A chitin deacetylase mutant, a recombinant expression vector, a recombinant engineering bacteria and a screening method thereof

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a chitin deacetylase mutant, a recombinant expression vector, a recombinant engineering bacterium and a screening method thereof. The present application firstly obtains two pairs of effective disulfide bond mutants by designing disulfide bonds of a starting template chitin deacetylase CdaBa. Four effective single-point mutants are obtained by performing rational single-point mutation on the starting template chitin deacetylase CdaBa. Finally, the two pairs of effective disulfide bond mutants and the four effective single-point mutants are combined to further screen an optimal mutant CdaBaM. The mutant is further applied to the preparation process of a recombinant vector and a recombinant strain. The residual enzyme activity of the mutant CdaBaM prepared by the method of the present application is 87.4% and 52.8% after 30 minutes and 60 minutes of heat treatment at 70 DEG C, which is 5.8 times and 10.4 times of that of the starting template chitin deacetylase CdaBa.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a chitin deacetylase mutant, a recombinant expression vector, a recombinant engineered bacterium, and a screening method thereof. Background Technology

[0002] Shrimp and crab are major aquatic products. In the processing industry, only the meat of shrimp and crab is usually extracted, while the rest is used as processing by-products. Studies have shown that shrimp and crab processing by-products mainly include chitin, protein, and inorganic salts, with chitin accounting for approximately 20%. Chitin, a polysaccharide composed of N-acetylglucosamine linked by β-1,4 glycosidic bonds, has limited application value due to its poor solubility. Research has found that after chitin is deacetylated to chitosan, its solubility is effectively improved, and it can be well dissolved in dilute acids, thus showing good application value in agriculture, food, and other fields.

[0003] Currently, the mainstream processing method for chitin deacetylation is the chemical concentrated alkali method, where chitin readily loses its acetyl groups under high-concentration alkaline conditions. While efficient, the chemical method has many drawbacks, such as damaging the chitin structure, difficulty in precisely controlling the deacetylation process, and severe environmental pollution from reaction emissions. Therefore, finding a green and efficient processing method is of great significance. Scientists both domestically and internationally have discovered that chitin deacetylases can directionally hydrolyze the acetyl groups of chitin to form chitosan. Compared to the chemical method, chitin deacetylases offer advantages such as being environmentally friendly, having mild reaction conditions, and allowing for controllable reaction processes and product structures. Previous research revealed that the Bacillus atrophaeus chitin deacetylase enzyme CdaBa exhibits good activity and can efficiently catalyze the conversion of chitin to chitosan. However, the poor thermal stability of CdaBa limits its industrial application. Therefore, improving the thermal stability of CdaBa can enhance its stability and catalytic performance, laying the foundation for its industrial application. Summary of the Invention

[0004] This invention focuses on the chitin deacetylase CdaBa from Bacillus atrophaeus. Through disulfide bond design, rational site-directed mutagenesis, and combinatorial mutagenesis, a mutant CdaBaM with improved thermal stability is obtained, laying the foundation for its next industrial application.

[0005] The specific solution adopted in this invention is as follows:

[0006] A chitin deacetylase mutant, CdaBaM, has the amino acid sequence shown in SEQ ID NO. 1.

[0007] GPRLEPIKESASLSEQMQKEKNKNSAKKQDEQKTSPEIGKVVYLTFDDGCHPAASEEIMNLLHKYNCKGTFFMLKPNIVQNPDCVKKMVESGHSVGSHGVTHKVFEIYKSPDSFATEMNDT LDFIKEYTKVNTHLIRAPYGSKPYITGPFREVVKRNQFNLWDWTVDSEDWKYTHGEFIKNTIQQVLNLVGKEPLVVLMHEKPMTAAYLGELLKYFRESGYECKAIDDSIKPVQFRFN(SEQ ID NO.1);

[0008] Preferably, the sequence encoding the amino acid is a polynucleotide sequence, as shown in SEQ ID NO. 2.

[0009] (SEQ ID NO.2);

[0010] The present invention also provides a recombinant expression vector pPICZαA-cdabam, comprising a polynucleotide sequence encoding the chitin deacetylases mutant CdaBaM.

[0011] The present invention also provides a recombinant engineered bacterium containing the recombinant expression vector pPICZαA-cdabam.

[0012] Preferably, the recombinant engineered bacteria uses Pichia pastoris as the host.

[0013] Preferably, the engineered Pichia pastoris strain includes Pichia pastoris X33.

[0014] The present invention also provides a method for screening the recombinant engineered bacteria, comprising the following steps:

[0015] S1. The expression vector pPICZαA-cdabam was linearized and then transformed into Pichia pastoris. The transformed transfectants after electroporation were plated on high-concentration YPDZ plates.

[0016] S2. Ninety-six recombinant transformants were selected for preliminary screening using the 24-well plate method.

[0017] S3. The high enzyme activity transformants obtained from the initial screening are then subjected to shake-flask culture for secondary screening.

[0018] S4. The transformants screened from shake-flask culture were verified by high-density fermentation.

[0019] Preferably, the concentration of bleomycin in the high-concentration YPDZ plate described in step S1 is greater than 500 mg / L.

