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

Abstract

The invention 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 chitin deacetylase mutant is obtained by mutating an amino acid sequence of chitin deacetylase AnCDA as follows: leucine (Leu) at the 119th site is mutated into threonine (Thr); and / or, the 139th site phenylalanine (Phe) is mutated into glycine (Gly); and / or, tyrosine (Tyr) at the 166th point is mutated into tryptophan (Trp). The stability of the obtained chitin deacetylase mutant under the acidic condition is remarkably improved, the half-life period t1 / 2 is prolonged by nearly 3 times when the pH value is 5.0, the optimal catalysis pH value is reduced to 5.0 from 6.0, and the chitin deacetylase mutant can be suitable for catalyzing chitin deacetylation reaction under the acidic condition to prepare chitosan and other chitin products and has good industrial application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to a chitin deacetylase mutant with improved acid resistance, a coding gene, a recombinant expression plasmid, a genetically engineered bacterium and a preparation method. Background Art

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

[0003] In production, the hot alkali method is commonly used to remove the acetyl group of chitin to prepare chitosan. The chitosan produced by this method has uneven deacetylation degree and molecular weight, and uses high concentration of sodium hydroxide, which seriously pollutes the environment. Chitin deacetylase can catalyze the hydrolysis of N-acetamide groups in chitin to produce chitosan. Compared with the chemical method, the product has controllable deacetylation degree, uniform molecular weight, mild reaction conditions, few by-products, and is environmentally friendly. Therefore, the chitin deacetylase-mediated biocatalysis method is the mainstream direction for preparing chitosan.

[0004] However, in the chitin deacetylase catalyzed chitosan preparation process, chitin as a substrate has a highly extended hydrogen bond semi-crystalline structure, so it is insoluble in water, and the heterogeneous system reduces the combination of deacetylase and substrate, resulting in poor deacetylase effect and low deacetylation rate. Based on this, the solubility of chitin is often increased by adding acid to the reaction solution. At the same time, the acetic acid produced by removing the acetyl group can further increase the acidity of the reaction solution. However, the chitin deacetylase as a protein is unstable in acid solution and has a short half-life, which greatly limits its application. Therefore, in order 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 the present invention is to solve the defect of low acid resistance faced by wild-type chitin deacetylase in the production and application process, and to obtain chitin deacetylase mutants with higher acid tolerance through enzyme engineering methods, thereby enhancing its applicability in industrial production.

[0006] In order to achieve the above object, the present invention provides a chitin deacetylase mutant with improved acid resistance, wherein the chitin deacetylase is chitin deacetylase AnCDA derived from Aspergillus niger with NCBI number XP_001395517.1, and the chitin deacetylase mutant is obtained by subjecting the amino acid sequence of the chitin deacetylase AnCDA to the following mutations:

[0007] Leucine (Leu) at position 119 was mutated to threonine (Thr);

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

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

[0010] It should be noted that, in the above three sites, one mutation occurs, or two mutations occur, or all three mutations occur, and the acid resistance of the corresponding chitin deacetylase mutant is improved compared to the wild type (i.e., chitin deacetylase AnCDA with NCBI number XP_001395517.1 from Aspergillus niger). Further, among the above-mentioned multiple chitin deacetylase mutants, the acid resistance of the chitin deacetylase mutant in which all three sites are mutated is higher, and its specific enzyme activity and residual enzyme activity after acid treatment are more obvious than the wild type.

[0011] Based on this, the present invention further prefers that the chitin deacetylase mutant is a chitin deacetylase mutant with mutations at three sites, that is, the preferred chitin deacetylase mutant here is based on the chitin deacetylase AnCDA with an amino acid sequence as shown in SEQ ID No: 1, and its 119th, 139th and 166th positions are mutated according to the above mutation method. Specifically, the amino acid sequence of the mutant after the triple mutation is shown in SEQ ID No: 3.

[0012] The present invention also provides a gene encoding the chitin deacetylase mutant as described above, wherein the gene encoding the chitin deacetylase mutant is obtained by subjecting the nucleotide sequence shown in SEQ ID No: 2 to the following mutation:

[0013] The 355th position was mutated from C to A, and the 356th position was mutated from T to C;

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

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

[0016] That is, the codons encoding the corresponding mutant amino acids also undergo corresponding mutations. Similarly, the coding gene of the chitin deacetylase mutant obtained based on the base mutation in one codon, two codons or three codons also has a variety. Preferably, here, three codons all mutate, and correspondingly, the nucleotide sequence of the coding gene of the chitin deacetylase mutant is as 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 described above.

