined mutant strain WS9-pHY300PLK-P SpoVG -SP YqxI -BlChiA E336K / A472T as an example:

[0072] Using the site-directed mutagenesis method, with the recombinant plasmid pHY300PLK-P SpoVG -SP YqxIUsing -BlChiA as a template, primers F2 and R2 (Table 1) were designed to mutate the glutamic acid residue at position 336 of chitinase to a lysine residue, and PCR amplification was performed on the plasmid backbone of pHY300PLK-P SpoVG -SP YqxI -BlChiA. DpnⅠ was added and the template was fully digested in a 37°C water bath. An appropriate amount of the product was taken to transform competent cells of B. subtilis SCK6, and positive transformants were picked to extract plasmids and sequence them. Using the plasmid pHY300PLK-P SpoVG -SP YqxI -BlChiA E336K with correct sequencing as a template, primers F3 and R3 (Table 1) were designed to mutate the alanine residue at position 472 to a threonine residue, and PCR amplification was performed on the plasmid backbone of pHY300PLK-P SpoVG -SP YqxI -BlChiA E336K . DpnⅠ was added and the template was fully digested in a 37°C water bath. An appropriate amount of the product was taken to transform competent cells of B. subtilis SCK6, and positive transformants were picked to extract plasmids and sequence them. The plasmid pHY300PLK-P SpoVG -SP YqxI -BlChiA E336K / A472T with correct sequencing as above was transformed into the host bacterium Bacillus subtilis WS9, and the resulting mutant strain was named WS9-pHY300PLK-P SpoVG -SP YqxI -BlChiA E336K / A472T .

[0073] Table 3 PCR reaction system

[0074]

[0075] 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 at 1 kb / min for 25 cycles, extension at 72°C for 10 min, and incubation at 4°C.

[0076] The above recombinant bacteria were respectively subjected to shake flask fermentation, and the enzyme activity of the crude enzyme solution in the fermentation supernatant was measured.

[0077] Enzyme activity detection results: The enzyme activity of the fermentation supernatant of the engineered bacterium WS9-pHY300PLK-P SpoVG -SP YqxI -BlChiA E336K was 1.64 U / mL, and that of WS9-pHY300PLK-P SpoVG -SP YqxI -BlChiA A472TThe enzyme activity of the fermentation supernatant of the engineered bacteria was 1.66 U / mL, WS9-pHY300PLK-P SpoVG -SP YqxI -BlChiA E336K / A472T The enzyme activity of the fermentation supernatant of the engineered bacteria was the highest, at 2.67 U / mL. After purification, its specific activity was measured. The specific activity of the mutant E336K / A472T was unchanged compared with that of the wild-type chitinase expressed by WS9-pHY300PLK-P SpoVG -SP YqxI -BlChiA

[0078] Example 4: Characterization of the enzymatic properties of chitinase mutant E336K / A472T

[0079] The DNS method was used to determine the optimal reaction pH, pH stability, optimal reaction temperature, and thermal stability of the recombinant enzyme

[0080] The range for determining the optimal reaction pH and pH stability was pH 3.0 - 10.0. The pH stability was measured after incubating the enzyme solution at 4°C for 24 h

[0081] The optimal reaction temperature of the recombinant enzyme was determined at 30 - 70°C. After incubating at its optimal reaction temperature for 3 h, samples were taken every 30 min to measure the thermal stability of the recombinant enzyme

[0082] When determining the optimal reaction pH and optimal reaction temperature of the recombinant enzyme, the highest measured enzyme activity 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

[0083] When determining the pH stability and thermal 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 remaining enzyme activity

[0084] Compared with the wild-type, the chitinase mutant E336K / A472T had better acid tolerance. The optimal reaction pH decreased to 5.0 and it had higher enzyme activity under acidic conditions. The mutant had better stability in the range of pH 3.0 - pH 6.0, with a residual enzyme activity of over 90%, indicating an enhanced adaptation to acidic environments. At the same time, the mutant had the same high optimal reaction temperature as the wild-type, which was beneficial for the industrial production and application of chitinase BlChiA

[0085] Comparative Example 1:

[0086] The genetically engineered bacterium WS9-pHY300PLK-P constructed as in Example 3 SpoVG -SP YqxI -BlChiA E336K and WS9-pHY300PLK-PSpoVG -SP YqxI -BlChiA A472T Expression, purification, and enzymatic property characterization were carried out in the same manner. The results showed that the optimal pH of chitinase mutant E336K was 6.0. When its pH stability was measured, it was found that the residual enzyme activity of mutant E336K was above 90% at pH 5.0 - pH 8.0; the optimal pH of mutant A472T was 5.0. When its pH stability was measured, it was found that the residual enzyme activity of mutant A472T was above 90% at pH 4.0 - pH 8.0. There was no significant difference in the pH stability between mutant E336K and A472T and the wild type.

[0087] 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 BlChiA mutant, characterized in that, the mutant is: glutamic acid at position 336 of the amino acid shown in SEQ ID NO.4 is mutated to lysine, and alanine at position 472 is mutated to threonine.

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

3. A recombinant vector carrying the gene according to claim 2, characterized in that, The vector is a pHY300PLK series vector; the vector contains a promoter P with the nucleotide sequence shown in SEQ ID NO.5 SpoVG , and an SP encoded by the nucleotide sequence shown in SEQ ID NO.6 YqxI as the signal peptide.

4. A recombinant Bacillus subtilis expressing chitinase BlChiA, characterized in that, using Bacillus subtilis WS9 as the host and carrying the recombinant vector according to claim 3.

5. A method for expressing chitinase, characterized in that, fermentation is carried out using the recombinant Bacillus subtilis according to claim 4.

6. The method for expressing chitinase according to claim 5, characterized in that, The recombinant Bacillus subtilis cultured under the conditions of 30-38°C and 180-220 rpm for 6-12 h was transferred into the fermentation medium at an inoculum amount of 1-10%, and cultured at 30-37°C until OD 600 reached 0.6-0.8, and fermented at 30-35°C and 200-220 rpm for 36-60 h.

7. The application of the chitinase BlChiA mutant according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, the recombinant Bacillus subtilis according to claim 4, or the method for expressing chitinase according to claim 5 or 6 in chitin degradation.

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