Endochitinase mutant, encoding gene and application thereof

Abstract

The application belongs to the field of enzymatic engineering and computer-aided biological technology, and particularly relates to an endochitinase mutant, a coding gene thereof and application, and an amino acid sequence of the endochitinase mutant is shown as SEQ ID NO. 4 or SEQ ID NO. 6. In order to solve the problems of low catalytic efficiency and poor stability of natural endochitinase, the application obtains N197R and N197RCBM14 fusion mutants by computer-aided rational design on wild type ThChi8. Experiments prove that the specific activity of the key mutant N197RCBM14 is increased to 268.25% of the wild type. The mutant gene can be cloned into a pET-29a(+) vector and expressed in Escherichia coli Rosetta (DE3), and the secreted enzyme preparation can efficiently degrade chitin to produce chitooligosaccharides, and has a wide application prospect in the fields of biomass conversion, agriculture and medicine.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and computer-aided biotechnology, specifically involving two optimized endochondrinase mutants and their encoding genes and applications, particularly suitable for chitin degradation and biomass conversion. Background Technology

[0002] Chitin, a high-molecular-weight polymer composed of N-acetylglucosamine (GlcNAc) linked by β-1,4-glycosidic bonds, is the second most abundant renewable resource in nature, second only to cellulose. Its degradation product, chitosan oligosaccharides (COS), possesses unique physiological activities, is small in molecular weight, highly soluble, and easily absorbed, showing broad application prospects in biomedicine, food, cosmetics, textiles, and agriculture. However, natural chitin is extremely stable and poorly soluble in water and common acidic and alkaline solvents, posing a significant challenge to its efficient degradation and utilization. Therefore, developing enzyme preparations capable of efficiently hydrolyzing chitin has become a key technological bottleneck for achieving the high-value utilization of chitin resources.

[0003] To overcome the performance limitations of natural enzymes, rational protein design techniques have emerged and developed rapidly. Traditional enzyme engineering methods, such as directed evolution, while not relying on detailed knowledge of protein structures, require large-scale construction of random mutant libraries and high-throughput screening, which is labor-intensive and somewhat unpredictable.

[0004] Furthermore, naturally derived endochondrinases, especially those with good thermal stability and acid tolerance, often suffer from low enzyme activity, low catalytic efficiency, or incompatibility between optimal reaction conditions and industrial applications, severely limiting their industrial application. For example, while early directed evolution strategies such as error-prone PCR could improve enzyme activity, the process was lengthy and the improvement in key properties such as thermal stability was limited. Summary of the Invention

[0005] This invention protects a chitinase mutant gene, which is designed by alanine scanning, saturation mutagenesis and PyRosetta calculation of the wild-type chitinase ThChi8 gene (sequence shown in SEQ ID NO.1). Key mutants include N197R (cDNA sequence shown in SEQ ID NO.3) and N197RCBM14 (cDNA sequence shown in SEQ ID NO.5), etc., wherein the specific activity of mutant N197RCBM14 is 268.25% of that of wild type.

[0006] This invention also protects the application of the above-mentioned endochondrinase mutant in chitin degradation and biomass conversion.

[0007] The purpose of this invention is to provide an endogenous chitinase mutant derived from NJAU4742, which improves its catalytic efficiency and / or stability through semi-rational design, thereby enhancing its ability to decompose chitin and making it suitable for biomass conversion applications.

[0008] The present invention also protects the protein encoded by the mutant gene (wherein the amino acid sequence of N197R is shown in SEQ ID NO.4 and the amino acid sequence of N197RCBM14 is shown in SEQ ID NO.6), as well as the recombinant expression vector and engineered bacteria containing the gene.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] An endochondinoxinase mutant, wherein the endochondinoxinase mutant is an N197R single-point mutant or an N197RCBM14 fusion mutant; wherein, the N197R is obtained by mutating the 197th amino acid residue of wild-type endochondinoxinase ThChi8 from asparagine (Asn) to arginine (Arg); the N197RCBM14 is obtained by introducing the N197R point mutation, replacing the domain of wild-type ThChi8 with the carbohydrate-binding module CBM14 and fusing it with a linker peptide; the amino acid sequence of the wild-type endochondinoxinase ThChi8 is shown in SEQ ID NO.2.