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

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

[0020] Specifically, the mutation primer pair here includes 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.

[0021] Preferably, the mutation primer pair here includes a first primer pair, a second primer pair and a third primer pair to achieve mutation of three codons in the gene encoding the chitin deacetylase mutant, thereby obtaining a chitin deacetylase mutant in which the 119th, 139th and 166th amino acids are mutated.

[0022] Through the above technical solution, the beneficial effects of the present invention are:

[0023] Compared with the prior art, the present invention uses Escherichia coli as an expression host, recombinantly expresses the chitin deacetylase AnCDA gene derived from Aspergillus Niger, and performs the following mutations: 119 leucine (Leu) is mutated to threonine (Thr), 139 phenylalanine (Phe) is mutated to glycine (Gly), 166 tyrosine (Tyr) is mutated to tryptophan (Trp). Compared with the wild enzyme, the mutant chitin deacetylase (i.e. the chitin deacetylase mutant obtained by the present invention) has significantly improved stability under acidic conditions, the half-life t1 / 2 is extended by nearly 3 times when the pH is 5.0, and the optimal catalytic pH is reduced from 6.0 to 5.0. The chitin deacetylase mutant provided by the present invention is suitable for catalyzing the chitin deacetylation reaction under acidic conditions to prepare chitosan and other chitin products, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention, but do not constitute a limitation of the present invention. In the accompanying drawings:

[0025] Figure 1 It is a test result diagram of the optimal pH of the wild enzyme in Test Example 1 and the chitin deacetylase mutant (triple mutation) provided by the present invention;

[0026] Figure 2 It is a test result diagram of the pH stability of the wild enzyme in Test Example 2 and the chitin deacetylase mutant (triple mutation) provided by the present invention;

[0027] Figure 3 It is a curve diagram showing the change of enzyme activity over time of the wild enzyme in detection example 3 and the chitin deacetylase mutant (triple mutation) provided by the present invention in an acidic environment. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0029] In the following examples, Escherichia coli BL21 (DE3) was purchased from Bao Biotechnology (Dalian) Co., Ltd. with a serial number of 9126; pET-28a was purchased from Sangon Biotechnology (Shanghai) Co., Ltd. with a serial number of B540183; p-nitroacetanilide was purchased from Aladdin Biochemical Technology (Shanghai) Co., Ltd. with a serial number of N159511; and the remaining reagents and raw materials were conventional commercial products.

[0030] LB liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH adjusted to 7.0, steam sterilized under high pressure for 20 min, and set aside.

[0031] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, pH adjusted to 7.0, steam sterilized under high pressure for 20 min, and set aside.

[0032] The construction methods of the mutant PCR and mutant chitin deacetylase recombinant bacteria (i.e., genetically engineered bacteria) involved in the following examples are as follows:

[0033] PCR reaction system: 5× Prime STAR Buffer (Mg 2+Plus) 10 μL, dNTP Mix (each nucleotide 2.5mM), mutant primer 0.5 μL, template (recombinant plasmid) 0.5 μL, Prime STAR DNA polymerase 0.5 μL, add water to 50 μL; PCR reaction conditions are: 95 ° C pre-denaturation 5min, after 25 cycles (95 ° C, 15s, 55 ° C, 15s, 72 ° C, 7min), and finally 72 ° C for another 10min. The mutant primer pairs used are shown in Table 1. The underlined part in Table 1 represents the codon for encoding the mutant amino acid, and NNS represents a non-single codon that can encode different amino acid types, that is, SEQ ID No: 11-SEQ ID No: 16 is not a single determined primer sequence, and it can achieve the effect of encoding different amino acids at this site by adjusting the different bases referred to by NNS.

