A chitinase mutant ChiTgM and its applications

Abstract

This invention belongs to the fields of genetic engineering and enzyme engineering, specifically relating to a chitinase mutant ChiTgM and its applications. This invention obtains the chitinase ChiTg through gene cloning, achieves heterologous expression of it in Pichia pastoris through codon optimization, and obtains the chitinase mutant ChiTgM through site-directed mutagenesis and combinatorial mutagenesis. Analysis of the enzymatic characteristics of chitinase ChiTg and the mutant ChiTgM shows that, compared to the starting template ChiTg, the chitinase mutant ChiTgM provided by this invention exhibits a 1.81-fold increase in specific activity and demonstrates excellent hydrolytic properties. Its application in the enzymatic preparation of chitin oligosaccharides can efficiently hydrolyze colloidal chitin, laying the foundation for its further industrial application.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering, and specifically relates to a chitinase mutant ChiTgM and its applications. Background Technology

[0002] Chitin oligosaccharides, as hydrolysis products of chitin, have been shown to have potential applications as biostimulants in agricultural planting. Reported studies have confirmed their important roles in several areas: (1) enhancing crop immunity, thereby increasing their resistance to pests and diseases; (2) regulating and activating crop growth factors to increase yield and income; and (3) reducing the use of pesticides and fertilizers, promoting green agriculture. Currently, the preparation of chitin oligosaccharides mainly relies on chemical methods, using strong acids to degrade chitin into chitin oligosaccharides. This process suffers from drawbacks such as numerous byproducts, incomplete final product structure, and environmental pollution. Therefore, a greener and more environmentally friendly method is needed for the preparation of chitin oligosaccharides.

[0003] Chitinases, as specific enzymes for chitin degradation, hydrolyze the glycosidic bonds within chitin to form chitin oligosaccharides with varying degrees of polymerization. According to reported literature, current chitinases suffer from drawbacks such as low catalytic activity and high production costs. Therefore, developing and screening highly efficient chitinases is of great significance.

[0004] This patent describes the acquisition of the chitinase ChiTg from *Trichoderma gesimsica* through gene cloning, its heterologous expression in *Pichia pastoris* through codon optimization, and the generation of the mutant ChiTgM with enhanced specific activity through site-directed and combinatorial mutagenesis. Furthermore, the enzymatic characteristics of chitinase ChiTg and the mutant ChiTgM were analyzed, and the mutant ChiTgM was applied to the enzymatic preparation of chitin oligosaccharides. The results of this patent demonstrate that the mutant ChiTgM can efficiently hydrolyze colloidal chitin, laying the foundation for its future industrial application. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a chitinase mutant, ChiTgM, and its applications. This invention obtains the chitg encoding gene for chitinase ChiTg based on homology RT-PCR; optimizes the codon bias of Pichia pastoris to obtain the chitgs gene, which does not contain the chitinase ChiTg's own signal peptide; obtains the three-dimensional conformation of chitinase ChiTg based on homology modeling; and identifies key amino acids in the catalytic activity region of chitinase ChiTg through molecular docking analysis; and obtains the chitinase mutant ChiTgM with enhanced enzyme catalytic activity through site-directed mutagenesis and combinatorial mutagenesis. Compared to the specific activity of chitinase ChiTg, the chitinase mutant ChiTgM obtained in this invention (specific activity 51.2 U / mg) is 1.81 times that of chitinase ChiTg (specific activity 28.3 U / mg). The chitinase mutant ChiTgM obtained in this invention can efficiently hydrolyze colloidal chitin to prepare chitin oligosaccharides, laying the foundation for its further industrial application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first objective of this invention is to provide a chitinase mutant ChiTgM, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] SPVAASNVSVEKRASGYANAVYFTNWGIYGRNFQPQNLVASDITHVIYSFMNFQADGTVVSGRAYADYQKHYDDDSWNDVGNNAYGCVKQLFKLKKANRNL KVMLSIGGWTWSTNFPSAASTDANRKNFAKTAITFMKDWGFDGIDVDWEYPADDTQATNMVLLLKEIRSQLDAYAAQYAPGYHFLLSIAAPARPEHYSFLHM SDLGQVLDYVNLMAYDYAGKWSSYSGHDANLFANPSNPNSSPYNTDQAIKDYIKGGVPASKIVLGMPIYGRSFESTGGIGQTYSGIGSGSWERGIWDYKVL PKAGATVQYDSVAQAYYSYDPSSKELISFDTPDMINTKVSHLKNDGLTGSMFWKAFANKTGKDSLIGTSHRALGSLDSTQNLLSYPNSQKDFIRNGLN (SEQ ID NO.1)

[0009] Preferably, the sequence encoding the amino acid of the chitinase mutant ChiTgM is a polynucleotide sequence, as shown in SEQ ID NO.2.