[0020] The objective of this invention is achieved through the following technical solutions: (1) Analyzing the three-dimensional conformation of chitin deacetylas CdaBa, finding the flexible region of the protein, and obtaining an effective disulfide bond mutant through construction and screening; (2) Analyzing the three-dimensional conformation of chitin deacetylas CdaBa, rationally designing site-directed mutants, and obtaining an effective single-point mutant; (3) Combining the effective disulfide bond mutant and the single-point mutant to obtain the optimal mutant CdaBaM; (4) Efficiently preparing mutant CdaBaM in Pichia pastoris; (5) Characterizing the characteristics of mutant CdaBaM.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] This invention uses the chitin deacetylase CdaBa as a starting template and obtains the optimal mutant CdaBaM through disulfide bond design, single-point mutagenesis, and combinatorial mutagenesis. The mutant CdaBaM prepared using this method exhibits residual enzyme activities of 87.4% and 52.8% after heat treatment at 70℃ for 30 and 60 minutes, respectively, which are 5.8 times and 10.4 times that of the starting template chitin deacetylase CdaBa. Furthermore, the high-efficiency expression of the mutant CdaBaM in Pichia pastoris X33 lays a solid foundation for its future industrial application. Attached Figure Description

[0023] Figure 1 Three-dimensional conformation (A) and conformational evaluation diagram (B) of chitin deacetylases CdaBa;

[0024] Figure 2 The optimal reaction temperature (A) and thermal stability diagram (B) for the effective disulfide bond mutant;

[0025] Figure 3 The optimal reaction temperature (A) and thermal stability diagram (B) for the effective single-point mutant;

[0026] Figure 4 High-density fermentation curve of recombinant engineered strain C28 (A) and protein electrophoresis of fermentation broth at different times (B);

[0027] Figure 5 The optimal reaction temperature and thermal stability diagrams (A and B) for the mutant CdaBaM are shown. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Molecular biology experimental methods not specifically described in the following embodiments are all performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions; the reagents and biological materials mentioned are commercially available unless otherwise specified. The experimental materials and reagents involved in the embodiments of the present invention are as follows:

[0029] 1. Strains and vectors:

[0030] Escherichia coli strains Top10 and Pichia pastoris X33 were obtained commercially. The expression vector pPICZαA-cdaba was constructed in previous experiments, and the construction process is roughly as follows:

[0031] (1) The previously obtained Bacillus atrophus (purchased from China Industrial Microbial Culture Collection Center, strain number CICC21608) was inoculated into LB liquid medium and cultured at 37℃ and 200rpm for 1 day; (2) 1mL of the cultured bacterial solution was heat-treated at 95℃ for 10 minutes and used as a PCR amplification template; (3) Using the heat-treated bacterial solution as a template, PCR amplification was performed using primers cdaba-fw and cdaba-rev. The PCR amplification system is shown in Table 1. The PCR amplification program is as follows: 95℃ for 5 minutes, 94℃ for 2 minutes, 95℃ for 3 minutes, 95℃ for 4 minutes, 95℃ for 5 ... 30 seconds, 55℃ for 30 seconds, 72℃ for 60 seconds, 30 cycles, to obtain the gene cdaba by PCR amplification; (4) The gene cdaba and expression vector pPICZαA were digested with restriction endonucleases EcoRI and NotI, respectively. The digested cdaba and expression vector pPICZαA were purified and recovered using a DNA product purification kit (#DP204, Tiangen Biotech (Beijing) Co., Ltd.). The purification and recovery process is as follows: the target product was cut into a gel and placed in a 2ml centrifuge tube; the sol was added and reacted at 60℃ for 10 minutes. Add the sol liquid from the second step to the collection tube, centrifuge at 10,000 rpm for 1 minute; wash twice with 75% ethanol and air dry; add 50 μL of water, centrifuge for 3 minutes, and it is ready; (5) perform the ligation reaction of the enzyme-digested gene cdaba and the expression vector pPICZαA. The ligation reaction system is shown in Table 2. After ligation reaction at 4℃ for 16 hours, transfer to E. coli top10, spread evenly on LBZ solid plates, and incubate at 37℃ for 4 days; (6) screen positive transformants by bacterial PCR. The screening process is as follows: pick up a single transformant with a high-pressure sterilized toothpick. The colonies were placed in 500 μL of LBZ medium and cultured at 37°C and 200 rpm for 4 hours. 2 μL of bacterial solution was taken as a template for PCR. The PCR reaction system is shown in Table 3. The primers used for bacterial solution PCR were 5'AOX-fw and 3'AOX-rev. The PCR amplification conditions were 95°C for 5 minutes, 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds for 33 cycles. After electrophoresis, the results were observed, and the colonies on the plates corresponding to the positive results were sequenced. (7) Based on the sequencing results, the expression vector pPICZαA-cdaba was finally obtained.

[0032] Table 1. Bacterial PCR Reaction System

[0033]

[0034]

[0035] Table 2 Connection Reaction System

[0036] Gene cdaba 7 Expression vector pPICZαA 1.5 T4 ligase 0.5 reaction buffer 1

[0037] Table 3. Bacterial PCR Reaction System

[0038] Taq enzyme MIX 25 Primer 5'AOX-fw 0.5 Primer 3'AOX-rev 0.5 bacterial solution 2 Sterilized water 22

[0039] The gene sequence information for the vector pPICZαA can be found at: https: / / www.thermofisher.cn / order / catalog / product / V19520?ICID=search-product. The gene sequence information for the expression vector pPICZαA-cdaba is shown below. The double underlined lines represent the 5'AOX1 promoter sequence, the dashed underlined lines represent the α signal peptide sequence, the wavy underlined lines represent the cdaba gene sequence, and the straight underlined lines represent the Zeocin resistance gene sequence.

[0040]

[0041]

[0042]

[0043]

[0044] 2. Experimental Materials

[0045] p-Nitroacetanilide (S30072-5g) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Q5 high-fidelity Taq enzyme MIX was purchased from NEB; plasmid extraction kit (#DP103-03) and gel purification kit (#DP209-02) were purchased from Tiangen Biotech (Beijing) Co., Ltd.; Taq enzyme MIX ( MAX PCR Master Mix was purchased from Baori Biotechnology (Beijing) Co., Ltd.; Zeocin was purchased from Invitrogen, USA.

[0046] 3. Culture medium

[0047] The culture medium for *E. coli* was LB medium: 1% (w / v) peptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0. LBZ was LB medium supplemented with 25 μg / mL Zeocin (bleomycin, Invitrogen, USA).