[0034]

[0035]

[0036] After the PCR reaction product was analyzed by 0.96% agarose gel electrophoresis and the PCR was positive, 20 μL of the PCR reaction solution was taken, 1 μL of DpnⅠ was added, and the template plasmid was removed by enzyme digestion at 37°C for 3 h, inactivated at 65°C for 10 min, transformed into E. coli BL21 (DE3) competent cells, coated with LB agar plates containing kanamycin (final concentration of 50 μg / mL), and incubated at 37°C overnight; sequencing was confirmed by Sangon Biotechnology (Shanghai) Co., Ltd. to obtain the mutant chitin deacetylase recombinant bacteria.

[0037] The detection method involved in the following embodiments is as follows:

[0038] Chitin deacetylase activity detection method:

[0039] Take 1mL of 200mg / L p-nitroacetanilide solution and 3mL of 0.05mol / L pH7.0 phosphate buffer in a 25mL test tube, keep warm in a 50℃ constant temperature water bath for 10min, add 1mL of enzyme solution, shake well and react at 50℃ for 15min, finally terminate the reaction in a boiling water bath, centrifuge at 6000r / min for 10min, and measure the absorbance of the supernatant at 400nm. Add 1mL of the same concentration of enzyme solution inactivated in a boiling water bath for 10min as a control. Definition of chitin deacetylase activity: Under the above reaction conditions, the amount of enzyme required to produce 1μg of nitrophenylamine per hour is defined as 1 enzyme activity unit (U / ml).

[0040] Protein concentration determination method:

[0041] A protein standard curve was prepared according to the instructions of the protein concentration determination kit and the protein concentration of the pure enzyme was determined. The protein concentration of each sample was calculated based on the absorbance of the sample at A595 nm on the microplate reader and the standard curve.

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

[0043] Specific enzyme activity (U / mg) = enzyme activity (U / mL) / protein content (mg / mL)

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

[0045] Chitin deacetylase was dissolved in 0.05 mol / L citric acid 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 above enzyme activity determination method; chitin deacetylase containing the same concentration but not acid-treated was used as a control to calculate the relative residual enzyme activity.

[0046] Take 1mL of 200mg / L p-nitroacetanilide solution and 3mL of 0.05mol / L pH7.0 phosphate buffer in a 25mL test tube, keep warm in a 40℃ constant temperature water bath for 10min, add 1mL of enzyme solution, shake well and react at 40℃ for 15min, finally terminate the reaction in a boiling water bath, centrifuge at 6000r / min for 10min, and measure the absorbance of the supernatant at 400nm. Use 1mL of enzyme solution with the same concentration inactivated in a boiling water bath for 10min as a control.

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

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

[0049] The chitin deacetylase AnCDA gene from Aspergillus niger with NCBI number 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. The above enzyme gene was inserted between the two restriction endonuclease sites NcoⅠ and XhoⅠ of the multiple cloning site of the pET-28a vector to construct a recombinant expression plasmid pET-28a-ancda, which was transformed into Escherichia coli E. coli BL21 (DE3) competent cells, coated with LB plates containing 50 μg / mL kanamycin (i.e., the LB solid medium prepared as mentioned above), and cultured at 37°C overnight, and single colony clones were randomly picked for colony PCR identification, and positive clones were sequenced and verified to obtain genetically engineered bacteria E. coli BL21 (DE3) / pET-28a-ancda.

[0050] 2. Inducible expression of chitin deacetylase AnCDA

[0051] The genetically engineered bacteria E. coli BL21 (DE3) / pET-28a-ancda was inoculated into LB medium with a final concentration of 50 μg / mL kanamycin, and cultured at 37 ° C, 180 rpm for 10 hours to obtain a seed solution, and the seed solution was transferred to a fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 4%, and cultured at 37 ° C, 180 rpm until the bacterial cell concentration OD600 was 0.8, and lactose was added at a final concentration of 6 g / L, and induced culture was carried out at 28 ° C, 150 rpm for 12 hours, centrifuged, 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., only one site mutated)

[0053] 1. Preparation of single mutants

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

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

[0056] The strains and wild-type strains (58 strains in total) in the above-mentioned single-site mutant strain library were inoculated into LB medium containing kanamycin at a final concentration of 50 μg / mL, respectively, and cultured and induced for expression as described in Example 1. The cells obtained by centrifugation were suspended 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, ultrasonically disrupted at a power of 300 W for 10 min, and centrifuged at 12000 r / min and 4° C. for 20 min to remove cell debris. The supernatant was collected and loaded into a Ni-NTA affinity chromatography preloaded gravity column (5 mL) equilibrated with buffer A. The column was washed with 25 mL of buffer A to remove unbound proteins, and then washed with buffer Chitin deacetylase protein was eluted with B (0.02 mol / L, pH 8.0 phosphate buffer containing 0.50 mol / L NaCl and 0.50 mol / L imidazole) and dialyzed overnight in phosphate buffer (0.02 mol / L, pH 8.0). The above purification steps were all carried out at 0-4°C to obtain wild-type and mutant chitin deacetylase protein solutions.