[0010]

[0011] A second objective of this invention is to provide a recombinant expression vector pPICZαA-chitgm containing the polynucleotide sequence described above.

[0012] A third objective of this invention is to provide a recombinant bacterium comprising the recombinant expression vector pPICZαA-chitgm as described above.

[0013] Preferably, the recombinant bacteria uses Pichia pastoris engineered strains as the host, and the Pichia pastoris engineered strains include Pichia pastoris X33.

[0014] Another object of the present invention is to provide an application of the chitinase mutant ChiTgM as described above in the preparation of chitin oligosaccharides.

[0015] Preferably, the chitin oligosaccharide is prepared by hydrolyzing chitin using the chitinase mutant ChiTgM.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The chitinase ChiTg from *Trichoderma gesimsica* was obtained through gene cloning, and its heterologous expression in *Pichia pastoris* was achieved through codon optimization. Through site-directed and combinatorial mutagenesis, a mutant ChiTgM with enhanced specific activity was obtained, the nucleotide sequence of which is shown in SEQ ID NO.2. Further analysis of the enzymatic characteristics of the chitinase mutant ChiTgM obtained in this invention revealed that its specific activity is 51.2 U / mg, which is 1.81 times that of chitinase ChiTg (specific activity of 28.3 U / mg), significantly improving the catalytic activity of the chitinase. Attached Figure Description

[0017] Figure 1 Evaluation diagram of the three-dimensional conformation of chitinase ChiTg;

[0018] Figure 2 Three-dimensional conformations of chitinase ChiTg and its mutant ChiTgM;

[0019] Figure 3 The optimal reaction temperature and thermal stability of the mutant ChiTgM and the chitinase ChiTg are shown in the figure.

[0020] Figure 4 Graphs showing the optimal reaction pH and pH stability of mutant ChiTgM and chitinase ChiTg;

[0021] Figure 5 The diagram shows the analysis of the mutant ChiTgM and the hydrolysis products of chitinase ChiTg. Detailed Implementation

[0022] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments.

[0023] 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. Experimental materials and reagents involved in the present invention:

[0024] 1. Strains and vectors

[0025] The filamentous fungus *Trichoderma gems* R1 was purchased from the China General Microbiological Culture Collection Center (strain number, CGMCC3.12961), identified by ITS, and stored in a laboratory freezer at -60℃. *Pichia pastoris* X33, *Escherichia coli* strain Top10, plasmid pMD20T, and expression vector pPICZαA were all purchased commercially.

[0026] 2. Enzymes and kits

[0027] High-fidelity Taq enzyme PrimeSTAR® HS (Premix), TA cloning amplification Taq enzyme EmeraldAmp GTPCRMaster Mix, reverse transcription kit RNA to cDNA EcoDry Premix, and restriction endonucleases (SacI, EcoRI, and NotI) were all purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; fungal total RNA extraction kit (DP419), plasmid extraction kit (#DP103-03), and gel purification kit (#DP209-02) were all purchased from Tiangen Biotech (Beijing) Co., Ltd.; Zeocin was purchased from Invitrogen. Chitin was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; chitin oligosaccharide kit (SCNKA) was purchased from Qingdao Bozhi Huili Biotechnology Co., Ltd.; other chemical reagents were purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0028] 3. Culture medium

[0029] The Trichoderma galvanicum culture medium is: liquid PDA medium, 200 g potato, 20 g glucose, and 1000 ml distilled water.

[0030] The culture medium for *E. coli* was LB (1% (w / v) peptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0). LBZ was LB medium with 25 μg / mL Zeocin (bleomycin).

[0031] Yeast culture medium was YPD (1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose). Yeast selection medium was YPDZ (YPD + different concentrations of zeocin).

[0032] Yeast induction medium BMGY (1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 1% glycerol (v / v)) and BMMY medium (the composition was the same as BMGY except that 0.5% (v / v) methanol was used instead of glycerol).

[0033] Note: YNB stands for Yeast Nitrogen Base; Biotin stands for Biotin.

