Chitin deacetylase mutant with improved acid resistance, encoding gene, recombinant expression plasmid, genetically engineered bacteria and preparation method

Abstract

The application discloses a chitin deacetylase mutant with improved acid resistance, a coding gene, a recombinant expression plasmid, a genetically engineered bacterium and a preparation method. The amino acid sequence of the chitin deacetylase AnCDA is subjected to the following mutations: the 119th leucine (Leu) is mutated into threonine (Thr); and / or, the 139th phenylalanine (Phe) is mutated into glycine (Gly); and / or, the 166th tyrosine (Tyr) is mutated into tryptophan (Trp). The chitin deacetylase mutant obtained in the application has significantly improved stability under acidic conditions, the half-life t1 / 2 is prolonged by nearly 3 times at pH 5.0, the optimal catalytic pH is reduced from 6.0 to 5.0, and the chitin deacetylase mutant can be applied to catalyzing chitin deacetylation under acidic conditions to prepare chitosan and other chitin products, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, specifically to a chitin deacetylase mutant with enhanced acid resistance, the encoding gene, a recombinant expression plasmid, genetically engineered bacteria, and a preparation method. Background Technology

[0002] Chitosan is a product of partial deacetylation of chitin. Due to its good biodegradability, biocompatibility, non-toxicity and antibacterial properties, it has been widely used in many fields such as food, medicine, medical equipment, agriculture and environmental protection.

[0003] In production, chitosan is commonly prepared by removing acetyl groups from chitin using a hot alkaline method. However, this method produces chitosan with inconsistent degrees of deacetylation and molecular weight, and it uses high concentrations of sodium hydroxide, causing significant environmental pollution. Chitosan deacetylase can catalyze the hydrolysis of N-acetamide groups in chitin to generate chitosan. Compared to chemical methods, this method offers controllable degrees of deacetylation, uniform molecular weight, milder reaction conditions, fewer byproducts, and is more environmentally friendly. Therefore, the biocatalytic method mediated by chitosan deacetylase is the mainstream approach for chitosan production.

[0004] However, in the preparation of chitosan catalyzed by chitin deacetylase, chitin, as the substrate, has a highly extended hydrogen-bonded semi-crystalline structure, making it poorly soluble in water. The heterogeneous system reduces the binding between the deacetylase and the substrate, resulting in poor deacetylase activity and a low deacetylation rate. Therefore, acid is often added to the reaction solution to improve chitin solubility. Simultaneously, the acetic acid produced by deacetylation further increases the acidity of the reaction solution. However, chitin deacetylase, as a protein, is unstable in acidic solutions and has a short half-life, greatly limiting its application. Therefore, to realize the industrial application of chitin deacetylase, its acid resistance must be improved. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to solve the defect of low acid resistance of wild-type chitin deacetylase in production and application. By using enzyme engineering methods to molecularly modify the enzyme, a chitin deacetylase mutant with higher acid resistance can be obtained, thereby enhancing its applicability in industrial production.

[0006] To achieve the above objectives, the present invention provides a chitin deacetylase mutant with enhanced acid resistance. The chitin deacetylase is chitin deacetylase AnCDA derived from Aspergillus niger with NCBI number XP_001395517.1. The chitin deacetylase mutant is obtained by mutating the amino acid sequence of chitin deacetylase AnCDA as follows:

[0007] The leucine residue at position 119 (Leu) is mutated to threonine (Thr);

[0008] And / or, the phenylalanine (Phe) at position 139 is mutated to glycine (Gly);

[0009] And / or, the 166th tyrosine (Tyr) is mutated to tryptophan (Trp).

[0010] It should be noted that whether a mutation occurs at one, two, or all three sites, the corresponding chitin deacetylase mutant exhibits improved acid resistance compared to the wild type (i.e., the chitin deacetylase AnCDA derived from Aspergillus niger, NCBI number XP_001395517.1). Furthermore, among the various chitin deacetylase mutants mentioned above, the chitin deacetylase mutant with mutations at all three sites shows a greater degree of improvement in acid resistance, with significantly higher specific enzyme activity and residual enzyme activity after acid treatment compared to the wild type.