[0048] The yeast culture medium was YPD medium: 1% (w / v) yeast extract, 2% (w / v) peptone, and 2% (w / v) glucose. The yeast selection medium was YPDZ (YPD + 300 mg / L zeocin).

[0049] BMGY yeast culture medium: 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 1% glycerol (v / v). Note: YNB (Shanghai Yuanye Biotechnology Co., Ltd.) is yeast nitrogen base; Biotin is biotin (Shanghai Yuanye Biotechnology Co., Ltd.).

[0050] Yeast induction medium BMMY: 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 1% methanol (v / v). Note: YNB (Shanghai Yuanye Biotechnology Co., Ltd.) is yeast nitrogen base; Biotin is biotin (Shanghai Yuanye Biotechnology Co., Ltd.).

[0051] High-density yeast fermentation BSM medium: 85% H3PO4 26.7ml / L, CaSO4·2H2O 0.93g / L, K2SO4 18.2g / L, MgSO4·2H2O 14.9g / L, KOH 4.13g / L, glycerol 40g / L, PMT1 trace element solution 4.0ml / L.

[0052] PMT1 trace element solution: CuSO4·5H2O 6.0g / L, KI 0.088g / L, MnSO4·H2O 3.0g / L, Na2MoO4·2H2O 0.2g / L, H3BO3 0.02g / L, CoCl2·6H2O 0.5g / L, ZnCl2 20.0g / L, FeSO4·7H2O 65.0g / L, Biotin 0.2g / L.

[0053] 4. Reagents used for chitin deacetylase activity assay

[0054] p-Nitroacetanilide: 200 mg / L aqueous solution of p-nitroacetanilide; 0.05 mol / L pH 7.0 phosphate buffer.

[0055] Example 1: Three-dimensional modeling and bioinformatics analysis of chitin deacetylases CdaBa

[0056] This invention aims to improve the thermostability of recombinant chitin deacetylases CdaBa through different rational protein design methods. Rational protein design must be based on the three-dimensional structure of the enzyme protein; therefore, it is first necessary to simulate and construct the three-dimensional conformation of the chitin deacetylase CdaBa. Using the three-dimensional structure of Bacillus cereus chitin deacetylase as a template (PDB database accession number: 5nc9.1.A), the three-dimensional conformation of the chitin deacetylase CdaBa was obtained using the modeling software Modeller 10.4 (https: / / salilab.org / modeller / ). Figure 1 A) The obtained 3D conformation was evaluated using the online software SAVES v6.0 (https: / / saves.mbi.ucla.edu / ). Figure 1 As shown in B, the Laplace plot results indicate that 90.6% of the amino acids are located in the optimal region, and 9.4% of the amino acids are located in other allowed regions, indicating that the obtained model conformation is correct and can be used for the next step of rational design analysis.

[0057] The chitin deacetylase CdaBa was analyzed using the molecular dynamics simulation software Gromacs 2019.06 (https: / / www.gromacs.org / ). During the simulation, the protein was subjected to an amber_ff14SB force field, and the small molecule ligand was subjected to a GAFF force field. The GAFF force field used a RESP potential, which was fitted using Multiwfn 3.6 software in conjunction with Gaussian 16.0 software. The protein / ligand model was placed in a cubic box with a minimum distance of 1.2 nm between the protein and the box. The box was filled with TIP3P water. The steepest descent method was used to minimize the system energy, and molecular dynamics simulations were performed at 50 ns intervals with a simulation step size of 2 fs, storing data every 10 ps. Analysis of simulation results revealed that the chitin deacetylase CdaBa possesses multiple flexible protein regions, such as the region from phenylalanine (position 46) to alanine (position 54), the region from tyrosine (position 65) to methionine (position 73), the region from serine (position 97) to serine (position 113), the region from threonine (position 129) to isoleucine (position 146), the region from glutamine (position 158) to aspartic acid (position 170), and the region from serine (position 219) to phenylalanine (position 235). These flexible protein regions of CdaBa are prone to structural changes under high-temperature conditions, leading to denaturation and inactivation. Therefore, increasing the rigidity of these flexible regions can effectively improve its thermal stability.

[0058] Example 2: Rational design of disulfide bonds improves the thermal stability of chitin deacetylase CdaBa.

[0059] Rational design of disulfide bonds in the flexible regions of proteins can effectively improve the thermal stability of recombinant proteins. Based on the simulation analysis results of Example 1, the targeted design of disulfide bonds in the flexible regions of the three-dimensional conformation of the chitin deacetylase CdaBa enhances its rigidity, thereby improving its thermal stability. Disulfide bond design analysis was performed using the online disulfide bond design software BRIDGED-Disulfide bondprediction (http: / / biodev.cea.fr / bridged / results.aspx?jobid=769) and Disulfideby Design (http: / / cptweb.cpt.wayne.edu / DbD2 / index.php). Finally, combining the design results from these two software programs, 12 pairs of disulfide bonds were selected for experiments. These 12 disulfide bonds are P50C-I84C, A54C-A206C, A67C-M223C, K68C-A225C, T70C-S94C, F72C-H98C, F72C-R137C, S97C-T121C, T101C-K103C, K103C-E106C, Y108C-Y145C, and F216C-Y221C.