[0057] 3. Screening of single mutant enzymes

[0058] The specific enzyme activities and acid resistance of the wild-type and mutant chitin deacetylase protein solutions were measured. As shown in Table 2, compared with the wild-type chitin deacetylase, the specific enzyme activities and acid resistance of L119T, F139G and Y166W were increased, and they were the optimal single mutants at the 119th, 139th and 166th positions of chitin deacetylase, respectively.

[0059]

[0060]

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

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

[0063] Table 3

[0064] Enzymes Specific enzyme activity (U / mg) Residual enzyme activity (%) Wild enzyme 1.27 32.61 L119T / F139G (double mutation) 2.12 43.23 L119T / Y166W (double mutation) 1.79 55.72 F139G / Y166W (double mutation) 1.55 61.25 L119T / F139G / Y166W (triple mutation) 1.84 71.53

[0065] Detection Example 1: Determination of the Optimal pH of Chitin Deacetylase AnCDA and Its Mutants

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

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

[0068] Take 1 mL of wild-type chitin deacetylase and its mutant L119T / F139G / Y166W (triple mutation), dissolve in 9 mL and 0.05 mol / L different buffers (pH 3.0-6.0 citric acid 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 above enzyme activity determination method; 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 residual enzyme activity of the mutant enzyme L119T / F139G / Y166W in the pH range of 3.0-8.0 can reach more than 70%, especially in the pH range of 5.0-7.0, the residual enzyme activity is more than 80%, which is significantly improved compared to about 60% of the wild-type enzyme. This shows that the mutant chitin deacetylase L119T / F139G / Y166W has a wide pH tolerance range, which is conducive to the application of chitin deacetylase.

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

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

[0071] The half-life (t1 / 2) was calculated by 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 , it can be calculated that at 25°C and pH 5.0, the half-life of the wild-type chitin deacetylase is about 0.46h, and the half-life of the mutant enzyme L119T / F139G / Y166W is about 1.45h, which is about 3 times that of the wild-type enzyme. It can be seen that the acid resistance of the chitin deacetylase mutant obtained by the mutation of the present invention is significantly enhanced.

[0073] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to 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 further describe various possible combinations.

[0075] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A chitin deacetylase mutant with improved acid resistance, wherein the chitin deacetylase is chitin deacetylase AnCDA derived from Aspergillus niger with NCBI number XP_001395517.1, and the amino acid sequence of the chitin deacetylase AnCDA is shown in SEQ ID No: 1, characterized in that: The chitin deacetylase mutant is obtained by subjecting the amino acid sequence of the chitin deacetylase AnCDA to the following mutation: Leucine (Leu) at position 119 was 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 in SEQ ID No:

3.

3. A gene encoding a chitin deacetylase mutant as claimed in claim 1, characterized in that: The encoding gene of the chitin deacetylase mutant is a nucleotide sequence as shown in SEQ ID No: 2 with the following mutation: The 355th position was mutated from C to A, and the 356th position was mutated from T to C; and / or, T at position 415 mutates to G, and T at position 416 mutates to G; And / or, A at position 497 mutates to G, and C at position 498 mutates to G.

4. The gene encoding the chitin deacetylase mutant according to claim 3, characterized in that The nucleotide sequence of the gene encoding the chitin deacetylase mutant is shown in SEQ ID No:

4.

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

6. A method for preparing a chitin deacetylase mutant according to claim 1, characterized in that: The preparation method is to design a mutation primer pair, perform site-directed mutation on the coding gene of the chitin deacetylase AnCDA, and then express the chitin deacetylase mutant.

7. The preparation method according to claim 6, characterized in that: The mutation primer pair includes 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 preparation method according to claim 6 or 7, characterized in that: The expression vector used in the expression process is Escherichia coli.

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