[0011] Based on this, the present invention further prefers a chitin deacetylase mutant with mutations at all three sites. That is, the preferred chitin deacetylase mutant here is based on the chitin deacetylase AnCDA with the amino acid sequence as shown in SEQ ID No:1, with mutations at positions 119, 139 and 166 in accordance with the above-mentioned mutation method. Specifically, the amino acid sequence of the mutant after the three mutations is shown in SEQ ID No:3.

[0012] The present invention also provides a coding gene for a chitin deacetylase mutant as described above, wherein the coding gene for the chitin deacetylase mutant is formed by a mutation in the nucleotide sequence shown in SEQ ID No:2 as follows:

[0013] The C at position 355 mutates to A, and the T at position 356 mutates to C;

[0014] And / or, the T at position 415 mutates to G, and the T at position 416 mutates to G;

[0015] And / or, the A at position 497 is mutated to G, and the C at position 498 is mutated to G.

[0016] That is, the codon encoding the corresponding mutated amino acid also undergoes a corresponding mutation. Similarly, there are various gene encoding chitin deacetase mutants obtained based on the base mutations in one, two, or three codons. Preferably, all three codons are mutated, and the corresponding nucleotide sequence of the gene encoding the chitin deacetase mutant is shown in SEQ ID No:4.

[0017] The present invention also provides a recombinant expression plasmid comprising the coding gene of the chitin deacetylase mutant as described above.

[0018] The present invention also provides a genetically engineered bacterium comprising the recombinant expression plasmid as described above.

[0019] The present invention further provides a method for preparing the chitin deacetylase mutant as described above. The method specifically includes: using a recombinant plasmid carrying the encoding gene of chitin deacetylase AnCDA from Aspergillus niger as a template, performing a whole plasmid PCR reaction, designing mutation primer pairs, constructing a site-directed mutant (i.e. obtaining the encoding gene of the chitin deacetylase mutant), and transforming it into Escherichia coli to obtain the chitin deacetylase mutant.

[0020] Specifically, the mutant primer pairs here include at least one of a first primer pair, a second primer pair, and a third primer pair, wherein the first primer pair is shown as SEQ ID No:5 and SEQ ID No:6, the second primer pair is shown as SEQ ID No:7 and SEQ ID No:8, and the third primer pair is shown as SEQ ID No:9 and SEQ ID No:10.

[0021] Preferably, the mutation primer pair here includes a first primer pair, a second primer pair, and a third primer pair to achieve mutations in three codons in the coding gene of the chitin deacetylase mutant, thereby obtaining a chitin deacetylase mutant with mutations in amino acids at positions 119, 139, and 166.

[0022] The beneficial effects of the present invention through the above technical solution are as follows:

[0023] Compared with existing technologies, this invention uses *E. coli* as the expression host to recombinantly express the chitin deacetylase AnCDA gene derived from *Aspergillus niger*, and performs the following mutations: leucine (Leu) at position 119 is mutated to threonine (Thr), phenylalanine (Phe) at position 139 is mutated to glycine (Gly), and tyrosine (Tyr) at position 166 is mutated to tryptophan (Trp). Compared with the wild-type enzyme, the mutant chitin deacetylase (i.e., the chitin deacetylase mutant obtained in this invention) exhibits significantly improved stability under acidic conditions, with a nearly 3-fold increase in half-life (t1 / 2) at pH 5.0, and the optimal catalytic pH decreasing from 6.0 to 5.0. The chitin deacetylase mutant provided by this invention is suitable for catalyzing the deacetylation reaction of chitin under acidic conditions to prepare chitosan and other chitin-based products, and has good prospects for industrial application. Attached Figure Description

[0024] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0025] Figure 1 The graph shows the detection results of the optimal pH for detecting the wild-type enzyme in Example 1 and the chitin deacetylase mutant (triple mutant) provided by the present invention.