[0060] Disulfide bond amplification primers were designed according to the cdaba gene sequence (primer sequence information is shown in Table 4). The construction process of expression vectors corresponding to different disulfide bond mutants is as follows (taking the expression vector pPICZαA-cdaba1 corresponding to mutant P50C-I84C as an example, and others are similar): (1) Using pPICZαA-cdaba as a template, PCR amplification was first performed using upstream and downstream primers P50C-fw and P50C-rev. The PCR amplification system is shown in Table 1. The PCR amplification program is as follows: 95℃ for 3 minutes, 95℃ for 30 seconds, 52℃ for 30 seconds, 72℃ for 60 seconds, 33℃ for 30 seconds, 33℃ for 30 seconds, 72℃ for 6 ... (2) Add 2 μL of restriction endonuclease DpnI to the successfully amplified PCR product, and digest it at 37℃ for 2 hours to remove the template vector pPICZαA-cdaba, thereby reducing the false positive rate of the transformant; (3) Purify and recover the digested PCR product. The purification and recovery process is the same as that for the digested cdaba and expression vector pPICZαA. Transfer the purified digested product into E. coli Top10; (4) The screening of E. coli transformants was performed using bacterial culture PCR. First, recombinant transformants were picked as single colonies and incubated at 37°C for 4 hours at 200 rpm. Then, 2 μL of the bacterial culture was used as a template for PCR amplification using primers 5'AOX-fw and 3'AOX-rev. The PCR amplification reaction system is shown in Table 3. The PCR amplification conditions were 95°C for 5 minutes, 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds, for 30 cycles. The correctly verified products were sequenced. Sequencing results identified the mutation site P50C, thus obtaining the mutant expression vector pPICZαA-cdaba-P50C; (4) Using the expression vector pPICZαA-cdaba-P50C as a template, the same method was used to construct the expression vector corresponding to the mutant P50C, except that the amplification primers were replaced with I84C-fw and I84C-rev, thus obtaining the disulfide bond mutant expression vector pPICZαA-P50C-I84C (abbreviated as pPICZαA-cdaba1).

[0061] The experiment ultimately yielded expression vectors corresponding to 12 pairs of disulfide bond mutants: pPICZαA-cdaba1 (mutant P50C-I84C), pPICZαA-cdaba2 (mutant A54C-A206C), pPICZαA-cdaba3 (mutant A67C-M223C), pPICZαA-cdaba4 (mutant K68C-A225C), pPICZαA-cdaba5 (mutant T70C-S94C), pPICZαA-cdaba6 (mutant F72C-H98C), pPICZαA-cdaba7 (mutant F72C-R137C), and pPICZαA-cdaba4 (mutant K68C-A225C). 8 (corresponding to mutant S97C-T121C) and pPICZαA-cdaba9 (corresponding to mutant T101C-K103C), pPICZαA-cdaba10 (corresponding to mutant K103C-E106C), pPICZαA-cdaba11 (corresponding to mutant Y108C-Y145C) and pPICZαA-cdaba12 (corresponding to mutant F216C-Y221C).

[0062] Table 4 Primer sequence list for disulfide bond mutants

[0063]

[0064]

[0065] After linearizing the expression vectors corresponding to 12 different disulfide mutants using the restriction endonuclease SacI, they were transformed into Pichia pastoris X33 cells by electroporation. The electroporation process was as follows: (1) Place Pichia pastoris X33 competent cells on ice for 20 minutes; (2) Add 200 ng of the linearized expression vectors corresponding to the different disulfide mutants, mix well, place on ice for 5 minutes, and then perform electroporation. The electroporation conditions were 1.5 kV and 400 ohms; (3) After electroporation for 2 minutes, immediately add 0.6 mL of pre-cooled 1M sorbitol to the cup and transfer the contents to a sterile centrifuge tube; (4) After standing at 30°C for 2 hours, spread the solution onto YPDZ plates and observe the transformants after culturing for 2-3 days.

[0066] The screening experiment for disulfide bond mutants is divided into two parts: preliminary screening and secondary screening. The preliminary screening experiment uses the 24-well plate method. The specific experimental steps are as follows: (1) Pick the recombinant transformants on the YPDZ plate one by one with a toothpick and transfer them to a 24-well plate containing 2 mL of BMGY medium in each well. Incubate overnight at 30°C and 200 rpm for 24 hours. After centrifugation at 4000 rpm to remove the supernatant, add 2 mL of BMMY medium and incubate at 30°C and 200 rpm for 24 hours. Measure the chitin deacetylase activity of the recombinant transformants.

[0067] The method for determining chitin deacetylase activity is as follows: Add 600 μL of 0.05 mol / L pH 7.0 phosphate buffer (pre-warmed at 50℃), 200 μL of 200 mg / L p-nitroacetanilide aqueous solution, and 200 μL of enzyme solution to a 2 mL centrifuge tube. Incubate at 50℃ for 10 minutes, then stop the reaction by boiling in a water bath. Centrifuge for 10 minutes and measure the absorbance of the supernatant at 410 nm. Use 200 μL of the same concentration of enzyme solution, inactivated by boiling in a water bath for 10 minutes, as a control. Enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μg of p-nitroacetanilide per hour under the above reaction conditions.

[0068] Based on the enzyme activity results, the enzyme thermal stability was further determined. The thermal stability test method is as follows: the chitin deacetylase fermentation enzyme solution was diluted 10 times with phosphate buffer (pH 7.0), and then incubated in a water bath at 60℃ for 30 minutes before the residual enzyme activity was determined. The sample without heat treatment was used as a control.

[0069] The preliminary screening results of 12 different disulfide bond mutants are shown in Table 5. As can be seen from Table 5, among the 12 rationally designed disulfide bond pairs, only mutants P50C-I84C and A67C-M223C can effectively improve the thermostability of chitin deacetylas CdaBa. After incubation in a water bath at 60℃ for 30 minutes, the remaining enzyme activities of the two mutants are 1.33 times and 1.61 times that of the starting template chitin deacetylas CdaBa, respectively.