[0011] The gene sequence encoding a wild-type endochondrinase ThChi8 is shown in SEQ ID NO.1.

[0012] The amino acid sequence of the endochondrinase mutant is shown in SEQ ID NO.4 or SEQ ID NO.6.

[0013] The gene encoding the above-mentioned endochondrinase mutant.

[0014] The encoding gene sequence of the endochondrinase mutant is shown in SEQ ID NO.3 or SEQ ID NO.5.

[0015] A recombinant vector contains the aforementioned endogenous chitinase mutant gene;

[0016] Preferably, the recombinant expression vector is pET-29a(+), the mutant sequence is obtained by whole-genome synthesis, and it is transformed into Escherichia coli Rosetta(DE3) for expression.

[0017] The expression vector for the recombinant vector is pET-29a(+).

[0018] A recombinant strain contains the endogenous chitinase mutant gene described above or the recombinant vector described above;

[0019] The recombinant strain was Escherichia coli Rosetta (DE3).

[0020] A method for producing an endochondrinase mutant as described above, the method comprising the following steps: constructing a gene encoding the endochondrinase mutant into an expression vector, transferring the expression vector into a host bacterium for expression, and obtaining the endochondrinase mutant from the host bacterium.

[0021] Application of any of the above-mentioned endochondrinase mutants in chitin degradation and biomass conversion.

[0022] The application of the described endochondrinase mutant in the degradation of chitin and biomass conversion under conditions of 40-60℃ and pH 4.0-6.0;

[0023] Preferably, the temperature is 40°C; and the pH is 4.0.

[0024] The described endochondrinase mutant is used to degrade colloidal chitin to generate chitin oligosaccharides.

[0025] Beneficial effects

[0026] This invention utilizes computer-aided rational design to obtain the ThChi8 mutant of endochondrinase, which exhibits significantly enhanced enzyme activity and stability. Experiments have confirmed that some mutants (such as N197RCBM14) show an enzyme activity 168.25% higher than the wild type, and demonstrate better stability at higher temperatures and over a wider pH range, providing a more efficient enzyme preparation for biomass conversion in industrial environments. Attached Figure Description

[0027] Figure 1 Expression, purification, and enzymatic properties of the ThChi8 mutant endochondrinase

[0028] A: Electrophoretic bands of ThChi8 and its mutants

[0029] B: Comparison of relative enzyme activities of ThChi8 and its mutants at different temperatures

[0030] C: Comparison of relative enzyme activities of ThChi8 and its mutants at different pH values

[0031] D: Comparison of the thermal stability of ThChi8 and its mutants at 50℃

[0032] E: Comparison of the thermal stability of ThChi8 and its mutants at 60℃

[0033] Figure 2 ThChi8 structural modeling and molecular docking

[0034] A: ThChi8 structural modeling

[0035] B: Conformation of the ThChi8 molecular docking complex

[0036] Figure 3 Alanine scan of PyRosetta mutant

[0037] Figure 4 PyRosetta mutant saturation mutation results diagram

[0038] Figure 5 Product analysis results of ThChi8 and its mutants

[0039] A: ThChi8 product analysis results

[0040] B: ThChi8N197R product analysis results

[0041] C: ThChi8N197RCBM14 Product Analysis Results Detailed Implementation

[0042] The present invention will be further described below with reference to the embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out in accordance with known means in the art.

[0043] Example 1 Preparation of substrates for enzyme activation reaction

[0044] Colloidal chitin is mainly prepared using an acid hydrolysis precipitation method. Chitin raw material is mixed with concentrated hydrochloric acid (approximately 20-40 mL of 35% concentrated hydrochloric acid per 1 g of chitin powder), stirred for 2 hours in an ice bath at low temperature (2-4℃), and then allowed to stand for 24 hours to fully dissolve. Subsequently, the resulting acid hydrolysate is slowly poured into a large volume of ice-pre-cooled precipitant (50% ethanol) (the volume should be 5 times the volume of the acid hydrolysate), and stirred to precipitate the colloidal chitin. The precipitate is collected by centrifugation at 10,000 rpm for 15 minutes, washed repeatedly with distilled water until the supernatant is neutral, and finally resuspended in deionized water to obtain a colloidal chitin suspension.