[0026] Figure 2 This is a graph showing the detection results of pH stability of the wild-type enzyme in Example 2 and the chitin deacetylase mutant (triple mutant) provided by the present invention;

[0027] Figure 3 This is a graph showing the change in enzyme activity over time in an acidic environment between the wild-type enzyme in Example 3 and the chitin deacetylase mutant (triple mutant) provided by the present invention. Detailed Implementation

[0028] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0029] In the following examples, E. coli BL21(DE3) was purchased from Takara Bio Engineering (Dalian) Co., Ltd., product number 9126; pET-28a was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B540183; p-nitroacetanilide was purchased from Aladdin Biotech (Shanghai) Co., Ltd., product number N159511; and all other reagents and raw materials were conventional commercial products.

[0030] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH adjusted to 7.0, autoclaved for 20 min, ready for use.

[0031] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, pH adjusted to 7.0, autoclaved for 20 min, ready for use.

[0032] The following examples illustrate the methods for constructing mutant PCR and mutant chitin deacetase recombinant bacteria (i.e., genetically engineered bacteria):

[0033] The PCR reaction system was: 5×Prime STAR Buffer (Mg 2+10 μL of Plus, dNTP Mix (2.5 mM for each nucleotide), 0.5 μL of mutant primer, 0.5 μL of template (recombinant plasmid), 0.5 μL of Prime STAR DNA polymerase, and water to a final volume of 50 μL; PCR reaction conditions: 95℃ pre-denaturation for 5 min, followed by 25 cycles (95℃, 15 s; 55℃, 15 s; 72℃, 7 min), and a final extension at 72℃ for 10 min. The mutant primer pairs used are shown in Table 1. The underlined portions in Table 1 indicate the codons used to encode the mutant amino acids, and NNS indicates non-single codons that can encode different amino acid types. That is, SEQ ID No:11-SEQ ID No:16 are not single, definitive primer sequences; different bases referred to by NNS can be used to encode different amino acids at that site.

[0034]

[0035]

[0036] After the PCR product was positive by 0.96% agarose gel electrophoresis, 20 μL of the PCR reaction solution was taken and 1 μL of DpnⅠ was added. The template plasmid was removed by enzyme digestion at 37℃ for 3 h, and the plasmid was inactivated at 65℃ for 10 min. The plasmid was then transformed into E. coli BL21(DE3) competent cells and plated on LB agar plates containing kanamycin (final concentration of 50 μg / mL) and incubated overnight at 37℃. Sequencing by Sangon Biotech (Shanghai) Co., Ltd. confirmed that the mutated chitin deacetylase recombinant bacteria was obtained.

[0037] The detection methods involved in the following embodiments are as follows:

[0038] Methods for detecting chitin deacetylase activity:

[0039] Take 1 mL of 200 mg / L p-nitroacetanilide solution and 3 mL of 0.05 mol / L pH 7.0 phosphate buffer into a 25 mL test tube. Incubate at 50°C for 10 min. Add 1 mL of enzyme solution, shake well, and incubate at 50°C for 15 min for enzymatic reaction. Finally, terminate the reaction by boiling in a water bath, centrifuge at 6000 rpm for 10 min, and measure the absorbance of the supernatant at 400 nm. Use 1 mL of the same concentration of enzyme solution inactivated by boiling in a water bath for 10 min as a control. Chitin deacetylase activity is defined as the amount of enzyme required to produce 1 μg of nitrophenylamine per hour under the above reaction conditions, defined as 1 unit of enzyme activity (U / mL).

[0040] Protein concentration determination method:

[0041] Prepare a protein standard curve according to the instructions of the protein concentration assay kit and determine the protein concentration of the purified enzyme. Calculate the protein concentration of each sample based on the absorbance of the microplate reader at 595 nm and the standard curve.