[0070] Table 5. Fermentation enzyme activity and thermal stability of recombinant bacteria with different disulfide bond mutants

[0071]

[0072]

[0073] The mutants P50C-I84C and A67C-M223C, which showed significant effects in the initial screening, and the starting template chitin deacetylase CdaBa were subjected to secondary screening. The secondary screening process was roughly as follows: (1) The recombinant engineered bacteria corresponding to the mutants P50C-I84C, A67C-M223C and chitin deacetylase CdaBa were respectively picked into 250mL shake flasks containing 50mL BMGY medium and cultured at 30℃ and 200rpm until OD. 600 (2) Centrifuge the cultured bacterial solution, remove the supernatant, and transfer it to a 500mL shake flask containing 100mL BMMY medium. The initial OD of the bacterial solution is 6.0. 600 The value was 1.0, and the culture was carried out at 30℃ and 200rpm. During the culture, methanol was added at a ratio of 0.5% (v / v) every 24 hours for induced culture for 5 days; (3) The cultured fermentation broth was centrifuged to collect the supernatant, and the collected supernatant fermentation broth was concentrated using a 10kDa ultrafiltration tube; (4) The concentrated fermentation broth was purified according to the Ni-IDA protein purification kit (Shanghai Sangon). The purification process is as follows: First, the fermentation broth was centrifuged in a shake flask and the supernatant was collected; the collected supernatant fermentation broth was concentrated using a 10kDa ultrafiltration tube; the concentrated supernatant fermentation broth was purified according to the Ni-IDA protein purification kit (Shanghai Sangon); (5) The optimal reaction temperature and thermal stability of the purified recombinant chitin deacetylase CdaBa, mutant P50C-I84C and A67C-M223C were determined.

[0074] The optimal reaction temperatures for chitin deacetylases CdaBa, P50C-I84C, and A67C-M223C were determined as follows: Enzyme activity was measured at different temperatures ranging from 30℃ to 80℃ under pH 7.0 conditions, with the enzyme activity at the highest temperature being taken as 100%, and the relative enzyme activity at other temperatures was calculated.

[0075] The experimental results of the optimal reaction temperature for chitin deacetylases CdaBa, mutants P50C-I84C and A67C-M223C are as follows: Figure 2 As shown in Figure A, the optimal reaction temperature for chitin deacetylase CdaBa, mutant P50C-I84C, and A67C-M223C is 50℃. The relative enzyme activities of mutants P50C-I84C and A67C-M223C are higher than those of chitin deacetylase CdaBa under high temperature conditions (50℃-80℃).

[0076] The thermal stability of chitin deacetylases CdaBa, mutants P50C-I84C and A67C-M223C was determined as follows: after water bath heat treatment at different temperatures from 50℃ to 80℃ for 30 minutes, the residual enzyme activity was measured. The enzyme activity of the sample without heat treatment was taken as 100%, and the relative residual enzyme activity at other temperatures was calculated.

[0077] The thermal stability of chitin deacetylase CdaBa, mutants P50C-I84C and A67C-M223C is as follows: Figure 2 As shown in Figure B, when the heat treatment temperature is greater than 50℃, the residual enzyme activities of mutants P50C-I84C and A67C-M223C after water bath treatment for 30 minutes are significantly higher than those of the starting template chitin deacetylases CdaBa. At a heat treatment temperature of 70℃, the residual enzyme activities of mutants P50C-I84C and A67C-M223C are 38.1% and 51.3%, respectively, which are 2.5 and 3.4 times the residual enzyme activities of chitin deacetylases CdaBa, respectively. At a heat treatment temperature of 80℃, the residual enzyme activity of chitin deacetylases CdaBa is only 5.1%, while the residual enzyme activities of mutants P50C-I84C and A67C-M223C are 13.3% and 21.3%, respectively.

[0078] Example 3: Point mutation enhances the thermal stability of chitin deacetylase CdaBa

[0079] Previous studies have shown that site-directed mutagenesis of proteins can improve their thermal stability, and synergistic effects between different amino acids can further enhance protein stability. FireProt (https: / / loschmidt.chemi.muni.cz / fireprotweb / ), developed by a research team in the Department of Biology at the University of Masakli, is an online mutation analysis software. Using FireProt for predictive analysis, target mutants can be identified, thereby improving the thermal stability of the target protein. Mutation prediction analysis of the chitin deacetylas CdaBa using FireProt ultimately selected 15 target single-point mutants. The prediction results from FireProt show that these 15 single-point mutants improve the stability of the chitin deacetylas CdaBa by reducing its three-dimensional conformational free energy. The 15 single-point mutants and their corresponding free energies are: K75W (-2.16 kcal / mol), V79I (-2.47 kcal / mol), S105F (-2.15 kcal / mol), D112N (-3.28 kcal / mol), N119K (-2.11 kcal / mol), D120I (-3.92 kcal / mol), D123Y (-2.07 kcal / mol), E 127Y(-2.09kcal / mol), T128Y(-3.91kcal / mol), K130G(-3.23kcal / mol), N160Y(-2.59kcal / mol), E177G (-5.48kcal / mol), N187L (-2.04kcal / mol), K202W (-3.35kcal / mol) and T204M (-3.09kcal / mol).

[0080] Primers for amplifying single-point mutants were designed based on the cdaba gene sequence (primer sequence information is shown in Table 6). The construction process of expression vectors corresponding to different single-point mutants was the same as the construction method of disulfide bond mutants in Example 2, except that the amplification primers were replaced with the primers corresponding to each single-point mutant. Through experiments, 15 expression vectors corresponding to single-point mutants were finally obtained, namely pPICZαA-cdabas1 (corresponding to mutant K75W), pPICZαA-cdabas2 (corresponding to mutant V79I), pPICZαA-cdabas3 (corresponding to mutant S105F), pPICZαA-cdabas4 (corresponding to mutant D112N), pPICZαA-cdabas5 (corresponding to mutant N119K), pPICZαA-cdabas6 (corresponding to mutant D120I), pPICZαA-cdabas7 (corresponding to mutant D123Y), pPICZαA-cdabas6 (corresponding to mutant D120I), pPICZαA-cdabas7 (corresponding to mutant D123Y), pPICZαA-cdabas6 (corresponding to mutant D120I), pPICZαA-cdabas6 (corresponding to mutant D123Y ...6 (corresponding to mutant D120I), pPICZαA-cdabas6 (corresponding to mutant D120I), pPICZαA-cdabas6 (corresponding to mutant D120I), pPICZαA-cdabas6 ( ZαA-cdabas8 (corresponding to mutant E127Y), pPICZαA-cdabas9 (corresponding to mutant T128Y), pPICZαA-cdabas10 (corresponding to mutant K130G), pPICZαA-cdabas11 (corresponding to mutant N160Y), pPICZαA-cdabas12 (corresponding to mutant E177G), pPICZαA-cdabas13 (corresponding to mutant N187L), pPICZαA-cdabas14 (corresponding to mutant K202W), and pPICZαA-cdabas15 (corresponding to mutant T204M).