[0045] Example 2 Construction of enzyme mutants

[0046] This study employed an integrated computational and experimental strategy to investigate a Trichoderma strain, T. guizhouense NJAU4742.

[0047] The endochondinokinase ThChi8 was structurally elucidated and molecularly modified. The signal peptide of ThChi8 was detected and cleaved using SignalP 4.1 Server (http: / / www.cbs.dtu.dk / services / SignalP / ). Laboratory-preserved E. coli DH5α strain was inoculated into glass tubes containing 5 mL of liquid LB medium and incubated at 37°C and 180 r·min. -1 After activation, the sample was transferred to 50 mL of liquid LB medium for further culture. After culturing, plasmid pET-29a(+) was extracted using a plasmid extraction kit (Axygen, AP-MN-P-50), and its concentration was determined. The plasmid pET-29a(+) and the target gene fragment were mixed with restriction endonucleases EcoRI and KpnI in a specific ratio, and reacted in a water bath for 20 h. The digestion products were then recovered. The two digestion products were mixed in a specific ratio (plasmid:target gene = 1:10) and ligated using T4 ligase in a 16℃ water bath for 16 h. The fusion fragment was verified by agarose gel electrophoresis after the reaction. The fusion fragment was stored at -4℃ and further sequenced by Anhui General Biotechnology Co., Ltd. Subsequently, based on the amino acid sequence of the wild-type ThChi8, its three-dimensional structure was predicted using AlphaFold3 (v2025.06), a tool capable of accurately simulating protein conformation. The obtained ThChi8 three-dimensional structure model is shown below. Figure 2 As shown in Figure A. Subsequently, molecular docking technology was used to explore the interaction mechanism between ThChi8 and the substrate. Using chitosan as the substrate ligand, energy minimization optimization was performed using Avogadro 1.2.0 (MMFF94 force field), and then the ligand was docked into the predicted active pocket of ThChi8 using AutoDock software. The optimal binding mode was screened by the binding free energy (ΔG), and key interactions such as hydrogen bonding and hydrophobic interactions were analyzed. The conformation of the docked ThChi8-substrate complex is shown in Figure A. Figure 2 As shown in B in the diagram.

[0048] To rationally design high-performance mutants, alanine scanning and saturation mutagenesis were further conducted using the PyRosetta-4 suite. Systematic mutations were performed on residues with a substrate binding pocket distance ≤ 5 Å, and the binding free energy change (ΔΔG) was calculated to screen for potentially beneficial mutations with ΔΔG < -0.5 kcal / mol. The calculation results for alanine scanning are shown below. Figure 3 As shown in the figure. Further saturation mutagenesis analysis was performed on the key sites identified through screening to compare the effects of different amino acid substitutions on the binding free energy. The results are shown in the figure. Figure 4 As shown.

[0049] Based on the PyRosetta calculations above, the binding free energy (ΔG_mut) and relative change (ΔΔG) of some representative mutants were summarized, and the results are shown in Table 1.

[0050] Table 1. Changes in binding free energy and stability of mutants

[0051]

[0052] As shown in Table 1, the R substitution at position 197 (197-R) has a stabilization trend with a negative ΔΔG. Therefore, N197R was selected as a candidate mutant for subsequent construction and experimental verification.

[0053] Meanwhile, to enhance the enzyme's binding ability to chitin, a domain substitution strategy was adopted: the original domain of the mutant N197R was replaced with the carbohydrate-binding module CBM14, and a fusion protein (such as N197RCBM14) was constructed using a natural linker peptide. All designed mutants were expressed and purified using an E. coli system, and their enzyme activity and thermostability were determined using the DNS method.