[0034] 4. Reagents and methods used for chitinase activity assay

[0035] Colloidal chitin: Weigh 10g of chitin and add it to 100mL of concentrated hydrochloric acid. Stir at 40℃ for 3 minutes to dissolve it. Add 1L of cold water at 5℃ to the solution, and the chitin will precipitate as a colloidal suspension. Filter with coarse filter paper, and wash the filtered colloidal precipitate with distilled water until neutral; DNS reagent (6.3‰ (w / v) 3,5-dinitrosalicylic acid; 18.2% (w / v) potassium sodium tartrate tetrahydrate; 5‰ (w / v) phenol; 5‰ (w / v) anhydrous sodium sulfite).

[0036] The method for determining chitinase activity is as follows: First, preheat colloidal chitin and enzyme solution separately at 45℃; add 500 μL of the preheated enzyme solution to a 10 mL glass test tube, then add 500 mL of colloidal chitin solution, react at 50℃ for 30 minutes, then add 2 mL of DNS reagent to terminate the reaction, and perform color development by boiling in a water bath at 100℃ for 10 minutes. After cooling, centrifuge and measure the absorbance at 540 nm. The enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol of acetylglucosamine per minute.

[0037] Example 1: Cloning and Analysis of the Chitinase ChiTg Gene

[0038] Since the chitinase gene sequence of *Trichoderma galbana* (gene accession number: XM_018810913.1) can be found in the NCBI database in the United States, the chitinase ChiTg encoding gene can be amplified using homologous RT-PCR. chitg .

[0039] The experimental procedure is roughly as follows: (1) First, Trichoderma gems R1 was inoculated into liquid PDA medium and cultured at 200 rpm and 30℃ for 3 days. Then, 1% colloidal chitin was added for induction culture for 24 hours; (2) Trichoderma gems mycelium was collected after induction culture, and total RNA was extracted using the RNAsimple total RNA extraction kit (DP419) from Tiangen Biotech (Beijing) Co., Ltd. Then, reverse transcription RT-PCR was performed to obtain total cDNA; (3) Using the obtained total cDNA as a template, primers were used to extract the total RNA. chitg- fw and chitg- PCR amplification was performed on the rev sample. The sequence information of the primers chitg-fw and chitg-rev is shown in SEQ ID NO.3-4 below; (4) After purifying and recovering the PCR amplification product, it was ligated into the vector pMD20T and then transformed into Escherichia coli Top10; (5) Positive transformants were obtained by bacterial PCR, and the plasmid of the positive transformants was extracted. The chitinase ChiTg encoding gene was finally obtained by enzyme digestion verification and sequencing. chitg.

[0040] Sequence information of primer chitg-fw: 5'-ATGTTGGGTT TCCTCGGAAAG-3' (SEQ ID NO.3);

[0041] Sequence information of primer chitg-rev: 5'-TTAGTTAAGACCGT TTCGGAT-3' (SEQ ID NO.4).

[0042] The purification and recovery process of PCR products is roughly as follows: (1) Cut the target product into a 2mL centrifuge tube; (2) Add the sol solution and react at 60℃ for 10 minutes; (3) Add the sol solution from the second step to the collection tube and centrifuge at 10000rpm for 1 minute; (4) Wash twice with 75% ethanol and air dry; (5) Add 50μL of water and centrifuge for 3 minutes.

[0043] The screening of E. coli transformants was performed using the bacterial culture PCR method. The specific steps of the bacterial culture PCR verification experiment were as follows: (1) A single colony was picked up with a high-pressure sterilized toothpick and placed in 500 μL of LBA medium and cultured at 37°C and 200 rpm for 4 hours; (2) 2 μL of bacterial culture was taken as the template for PCR. The PCR reaction system is shown in Table 1. The primers used for bacterial culture PCR were chitg-fw and chitg-rev. The sequence information of the primers chitg-fw and chitg-rev is shown in SEQ ID NO.3-4. The PCR amplification conditions were 94°C for 5 minutes, 94°C for 30 seconds, 50°C for 30 seconds, 72°C for 90 seconds, for 30 cycles. After electrophoresis, the results were observed, and the colonies on the plates corresponding to the positive results were sequenced.