[0081] Table 6 Primer sequences for single-point mutants

[0082]

[0083]

[0084] The construction process of single-point mutant recombinant bacteria is the same as that of disulfide bond mutant recombinant engineered bacteria in Example 2. First, the expression vectors corresponding to 15 different single-point mutants were linearized using the restriction endonuclease SacI, and then transformed into Pichia pastoris X33. The transformants were plated on YPDZ plates and screened after being cultured at 30°C for 4 days.

[0085] The screening process for single-point mutant recombinant engineered bacteria is the same as that for disulfide bond mutant recombinant engineered bacteria in Implementation 2, including preliminary screening and secondary screening.

[0086] The preliminary screening results are shown in Table 7. The mutants S105F, T128Y, N187L, and T204M can improve the thermostability of chitin deacetylas CdaBa. After heat treatment at 60℃ for 30 minutes, the remaining enzyme activities were 43.5%, 45.1%, 44.2%, and 43.7%, respectively. Compared with the starting template chitin deacetylas CdaBa, the thermostability was improved by 10.7%, 14.8%, 12.5%, and 11.2%, respectively.

[0087] Table 7. Fermentation enzyme activity and thermal stability of recombinant bacteria with different single-point mutants.

[0088]

[0089] The effective single-point mutants S105F, T128Y, N187L, and T204M were subjected to a secondary screening experiment. The secondary screening procedure was the same as that for the disulfide bond mutants in Example 2. Purified single-point mutants S105F, T128Y, N187L, and T204M were obtained through culture and purification experiments. The optimal reaction temperature and thermal stability of the purified single-point mutants S105F, T128Y, N187L, and T204M were determined, with the chitin deacetylase CdaBa serving as a control throughout the experiment.

[0090] The experimental results of the optimal reaction temperature for single-point mutants S105F, T128Y, N187L, T204M and the chitin deacetylase CdaBa are as follows: Figure 3 As shown in Figure A, the optimal reaction temperature for single-point mutants S105F, T128Y, N187L, T204M, and the chitin deacetylascase CdaBa is 50℃. Within the temperature range of 50℃-80℃, the relative enzyme activities of single-point mutants S105F, T128Y, N187L, and T204M are all higher than those of chitin deacetylascase CdaBa.

[0091] The thermostability results of single-point mutants S105F, T128Y, N187L, T204M and chitin deacetylas CdaBa are as follows: Figure 3 As shown in B, when the heat treatment temperature is greater than 60℃, the single-point mutants S105F, T128Y, N187L, and T204M exhibit significantly better thermostability than the starting template chitin deacetylases CdaBa. After treatment in a 70℃ water bath for 30 minutes, the residual enzyme activities of the single-point mutants S105F, T128Y, N187L, and T204M were 23.1%, 28.6%, 27.6%, and 22.8%, respectively, which are 1.54 times, 1.91 times, 1.84 times, and 1.52 times that of the chitin deacetylases CdaBa.

[0092] Example 4: Effective mutant combination mutation

[0093] Example 2 experimentally obtained two pairs of effective disulfide bond mutants, P50C-I84C and A67C-M223C. Example 3 experimentally obtained four effective single-point mutants, S105F, T128Y, N187L, and T204M. This section combines these effective mutants to further improve thermal stability.

[0094] First, the disulfide mutants P50C-I84C and A67C-M223C were combined with mutated, following the same experimental procedure as in Example 2. Using the expression vector pPICZαA-cdaba3 corresponding to the A67C-M223C mutant as a template, the expression vector pPICZαA-cdaba3-1 corresponding to the disulfide mutants (P50C-I84C and A67C-M223C) was finally obtained. The disulfide mutant expression vector pPICZαA-cdaba3-1 was linearized with the restriction endonuclease SacI and then transformed into Pichia pastoris X33. The screening of recombinant transformants, enzyme activity assays, and thermostability assays were all consistent with Example 2. The optimal reaction temperature for the disulfide mutant P50C-I84C / A67C-M223C was ultimately 55℃, an increase of 5℃ compared to the starting template chitin deacetylases CdaBa. Furthermore, after treatment in a 60°C water bath for 30 minutes, the residual enzyme activity of the didisulfide mutant P50C-I84C / A67C-M223C was 92.5%, which is 2.35 times the residual enzyme activity of the starting template chitin deacetylase CdaBa.