[0042] Specific activity assay method: Calculate specific activity according to the following formula:

[0043] Enzyme activity (U / mg) = Enzyme activity (U / mL) / Protein content (mg / mL)

[0044] The method for detecting the acid resistance of chitin deacetylase used in the following examples is as follows:

[0045] Chitin deacetylase was dissolved in 0.05 mol / L citrate buffer at pH 3.0 and incubated at 25°C for 0.5 h for acid treatment. The residual enzyme activity of the acid-treated chitin deacetylase was determined using the enzyme activity assay method described above. The relative residual enzyme activity was calculated by using chitin deacetylase of the same concentration but not acid-treated as a control.

[0046] Take 1 mL of 200 mg / L p-nitroacetanilide solution and 3 mL of 0.05 mol / L pH 7.0 phosphate buffer into a 25 mL test tube. Incubate at 40 °C for 10 min. Add 1 mL of enzyme solution, shake well, and incubate the reaction at 40 °C for 15 min. Finally, terminate the reaction by boiling in a water bath. Centrifuge at 6000 rpm for 10 min and measure the absorbance of the supernatant at 400 nm. Use 1 mL of enzyme solution of the same concentration, inactivated by boiling in a water bath for 10 min, as a control.

[0047] Example 1: Construction and expression of genetically engineered bacteria containing wild-type chitin deacetylase

[0048] 1. Construction of the genetically engineered strain E. coli BL21(DE3) / pET-28a-ancda

[0049] The chitin deacetase AnCDA gene from *Aspergillus niger* (NCBI ID XP_001395517.1) was codon-optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and its nucleotide sequence is shown in SEQ ID No:2. This enzyme gene was inserted between the NcoⅠ and XhoⅠ restriction endonuclease sites in the multiple cloning site of the pET-28a vector to construct the recombinant expression plasmid pET-28a-ancda. This plasmid was transformed into *E. coli* BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin (prepared using the aforementioned LB solid medium), and incubated overnight at 37°C. Single colonies were randomly selected for colony PCR identification, and positive clones were sequenced for verification, yielding the genetically engineered strain *E. coli* BL21(DE3) / pET-28a-ancda.

[0050] 2. Inducible expression of chitin deacetylase AnCDA

[0051] The genetically engineered E. coli BL21(DE3) / pET-28a-ancda strain was inoculated into LB medium with a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 10 h to obtain a seed culture. The seed culture was then transferred at a volume concentration of 4% to fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm until the bacterial cell concentration OD600 reached 0.8. Lactose was added to a final concentration of 6 g / L, and the culture was induced at 28°C and 150 rpm for 12 h. After centrifugation, the supernatant was discarded, and the precipitate was collected to obtain recombinant cells containing chitin deacetylase.

[0052] Example 2: Preparation, expression, and screening of chitin deacetylase single mutants (i.e., mutations occurring at only one site).

[0053] 1. Preparation of a single mutant

[0054] The chitin deacetylase AnCDA gene (NCBI Reference Sequence: XP_001395517.1) underwent homology modeling, and combined with sequence and three-dimensional structure analysis, potential sites in the enzyme molecule that might affect acid tolerance (Leu at position 119, Phe at position 139, and Tyr at position 166) were selected as mutation sites. Using the recombinant plasmid pET-28a-ancda as a template, primer pairs L119-F and L119-R (a pair of mutant primers shown in SEQ ID No: 11 and SEQ ID No: 12), F139-F and F139-R (a pair of mutant primers shown in SEQ ID No: 13 and SEQ ID No: 14), and Y166-F and Y166-R (SEQ ID No: 15 and SEQ ID No: 166) were used, respectively. Mutant PCR amplification was performed using the mutant primer pair shown in No. 16 to obtain single saturated mutant plasmids containing positions 119, 139, and 166, respectively (i.e., the mutant plasmid here has a mutation at position 119, position 139, or position 166; and the mutant amino acid at each site can be any one of 20 amino acids, meaning that a single mutation at each mutation site has 20 different mutant types). The template plasmid was removed by DpnI digestion and transformed into *E. coli*. BL21(DE3) competent cells were plated on LB agar plates containing 50 μg / mL kanamycin and cultured overnight at 37°C. Single colonies were obtained from the plates. Single colonies were randomly picked and inoculated into LB medium for seed culture. The bacterial strains were preserved, and plasmids were extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. All 57 species (excluding the wild type corresponding to each site, there are a total of 57 single-site mutants) of chitin deacetylase recombinant bacteria with single amino acid site mutants were obtained, forming a single-site mutant strain library.