[0044] Table 1. Bacterial PCR Reaction System

[0045]

[0046] Obtained through sequencing chitg Gene sequence, chitg The full length is 1275 bp, encoding 424 amino acids. chitg The gene sequence and the encoding amino acid sequence are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0047]

[0048] MLGFLGKSPAHRAATQATFESASPVAASNVSVEKRASGYANAVYFTNWGIYGRNFQPQNLVASDITHVIYSFMNFQADGTVVSGDAYADYQKHYDDDSWNDVGNNAY GCVKQLFKLKKANRNLKVMLSIGGWTWSTNFPSAASTDANRKNFAKTAITFMKDWGFDGIDVDWEYPADDTQATNMVLLLKEIRSQLDAYAAQYAPGYHFLLSIAAP AGPEHYSFLHMSDLGQVLDYVNLMAYDYAGSWSSYSGHDANLFANPSNPNSSPYNTDQAIKDYIKGGVPASKIVLGMPIYGRSFESTGGIGQTYSGIGSGSWEGGIW DYKVLPKAGATVQYDSVAQAYYSYDPSSKELISFDTPDMINTKVSHLKNDGLTGSMFWKAFANKTGKDSLIGTSHRALGSLDSTQNLLSYPNSQKDFIRNGLN (SEQ ID NO.6)

[0049] Example 2: Expression and Enzyme Kinetics of Chitinase ChiTg

[0050] Using Pichia pastoris as the host, chitinase ChiTg was recombinantly expressed. Because recombinant expression was performed in Pichia pastoris, the signal peptide of chitinase ChiTg needed to be removed. Predictive analysis using the online software SignalP-5.0 (see website: https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0) revealed that the first 22 amino acids of chitinase ChiTg were its signal peptide sequence. Furthermore, due to differences in codon preferences between *Trichoderma galbana* and Pichia pastoris, the chitinase ChiTg coding gene needed to be optimized according to the codon preferences of Pichia pastoris. The coding sequence without the signal peptide (named...) was then used. chitgs The code was submitted to an online codon optimization software (see: https: / / www.vectorbuilder.cn / tool / codon-optimization.html) for optimization. The optimization results are as follows. Figure 1 As shown, through optimization, chitgsThe expression fitness index of Pichia pastoris was increased from 0.56 to 0.82, and the GC content was adjusted from 54.2% to 48.6%, close to the optimal expression content of Pichia pastoris. A total of 238 bases were optimized during the optimization process, resulting in the optimized gene. chitgsp With original genes chitgs The similarity is 80.3%, and the optimized gene... chitgsp Its sequence is shown in SEQ ID NO.7.

[0051]

[0052] Optimized genes chitgsp Synthesized by Anhui General Biotechnology Co., Ltd., the synthesis process involves genes chitgsp Linked to the vector pPICZαA, thereby obtaining the expression vector pPICZαA- chitgsp The expression vector pPICZαA- chitgsp After linearization with the restriction endonuclease SacI, the product was transformed into Pichia pastoris X33 via electroporation. The transformation process was performed according to the Pichia pastoris instruction manual provided by Thermo Fisher Scientific (see: https: / / www.thermofisher.cn / order / catalog / product / V19520?ICID=search-product). The transformed product was evenly spread on YPDZ plates and incubated statically at 30°C for 3 to 5 days.

[0053] The transformants grown from static culture were screened. The experimental process was roughly as follows: (1) Each transformant was picked into a 100mL shake flask containing 10mL YPD medium and cultured at 30℃ and 200rpm for 24 hours before induction culture was started. During the induction culture, methanol was added at a ratio of 0.75% (v / v) every 24 hours. After 72 hours of culture, chitinase activity was measured. By screening 26 transformants, a dominant enzyme-active bacterium was obtained and named Tg-23.

[0054] The recombinant engineered bacteria Tg-23 was further cultured in 500 mL shake flasks. The experimental procedure was roughly as follows: (1) Each transformant was picked into a 250 mL shake flask containing 50 mL of BMGY medium and cultured at 30 °C and 200 rpm until OD. 600 (2) Centrifuge the cultured bacterial solution, remove the supernatant, and transfer it to a 500 mL shake flask containing 100 mL of BMMY medium. The initial OD of the bacterial solution is 6.0. 600 The culture was carried out at a value of 1.0, 30℃, and 200rpm. During the culture process, methanol was added at a ratio of 0.5% (v / v) every 24 hours for induction culture, and samples were taken at the same time for chitinase activity determination.

[0055] The purification process of recombinant chitinase ChiTg is roughly as follows: (1) First, the supernatant of the 500 mL shake flask fermentation broth in Example 2 was collected by centrifugation; (2) The collected supernatant fermentation broth was concentrated using a 10 kDa ultrafiltration tube; (3) The concentrated recombinant chitinase ChiTg was purified using the Ni-IDA protein purification kit (Shanghai Sangon Biotech); (4) The specific enzyme activity of the purified recombinant chitinase ChiTg was measured. Through purification and activity determination, the specific enzyme activity of recombinant chitinase ChiTg was found to be 28.3 U / mg.