[0095] Using disulfide bonds as templates, effective single-point mutants S105F, T128Y, N187L and T204M were superimposed respectively. The expression vectors pPICZαA-cdaba3-1-105 (corresponding to mutant P50C-I84C / A67C-M223C-S105F), pPICZαA-cdaba3-1-128 (corresponding to mutant P50C-I84C / A67C-M223C-T128Y), pPICZαA-cdaba3-1-187 (corresponding to mutant P50C-I84C / A67C-M223C-N187L), pPICZαA-cdaba3-1-204 (corresponding to mutant P50C-I84C / A67C-M223C-T204M), and pPICZαA-cdaba3-1-105-128 (corresponding to mutant P50C-I84C / A67C-M223C-S105F) were obtained experimentally. -T128Y), pPICZαA-cdaba3-1-105-187 (corresponding mutant P50C-I84C / A67C-M223C-S105F-N187L), pPICZαA-cdaba3-1-105-204 (corresponding mutant P50C-I84C / A67C-M223C-S105F-T204M), pPICZα A-cdaba3-1-128-187 (corresponding to mutant P50C-I84C / A67C-M223C-T128Y-N187L), pPICZαA-cdaba3-1-128-204 (corresponding to mutant P50C-I84C / A67C-M223C-T128Y-T204M), pPICZαA-cdaba3-1-187-204 (corresponding to mutant P50C-I84C / A67C-M223C-N187L-T204M), and pPICZαA-cdaba3-1-105-128-187-204 (P50C-I84C / A67C-M223C-S105F-T128Y-N187L-T204M).

[0096] The expression vectors of the combined mutants were linearized and transformed into Pichia pastoris X33. The screening and enzyme activity assays of the recombinant transformants were consistent with those in Example 2.

[0097] The thermal stability of different mutant combinations was determined using the same method as in Example 2, except that the heat treatment temperature was increased to 70°C. Residual enzyme activity was measured after 30 minutes of water bath treatment at 70°C. The starting template chitin deacetylases CdaBa served as a control throughout the experiment.

[0098] The enzyme activity and thermostability results of different mutant combinations are shown in Table 8. Table 8 shows that combining effective disulfide bond mutants and single-point mutations can further improve thermostability. Among them, the combined mutant P50C-I84C / A67C-M223C-S105F-T128Y-N187L-T204M showed the best effect, followed by mutants P50C-I84C / A67C-M223C-T128Y-N187L and P50C-I84C / A67C-M223C-S105F-N187L, respectively. The combined mutant P50C-I84C / A67C-M223C-S105F-T128Y-N187L-T204M had a residual enzyme activity of 87.2% after being treated in a water bath at 70℃ for 30 minutes, which is 5.69 times that of the starting template chitin deacetylas CdaBa.

[0099] For ease of writing, the mutant P50C-I84C / A67C-M223C-S105F-T128Y-N187L-T204M is abbreviated as CdaBaM, and its corresponding expression vector is abbreviated as pPICZαA-cdabam.

[0100] Table 8. Fermentation enzyme activity and temperature characteristics of the combined mutant recombinant bacteria.

[0101]

[0102]

[0103] Example 5: High-efficiency preparation of recombinant mutant CdaBaM

[0104] The recombinant mutant CdaBaM exhibits significantly improved thermal stability, thus broadening its value for industrial applications. To further enhance its industrial application prospects, the production cost of CdaBaM must be reduced.

[0105] Screening of recombinant engineered bacteria with high enzyme activity and high-density fermentation can effectively improve the expression level of recombinant proteins in Pichia pastoris. This section aims to achieve the efficient preparation of recombinant mutant CdaBaM through these two technologies.

[0106] The screening process for high enzyme activity recombinant engineered bacteria is as follows: (1) The expression vector pPICZαA-cdabam was linearized and transformed into Pichia pastoris. The transformation method (i.e., electroconversion) was carried out according to Example 2. The electroconverted transformants were plated on high concentration YPDZ plates (greater than 500 mg / L zeocin); (2) 96 recombinant transformants were selected by 24-well plate method for preliminary screening; (3) The high enzyme activity transformants obtained by preliminary screening were cultured in shake flasks for secondary screening; (4) The transformants screened by shake flask culture were verified by high-density fermentation.

[0107] Three recombinant engineered bacteria with high enzyme activity were obtained from 96 transformants through preliminary screening using 24-well plates and named C12, C28 and C78, ​​respectively. The enzyme activities of these three recombinant engineered bacteria were 11.2 U / mL, 12.5 U / mL and 10.8 U / mL, respectively.

[0108] The three high-enzyme-activity recombinant engineered bacteria C12, C28 and C78 obtained from the preliminary screening were cultured in shake flasks. The experimental procedure was as follows: (1) The corresponding recombinant engineered bacteria were inoculated into a 50 mL centrifuge tube containing 5 mL of BMGY medium and cultured at 30℃ and 220 rpm for 24 hours; (2) The cultured recombinant yeast engineered bacteria were inoculated into a 250 mL Erlenmeyer flask containing 50 mL of BMMY medium at an inoculation amount of 1% (v / v) and cultured in shake flasks at 30℃ and 220 rpm; (3) 1% (v / v) methanol was added every 24 hours for induction culture and samples were taken for enzyme activity determination.

[0109] After 120 hours of shake-flask induction culture, the fermentation enzyme activities of recombinant engineered bacteria C12, C28 and C78 were 35.6 U / mL, 41.2 U / mL and 33.5 U / mL, respectively.

[0110] The recombinant engineered strain C28 obtained through shake-flask screening was subjected to high-density fermentation. The high-density fermentation of the recombinant engineered strain was carried out in a 7L fermenter, and the specific process was roughly as follows: a single colony of recombinant yeast was inoculated into a 250mL Erlenmeyer flask containing 50mL of YPD medium and cultured at 30℃ with shaking at 200rpm for 16 hours. Then, the overnight cultured recombinant yeast was inoculated at a 1% (v / v) inoculation rate into a 500mL Erlenmeyer flask containing 100mL of YPD medium and cultured overnight at 30℃ with shaking at 200rpm until OD reached... 600 Greater than 10. The recombinant yeast strain, cultured overnight twice, was inoculated at a rate of 10% (v / v) into a 7L fermenter containing 3L of BSM medium. The culture conditions for the recombinant yeast strain in the 7L fermenter were: temperature 30℃, pH 5.0, stirring speed 500 rpm, and air flow rate 40 L / min. In the initial stage of culture, glycerol was used as the carbon source for cell growth. When the cell wet weight reached a certain level (approximately 180 g / L), the addition of glycerol was stopped, and after the glycerol was completely absorbed by the cells (dissolved oxygen rose rapidly), methanol induction was initiated. Enzyme activity and total protein were measured every 24 hours during fermentation.