[0055] 2. Expression and purification of single-site mutant enzymes and wild-type enzymes

[0056] The strains from the single-site mutant strain library and wild-type strains (a total of 58 strains) were inoculated into LB medium containing kanamycin at a final concentration of 50 μg / mL, and cultured and induced for expression as described in Example 1. The centrifuged cells were resuspended in buffer A (0.02 mol / L, pH 8.0 phosphate buffer containing 0.50 mol / L NaCl and 0.02 mol / L imidazole) to a wet cell concentration of 50 g / L. The cell suspension was placed in an ice bath and sonicated at 300 W for 10 min, followed by centrifugation at 12000 rpm and 4°C for 20 min to remove cell debris. The supernatant was collected and loaded onto a pre-packed gravity column (5 mL) of Ni-NTA affinity chromatography equilibrated with buffer A. The column was washed with 25 mL of buffer A to remove unbound proteins, and then... Chitin deacetylase protein was eluted with 0.02 mol / L, pH 8.0 phosphate buffer containing 0.50 mol / L NaCl and 0.50 mol / L imidazole, followed by overnight dialyzing in 0.02 mol / L, pH 8.0 phosphate buffer. All purification steps were performed at 0–4 °C to obtain wild-type and mutant chitin deacetylase protein solutions.

[0057] 3. Screening for single mutant enzymes

[0058] The specific enzyme activity and acid resistance of the above wild-type and mutant chitin deacetylase protein solutions were determined, as shown in Table 2. Compared with wild-type chitin deacetylase, L119T, F139G and Y166W showed increased specific enzyme activity and acid resistance, and were the best single mutants at positions 119, 139 and 166 of chitin deacetylase, respectively.

[0059]

[0060]

[0061] Example 3: Preparation, expression, and screening of chitin deacetylase combinatorial mutants (mutations at two or three sites).