[0056] The process of determining the kinetic parameters of the purified recombinant chitinase ChiTg is roughly as follows: (1) First, prepare colloidal chitin at different concentrations (1 mg / mL-10 mg / mL); (2) Measure the enzyme reaction rate of the purified recombinant chitinase ChiTg at different substrate concentrations; (3) Analyze the different enzyme reaction rates using the software Graph to obtain the Michaelis constant K. m and maximum reaction rate V max Experimental determination revealed the Michaelis constant K of recombinant chitinase ChiTg. m and maximum reaction rate V max The concentrations were 0.74 mg / mL and 31.2 µM / min / mg, respectively.

[0057] Example 3: Homology Modeling and Molecular Docking of Chitinase ChiTg

[0058] Example 2 revealed that the specific activity of the recombinant chitinase ChiTg was 28.3 U / mg, indicating a need for further improvement to lay the foundation for its industrial application. The specific activity of chitinase ChiTg can be further improved through rational protein design. Rational protein design must be based on the three-dimensional structure of the enzyme protein; therefore, it is necessary to simulate and construct the three-dimensional conformation of the recombinant chitinase ChiTg.

[0059] Using the three-dimensional structure of the *Trichoderma harzianum* chitinase Chi42 (PDB accession number: 6epb.1) as a template, the three-dimensional conformation of the chitinase ChiTg was obtained through analysis and modeling using the online software SWISS-MODEL server (see: https: / / swissmodel.expasy.org / interactive). The obtained three-dimensional conformation was evaluated using the online software SAVES v6.0 (see: https: / / saves.mbi.ucla.edu / ). Figure 1 As shown in Figure A, the Laplace plot results indicate that 92.6% of the amino acids are located in the optimal region, and 6.8% of the amino acids are located in other allowed regions, indicating that the model conformation obtained is correct. Figure 1B represents the QMEANDisCo Global assessment result. Generally, a score greater than 0.7 indicates a good model structure. The chitinase ChiTg three-dimensional model has an assessment score of 0.92, which is much higher than 0.7. Figure 1 C represents the 3D-1D score of the model. Generally, a score greater than 0.2 for 80% of amino acids indicates a good model; in the chitinase ChiTg 3D model, 92% of amino acid scores are greater than 0.2. (Summary) Figure 1 The results indicate that the obtained model can be used for further bioinformatics analysis.

[0060] Three-dimensional conformational diagrams of chitinase ChiTg and chitinhexasaccharide were obtained using the molecular docking software Autodock viner. Figure 2 A). Analysis of the docking model using the online software ProteinTools (see website: https: / / proteintools.uni-bayreuth.de / ) revealed that chitinase ChiTg primarily binds chitohexasaccharides to the catalytically active region via hydrogen bonds. Furthermore, the catalytically active region of chitinase ChiTg contains several key amino acids, such as tryptophan (W) at position 26, tyrosine (Y) at position 29, glycine (G) at position 30, arginine (R) at position 31, aspartic acid (D) at position 63, threonine (T) at position 111, glycine (G) at position 194, serine (S) at position 223, glutamic acid (E) at position 295, glycine (G) at position 296, isoleucine (I) at position 298, and tryptophan (W) at position 357.

[0061] By rationally mutating key amino acids in the active region of chitinase ChiTg, the hydrogen bonds between it and the substrate can be increased, thereby further enhancing its specific activity. The mutants to be constructed include: W26R, Y29K, G30K, D63R, T111K, G194R, S223K, E295R, G296R, I298K, and W357R.