[0111] The fermentation process curve of recombinant engineered bacteria C28 is as follows: Figure 4 As shown in Figure A, when the culture was induced for 168 hours, the fermentation enzyme activity reached its maximum (1562.6 U / mL), and the total protein concentration reached its maximum of 5.16 g / L.

[0112] In addition, the protein electrophoresis analysis results ( Figure 4B) indicates that the fermentation supernatant mainly contains the recombinant mutant CdaBaM, which is approximately 30 kDa in size.

[0113] Example 6 Characterization of recombinant mutant CdaBaM

[0114] The recombinant mutant CdaBaM was isolated and purified using the disulfide bond mutant purification method described in Example 2 (the specific process was the same as for the concentrated fermentation broth, referring to the Ni-IDA protein purification kit). The purified recombinant mutant CdaBaM was obtained experimentally. The characteristics of the recombinant mutant CdaBaM were characterized, including specific activity, enzyme kinetic parameters, and temperature characteristics. The starting template chitin deacetylases CdaBa were used as controls throughout the experiment.

[0115] The specific activities and enzyme kinetic parameters of the recombinant mutant CdaBaM and the chitinase deacetylas CdaBa are shown in Table 9. The specific activities of the recombinant mutant CdaBaM and the chitinase deacetylas CdaBa are 355 U / mg and 293 U / mg, respectively, and the Michaelis constant K is... m The concentrations were 2.3 mg / mL and 2.6 mg / mL, respectively, with a maximum reaction rate V. max The values ​​were 561.1 μM / min / mg and 506.8 μM / min / mg, respectively.

[0116] Table 9. Kinetic parameters of mutant CdaBaM and chitin deacetylation enzyme CdaBa.

[0117] Enzyme specific activity (U / mg) 355 293 <![CDATA[Michaelis constant K m (mg / mL)]]> 2.3 2.6 <![CDATA[Maximum reaction velocity V max (μM / min / mg)]]> 561.1 506.8

[0118] The optimal reaction temperature for the recombinant mutant CdaBaM and the chitinase deacetylase CdaBa was determined using the same method as that for the disulfide bond mutant in Example 2. The experimental results are as follows: Figure 5 As shown in Figure A.

[0119] Depend on Figure 5 As can be seen from A, the optimal reaction temperature of the starting template chitinase deacetylas CdaBa is 50℃, while the optimal reaction temperature of the recombinant mutant CdaBaM is 60℃, which is 10℃ higher than that of the chitinase deacetylas CdaBa.

[0120] The thermostability assays for the recombinant mutant CdaBaM and the chitinase deacetylas CdaBa were performed as follows: Residual enzyme activity was measured after incubation in a water bath at 60℃, 70℃, and 80℃ for 30 and 60 minutes, respectively. The enzyme activity of the untreated sample was taken as 100%, and the relative residual enzyme activity at other temperatures was calculated. The experimental results are as follows: Figure 5 As shown in B.

[0121] Depend on Figure 5As shown in Figure B, the recombinant mutant CdaBaM exhibits significantly better thermostability than the starting template chitinase deacetylase CdaBa within the temperature range of 60℃ to 80℃. After heat treatment at 60℃ for 30 minutes and 60 minutes, the residual enzyme activities of the recombinant mutant CdaBaM were 97.2% and 75.3%, respectively, while the residual enzyme activities of the chitinase deacetylase CdaBa under the same conditions were 41.6% and 18.9%, respectively.

[0122] When the temperature was raised to 70℃, after heat treatment of the recombinant mutant CdaBaM for 30 minutes and 60 minutes, the remaining enzyme activities were 87.4% and 52.8%, respectively, which were 5.8 times and 10.4 times that of the chitinase deacetylase CdaBa. Figure 5 B).

[0123] When the temperature was increased to 80℃, after heat treatment of the recombinant mutant CdaBaM for 30 minutes and 60 minutes, the remaining enzyme activities were 40.2% and 21.3%, respectively, which were 10.2 times and 18.5 times that of the chitinase deacetylase CdaBa. Figure 5 B).

Claims

1. A chitin deacetylase mutant, CdaBaM, characterized in that, The amino acid sequence of the mutant CdaBaM is shown in SEQ ID NO.

1.

2. The chitin deacetylase mutant CdaBaM as described in claim 1, characterized in that, The sequence encoding the amino acid is a polynucleotide sequence, as shown in SEQ ID NO.

2.

3. A recombinant expression vector pPICZαA-cdabam, characterized in that, It contains a polynucleotide sequence encoding the chitin deacetylase mutant CdaBaM of claim 2.

4. A recombinant engineered bacterium, characterized in that, It includes the recombinant expression vector pPICZαA-cdabam as described in claim 3.

5. The recombinant engineered bacteria as described in claim 4, characterized in that, The recombinant engineered bacteria used Pichia pastoris as the host.

6. The recombinant engineered bacteria as described in claim 5, characterized in that, The engineered Pichia pastoris strain includes Pichia pastoris X33.

7. A method for screening recombinant engineered bacteria as described in claim 4, characterized in that, Includes the following steps: S1. The expression vector pPICZαA-cdabam was linearized and then transformed into Pichia pastoris. The transformed transfectants after electroporation were plated on YPDZ plates. S2. Ninety-six recombinant transformants were selected for preliminary screening using the 24-well plate method. S3. The high enzyme activity transformants obtained from the initial screening are then subjected to shake-flask culture for secondary screening. S4. The transformants screened from shake-flask culture were verified by high-density fermentation.

8. The screening method as described in claim 7, characterized in that, The concentration of bleomycin in the YPDZ plate described in step S1 is greater than 500 mg / L.

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