[0062] Using the recombinant plasmid pET-28a-ancda-L119T extracted from the L119T mutant strain obtained in Example 2 (i.e., the single saturated mutant plasmid in which leucine L at position 119 is mutated to threonine T during the preparation of the single mutant in Example 2) as a template, primer pairs F139G-F and F139G-R (a pair of mutant primer pairs shown in SEQ ID No:7 and SEQ ID No:8), Y166W-F and Y166W-R (SEQ ID No:9 and SEQ ID No:8) from Table 1 were used respectively. Mutation PCR amplification was performed using the pair of mutant primers shown in SEQ ID No: 10 to obtain recombinant plasmids pET-28a-ancda-L119T / F139G and pET-28a-ancda-L119T / Y166W containing a dual-site combined mutant chitin deacetylase gene, respectively. Using the recombinant plasmid pET-28a-ancda-F139G extracted from the F139G-mutant bacteria obtained in Example 2 as a template, mutation PCR amplification was performed using primer pairs Y166W-F and Y166W-R (the pair of mutant primers shown in SEQ ID No: 9 and SEQ ID No: 10) to obtain the recombinant plasmid pET-28a-ancdk-F139G / Y166W containing a dual-site combined mutant chitin deacetylase gene. The PCR reaction solution containing the double-mutant recombinant plasmid was digested with DpnI to remove the template, transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin and cultured overnight at 37°C. Single colonies were obtained from the plates, and positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The chitin deacetylase double-mutant recombinant bacteria E. coli BL21 / pET-28a-ancda-L119T / F139G, E. coli BL21 / pET-28a-ancda-L119T / Y166W, and E. coli BL21 / pET-28a-ancda-F139G / Y166W were obtained. The double-mutant strains were preserved and plasmids were extracted. Using the double mutant plasmid pET-28a-ancda-L119T / F139G as a template, mutant PCR amplification was performed using primer pairs Y166W-F and Y166W-R (a pair of mutant primers shown in SEQ ID No:9 and SEQ ID No:10) in Table 1 to obtain the recombinant plasmid pET-28a-ancda-L119T / F139G / Y166W containing the three-site combined mutant chitin deacetylase gene.The PCR reaction solution containing the triple-mutant recombinant plasmid was digested with DpnI to remove the template, transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Single colonies were obtained, and positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain the triple-mutant recombinant chitin deacetylase strain E. coli BL21 / pET-28a-ancda-L119T / F139G / Y166W. The triple-mutant strain was preserved and the plasmid was extracted. The obtained double-mutant and triple-mutant strains were inoculated into LB medium, cultured, induced to express, and purified according to the above method. The specific activity and acid resistance of the combined mutant enzyme were measured and compared with wild-type chitin deacetylase. As shown in Table 3, compared with other single mutant enzymes, the combined mutants obtained better specific enzyme activity and acid resistance. Among them, the residual enzyme activity of the three-site combined mutant L119T / F139G / Y166W after acid treatment was still maintained at 73.31%, which is 2.28 times that of the wild enzyme and is also the highest among the mutant enzymes. At the same time, it maintained a high specific enzyme activity of 1.45 times that of the wild enzyme, making it the best mutant enzyme.

[0063] Table 3

[0064] enzymes Enzyme activity (U / mg) Residual enzyme activity (%) Wild enzymes 1.27 32.61 L119T / F139G (double mutation) 2.12 43.23 L119T / Y166W (double mutant) 1.79 55.72 F139G / Y166W (double mutation) 1.55 61.25 L119T / F139G / Y166W (three mutations) 1.84 71.53

[0065] Example 1: Determination of the optimal pH for chitin deacetylase AnCDA and its mutants

[0066] Take 1 mL each of the purified wild-type enzyme (i.e., chitin deacetylase AnCDA) and the mutant enzyme L119T / F139G / Y166W (triple mutant), and dissolve them in 9 mL of different 0.05 mol / L buffers (pH 3.0-6.0 citrate buffer; pH 6.5-8.0 phosphate buffer; pH 8.5-9.0 Tris-HCl buffer). Perform enzyme activity assays under different buffer conditions, and calculate the relative enzyme activity with the highest activity as 100%. Figure 1 As shown, after the above three-point combination mutation, the optimal pH of chitin deacetylase shifted from 6.0 to the acidic range, becoming 5.0.

[0067] Example 2: pH stability characterization of chitin deacetylase AnCDA and its mutants

[0068] Take 1 mL each of wild-type chitin deacetylase and its mutants L119T / F139G / Y166W (triple mutant), and dissolve them in 9 mL of different 0.05 mol / L buffer solutions (pH 3.0-6.0 citrate buffer; pH 6.0-8.0 phosphate buffer; pH 8.0-9.0 Tris-HCl buffer). Incubate at 25°C for 30 min, and then determine the residual enzyme activity of the acid-treated chitin deacetylase using the enzyme activity assay method described above. The enzyme activity of the untreated enzyme solution is defined as 100%, and the ratio of the enzyme activity of the treated enzyme solution to the enzyme activity of the untreated enzyme solution is defined as the residual enzyme activity. Figure 2 As shown, compared with the wild-type enzyme, the mutant enzyme L119T / F139G / Y166W retains over 70% of its residual enzyme activity within the pH range of 3.0-8.0, and particularly over 80% within the pH range of 5.0-7.0, a significant improvement over the approximately 60% of the wild-type enzyme. This indicates that the mutant chitin deacetylase L119T / F139G / Y166W has a wide pH tolerance range, which is beneficial for the application of chitin deacetylases.