[0062] Example 4 Single-point mutation

[0063] Primers were designed separately (primer sequences are shown in Table 2 SEQ ID NO. 8-29) to construct mutants. The construction of different mutants is roughly as follows (taking the W26R site as an example, and others are constructed similarly): using the expression vector pPICZαA- chitgspUsing W26R-fw and W26R-rev as templates, PCR amplification was performed. The PCR reaction system is shown in Table 3. The PCR amplification conditions were: 98℃ pre-denaturation for 30 seconds, 98℃ denaturation for 5 seconds, 55℃ annealing for 20 seconds, and 72℃ extension for 20 seconds, for 33 cycles. The PCR amplification results were detected by agarose gel electrophoresis. The PCR products were purified and recovered. The original plasmid was digested with the restriction endonuclease DpnI. The digested products were then transformed into E. coli Top10 using the heat shock method. The experimental procedure is roughly as follows: (1) Take out Escherichia coli Top10 competent cells from the -60℃ freezer and place them on ice for 20 minutes; (2) Add 10 μL of PCR product, place on ice for 10 minutes, heat shock at 42℃ for 90 seconds, continue to place on ice for 3 minutes, add 500 μL of LB medium, and incubate at 37℃ and 200 rpm for 1 hour; (3) Spread the bacterial solution after 1 hour of incubation evenly on LBZ solid plates, incubate at 37℃ for 18 hours, and then perform bacterial PCR. The bacterial PCR reaction system is shown in Table 4. The reaction conditions are as follows: 94℃, pre-denaturation for 4 minutes, 94℃ denaturation for 30 seconds, 50℃ annealing for 30 seconds, 72℃ extension for 90 seconds, and amplification for 33 cycles; among them, the primers used for bacterial PCR are 5'AOX-fw (GACTGGTTCCAATTGACAAGC) and 3'AOX-rev (GGCACCTGGCATTCTGA CATCC); (4) The recombinant transformant was verified by bacterial PCR. The plasmid of the verified transformant was extracted and sequenced. The expression vector pPICZαA-chitgsp-1 corresponding to the mutant W26R was successfully constructed by sequencing.

[0064] Eleven mutant expression vectors, pPICZαA-, were constructed using the same method. chitgsp- 1 (corresponding to mutant W26R), pPICZαA- chitgsp- 2 (mutant Y29K), pPICZαA- chitgsp- 3 (mutant G30K), pPICZαA- chitgsp- 4 (mutant D63R), pPICZαA- chitgsp- 5 (mutant T111K), pPICZαA- chitgsp- 6 (mutant G194R), pPICZαA- chitgsp- 7 (mutant S223K), pPICZαA- chitgsp- 8 (mutant E295R), pPICZαA- chitgsp- 9 (mutant G296R), pPICZαA- chitgsp- 10 (mutant I298K) and pPICZαA- chitgsp- 11 (mutant W357R).

[0065] Table 2 Single-point mutation primers

[0066]

[0067] Table 3 PCR amplification system for single-point mutants

[0068]

[0069] Table 4 Bacterial PCR Amplification System

[0070]

[0071] The 11 constructed mutant expression vectors were linearized with SacI and transformed into Pichia pastoris X33 via electroporation. To facilitate screening, the copy number of the gene corresponding to each chitinase mutant was controlled to single copy. Based on previous literature reports and our experimental experience, when the plasmid concentration was controlled at 80 ng, the target gene copy number in Pichia pastoris was always single copy. Therefore, the plasmid concentration for each mutant was controlled at 80 ng. Transformants were plated on YPDZ plates and incubated at 30°C for 4 days before screening. Due to the controlled plasmid concentration of 80 ng, only a few positive transformants (approximately 2-4) grew on each YPDZ plate. The screening of positive transformants was the same as in Example 1, and the enzyme activities of the recombinant bacteria corresponding to the 11 mutants are shown in Table 2.

[0072] As shown in Table 5, among the 11 mutants constructed, only the fermentation enzyme activities of the recombinant engineered bacteria corresponding to mutants D63R, G194R, S223K, and G296R were higher than those of the control, at 1.63 U / mL, 1.56 U / mL, 1.43 U / mL, and 1.48 U / mL, respectively, which were 23.5%, 18.2%, 8.3%, and 12.1% higher than the control (1.32 U / mL).

[0073] Table 5 Fermentation enzyme activities of different mutant recombinant bacteria

[0074]

[0075] Example 5, Combinatorial Mutation

[0076] Combinatorial mutations were performed using the positive mutants D63R, G194R, S223K, and G296R obtained in Example 4 to further enhance the specific activity of chitinase ChiTg. Since mutant D63R showed the most significant enhancement, it was used as a starting template to construct the combined mutants D63R / G194R, D63R / S223K, D63R / G296R, D63R / G194R / S223K, D63R / G194R / G296R, D63R / S223K / G296R, and D63R / G194R / S223K / G296R. The primers used in the experiments are shown in Table 2. The construction and screening process of mutants was the same as in Example 4. Table 6 shows that, through experiments, the recombinant strain corresponding to the combined mutant D63R / G194R / S223K / G296R exhibited the best performance under single-copy conditions, with a fermentation enzyme activity of 2.42 U / mL, which was 1.85 times and 1.49 times that of ChiTg and mutant D63R, respectively. Figure 2 As shown in section B, the mutant D63R / G194R / S223K / G296R and the substrate have additional hydrogen bonds, thereby enhancing their binding affinity to the substrate and hydrolysis efficiency. For ease of labeling, the mutant D63R / G194R / S223K / G296R is named the mutant ChiTgM.

[0077] Table 6 Fermentation enzyme activity of recombinant mutant bacteria

[0078]

[0079] Example 6: Purification and Enzyme Kinetic Parameter Determination of the ChiTgM Mutant

[0080] The purification and enzyme kinetic parameter determination procedures for the mutant ChiTgM were consistent with those in Example 2, and the experimental results are shown in Table 7. Table 7 shows that all enzyme kinetic parameters of the mutant ChiTgM are superior to those of the original chitinase ChiTg. The specific activity of the mutant ChiTgM is 51.2 U / mg, which is 1.81 times that of the original chitinase ChiTg. Furthermore, the Michaelis constant K of ChiTgM is... m The concentration was 0.42 mg / mL, indicating that it has better affinity and binding capacity for colloidal chitin compared to the original chitinase ChiTg (0.74 mg / mL). Because the mutant ChiTgM has better substrate binding capacity, its maximum reaction rate VM is higher. max Enzyme catalytic constant k cat And the enzyme specificity constant k cat / K mAll three were superior to the original chitinase ChiTg, being 1.85 times, 2.04 times, and 3.6 times that of the original chitinase ChiTg, respectively. The results in Table 7 indicate that the mutant ChiTgM has better substrate binding ability and catalytic activity, and is more suitable for industrial applications in the preparation of chitin oligosaccharides compared to the original chitinase ChiTg.

[0081] Table 7 Comparison of enzyme kinetic parameters between mutant ChiTgM and chitinase ChiTg

[0082]

[0083] Example 7: Temperature characteristics of mutant ChiTgM and chitinase ChiTg

[0084] The enzyme activities of the mutant ChiTgM and chitinase ChiTg were measured at different temperatures ranging from 35℃ to 60℃ under pH 6.0 conditions. The enzyme activity at the highest temperature was taken as 100%, and the relative enzyme activities at other temperatures were calculated. The experimental results are as follows: Figure 3 As shown in Figure A.

[0085] Depend on Figure 3 As shown in A, the optimal reaction temperature for both the mutant ChiTgM and the chitinase ChiTg is 45℃. Within the range of 30℃ to 45℃, the relative enzyme activity of the mutant ChiTgM is higher than that of the chitinase ChiTg.

[0086] The thermostability of the mutant ChiTgM and chitinase ChiTg was determined as follows: The diluted enzyme solution was heat-treated at different temperatures (30℃ to 55℃) for 1 hour, and the remaining enzyme activity was measured. The enzyme activity of the untreated sample was set as 100%. The remaining enzyme activity at different treatment temperatures was calculated. The experimental results are shown below. Figure 3 As shown in B.

[0087] Depend on Figure 3 As shown in B, the mutant ChiTgM and chitinase ChiTg exhibit good thermostability within the temperature range of 30℃ to 45℃, with residual enzyme activity greater than 90% in both cases. However, when the heat treatment temperature exceeds 50℃, the residual enzyme activity decreases sharply. After heat treatment at 55℃ for 1 hour, the residual enzyme activities of the mutant ChiTgM and chitinase ChiTg are only 8.4% and 7.5%, respectively.

[0088] Example 8: pH characteristics of mutant ChiTgM and chitinase ChiTg

[0089] The enzyme activities of mutant ChiTgM and chitinase ChiTg were measured at 45℃ in pH 4.0–8.0 (pH 4.0–6.0 was measured in 0.05 M sodium acetate buffer, and pH 7.0–8.0 was measured in 0.05 M Tris hydrochloric acid buffer). The enzyme activity at the highest pH was taken as 100%, and the relative enzyme activity at other pH values ​​was calculated. The experimental results are as follows: Figure 4 As shown in Figure A.

[0090] Depend on Figure 4 As shown in A, the optimal reaction pH for both the mutant ChiTgM and the chitinase ChiTg is 6.0, and their relative enzyme activities are similar at different pH values.