[0069] Example 3: Determination of the half-life of chitin deacetylase AnCDA and its mutant in pH 5.0 solution.

[0070] Determination of half-life (t1 / 2): The wild-type chitin deacetase AnCDA obtained in Example 2 and the purified enzyme solutions of the mutant L119T / F139G / Y166W (triple mutant) obtained in Example 3 were incubated in citrate buffer (0.05 mol / L, pH 5.0) for 3 h. Samples were taken at regular intervals to determine residual enzyme activity, and inactivation curves were plotted. Figure 3 As shown. From Figure 3 It can be seen that the catalytic activity of both wild-type chitin deacetylase and mutant L119T / F139G / Y166W decreased rapidly.

[0071] The half-life (t1 / 2) is calculated using the first-order inactivation equation: ln(residual enzyme activity) = -kDt, t1 / 2 = ln2 / kD (where kD represents the inactivation constant and t represents the incubation time).

[0072] like Figure 3 Calculations show that, at 25°C and pH 5.0, the half-life of the wild-type chitin deacetylase is approximately 0.46 h, while the half-life of the mutant enzyme L119T / F139G / Y166W is approximately 1.45 h, about three times that of the wild-type enzyme. This demonstrates that the chitin deacetylase mutant obtained through the mutation of this invention exhibits significantly enhanced acid resistance.

[0073] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0074] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0075] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. An acid resistance-improved chitin deacetylase mutant, the chitin deacetylase being chitin deacetylase of Aspergillus niger origin having NCBI accession number XP_001395517.1 An CDA, the chitin deacetylase An The amino acid sequence of the CDA is shown in SEQ ID No: 1, characterized in that, The chitin deacetylase mutant is a chitin deacetylase An The amino acid sequence of CDA was mutated as follows: Leucine (Leu) at position 119 is mutated to threonine (Thr); and / or, phenylalanine (Phe) at position 139 is mutated to glycine (Gly); and / or, tyrosine (Tyr) at position 166 is mutated to tryptophan (Trp).

2. The chitin deacetylase mutant according to claim 1, characterized in that, The amino acid sequence of the chitin deacetylase mutant is shown as SEQ ID No:

3.

3. A gene encoding the chitin deacetylase mutant of claim 1, wherein The coding gene of the chitin deacetylase mutant is mutated from the nucleotide sequence shown as SEQ ID No: 2 as follows: C at position 355 is mutated to A, and T at position 356 is mutated to C; and / or, T at position 415 is mutated to G, and T at position 416 is mutated to G; and / or, A at position 497 is mutated to G, and C at position 498 is mutated to G.

4. The chitin deacetylase mutant encoding gene according to claim 3, wherein, The nucleotide sequence of the coding gene of the chitin deacetylase mutant is shown as SEQ ID No:

4.

5. A recombinant expression plasmid or genetically engineered bacteria comprising the coding gene of the chitin deacetylase mutant according to claim 3 or 4.

6. A method for producing the chitin deacetylase mutant according to claim 1, which is characterized by, The preparation method is to design a pair of mutant primers, and the chitin deacetylase An The chitin deacetylase mutant is obtained by expressing the gene encoding CDA after site-directed mutation.

7. The preparation method according to claim 6, characterized in that, The mutant primer pair comprises at least one of a first primer pair, a second primer pair and a third primer pair, and the first primer pair is shown as SEQ ID No: 5 and SEQ ID No: 6, the second primer pair is shown as SEQ ID No: 7 and SEQ ID No: 8, and the third primer pair is shown as SEQ ID No: 9 and SEQ ID No:

10.

8. The production method according to claim 6 or 7, characterized by, The expression host is Escherichia coli.

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