[0091] The pH stability of the mutant ChiTgM and chitinase ChiTg was determined as follows: The enzyme solution was diluted with different pH buffers (0.05M sodium acetate buffer for pH 4.0 to 6.0, and 0.05M Tris hydrochloric acid buffer for pH 7.0 to 8.0), and the residual enzyme activity was measured after incubation at 25℃ for 4 hours. The enzyme activity of the untreated sample was set as 100%, and the residual enzyme activity at different treatment temperatures was calculated. The experimental results are shown below. Figure 4 As shown in B.

[0092] Depend on Figure 4 B indicates that the mutant ChiTgM and chitinase ChiTg have poor stability at pH 4.0, with remaining enzyme activities of 52% and 43%, respectively; within the pH range of 5.0 to 8.0, the mutant ChiTgM and chitinase ChiTg have good stability, with remaining enzyme activities greater than 80%.

[0093] Example 9: Effects of metal ions on the stability of mutant ChiTgM and chitinase ChiTg

[0094] The effects of metal ions on the stability of mutant ChiTgM and chitinase ChiTg were determined as follows: Mutant ChiTgM and chitinase ChiTg were added to buffer solutions containing 1 mM of different metal ions (pH 6.0), and incubated at room temperature for 4 hours before activity assays were performed. An untreated sample was used as a control, and the remaining enzyme activity was calculated. The experimental results are shown in Table 8.

[0095] As shown in Table 8, the mutant ChiTgM and chitinase ChiTg exhibit good stability under different metal ion treatment conditions, with residual enzyme activity greater than 73%.

[0096] Table 8. Effects of metal ions on the stability of mutant ChiTgM and chitinase ChiTg.

[0097]

[0098] Example 10: Hydrolysis experiment of mutant ChiTgM and chitinase ChiTg

[0099] Hydrolysis experiments were conducted using a 1% (w / v) colloidal chitin solution as the substrate. The experimental process was roughly as follows: (1) 50 mL of 1% colloidal chitin solution was added to a 250 mL shake flask, and the enzyme addition amount was 1 U / mL; (2) The reaction was terminated after 2 hours of hydrolysis at 200 rpm and 45 °C. The hydrolysis rate was measured and the composition of the hydrolysis products was analyzed.

[0100] The determination of hydrolysis rate is roughly as follows: (1) First, weigh the centrifuge tubes of different 2mL specifications respectively; (2) Then take 2mL of hydrolysis samples of different reaction times; (3) After centrifugation to remove the supernatant, put the centrifuge tube containing the precipitate into 100℃ and dry it to constant weight; (4) The weight after drying minus the weight of the centrifuge tube is the unhydrolyzed colloidal chitin; (5) The hydrolysis rate is calculated as follows: Hydrolysis rate = (theoretical colloidal chitin weight - unhydrolyzed colloidal chitin weight) / theoretical colloidal chitin weight.

[0101] The measurements showed that the hydrolysis rates of the mutant ChiTgM and the chitinase ChiTg were 75% and 43%, respectively, indicating that the mutant ChiTgM has better hydrolysis efficiency.

[0102] The composition of chitin oligosaccharides in the hydrolysis products of mutant ChiTgM and chitinase ChiTg was analyzed by thin-layer chromatography. The experimental procedure was as follows: (1) 2 μL of hydrolysis product and 2 μL of chitin oligosaccharide standard mixture were spotted onto a silica gel plate (Silica gel 60, Merck); the spotted silica gel plate was placed in an expansion tank for expansion. The expansion buffer was a mixture of n-butanol, methanol, ammonia and water (volume ratio of 5:4:2:1); (2) the expanded silica gel plate was removed from the expansion tank, dried, and then sprayed with a display agent (the display agent was 0.4 g diphenylamine, 0.4 mL aniline and 3 mL 85% phosphate dissolved in 20 mL acetone solution); (3) after drying, the silica gel plate was placed at 100 °C for high-temperature color development.

[0103] Depend on Figure 5 It is known that the products of the mutant ChiTgM and the chitinase ChiTg hydrolyzing colloidal chitin are mainly composed of chitobiose.

[0104] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A chitinase mutant ChiTgM, characterized in that, The amino acid sequence of the chitinase mutant ChiTgM is shown in SEQ ID NO.

1.

2. The use of the chitinase mutant ChiTgM as described in claim 1 in the preparation of chitin oligosaccharides.

3. The application according to claim 2, characterized in that, The chitin oligosaccharide was prepared by hydrolyzing chitin using the chitinase mutant ChiTgM.

Images