[0054] Example 3 Purification of enzyme mutants

[0055] This invention employs nickel affinity chromatography to purify wild-type and mutant (e.g., N197R, N197RCBM14) endonucleases carrying histidine tags (His-Tag), with consistent purification procedures. The specific steps are as follows: First, *E. coli* Rosetta(DE3) containing recombinant plasmids pET-29a(+)-ThChi8N197R (cDNA sequence shown in SEQ ID NO. 7) and pET-29a(+)-ThChi8N197RCBM14 (cDNA sequence shown in SEQ ID NO. 8) are cultured in LB medium containing appropriate amounts of antibiotics until OD500. 600 The concentration was set to 0.6-0.8, followed by the addition of 1 mM IPTG, and expression was induced overnight at 30°C. Bacterial cells were collected by centrifugation and resuspended in pre-cooled equilibration buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8.0). After high-pressure homogenization (total time 15 minutes) on ice, the cells were centrifuged at 18,000 rpm for 25 minutes at 4°C, and the supernatant was collected as the crude enzyme solution.

[0056] Purification was performed using a Ni Seplife FF affinity chromatography column. Before loading, the column was equilibrated with at least 5 column volumes (CV) of equilibration buffer. The crude enzyme solution was loaded at a flow rate of 0.5–1 mL / min, followed by washing with wash buffer containing 20–50 mM imidazole for 10–15 CV to remove non-specifically bound proteins. Finally, the target protein was collected using either step elution or a linear gradient elution (imidazole concentration 200–500 mM). Elution was performed at 4°C to maintain protein stability. The purified protein solution was transferred to storage buffer via ultrafiltration centrifuge tubes, and after concentration determination, stored at -80°C.

[0057] The purity and specificity of the purified protein were verified by SDS-PAGE (using a 12.5% ​​separating gel and a 4.5% stacking gel). Figure 1 As shown in Figure A (from left to right: wild-type ThChi8; mutant N197RCBM14; mutant N197R; mutant N197Y), both wild-type and mutant proteins show a single, clear band at the expected molecular weight, indicating that high-purity protein samples have been obtained and can be used for subsequent enzymatic property analysis.

[0058] Example 4: Effects of different temperatures on wild-type ThChi8 and mutants N197R and N197RCBM14

[0059] Take 50 μL of diluted enzyme solution containing 1 μM enzyme, add 450 μL of pH 5.0 20mM acetate-sodium acetate buffer to a 2 mL centrifuge tube, and preheat in a water bath at 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 10 min. Add 10 g·L⁻¹ of preheated solution at each temperature for 10 min. -1 500 μL of colloidal chitin solution was mixed well and then placed in a water bath at the corresponding temperature for 10 min. Immediately afterwards, 1 mL of 1 mol·L⁻¹ solution was added. -1 The reaction was terminated with Na₂CO₃, and the mixture was incubated at room temperature for 5 min. The absorbance (OD) was measured at 540 nm. Simultaneously, the heat-inactivated enzyme solution was treated in the same way as a blank control. Each treatment was performed in triplicate. The measured data were analyzed graphically. The comparison of relative enzyme activities between wild-type and mutant under different temperature conditions is shown below. Figure 1 As shown in B, the enzyme exhibits the highest activity at 40°C and maintains high activity in the 40-60°C range. At the optimal temperature, the two mutants increased their activity to 190.77% and 268.25% of the original enzyme activity, respectively.

[0060] Example 5: Effects of different pH buffers on wild-type ThChi8 and mutants N197R and N197RCBM14

[0061] Take 50 μL of diluted enzyme solution containing 1 μM enzyme, add 450 μL of Na₂HPO₄-citric acid buffer (pH 4.0, 5.0), Na₂HPO₄-KH₂PO₄ buffer (pH 5.0, 6.0, 7.0), or Tris-HCl buffer (pH 7.0, 8.0, 9.0) to a 2 mL centrifuge tube, preheat in a 50°C water bath for 10 min, add 500 μL of preheated 10 g / L colloidal chitin solution, mix well, and then incubate in a water bath for another 10 min. Immediately afterward, add 1 mL of 1 mol·L⁻¹ enzyme solution. -1 The reaction was terminated with Na₂CO₃, and the solution was incubated at room temperature for 5 min. The absorbance (OD) was measured at 540 nm. Simultaneously, the heat-inactivated enzyme solution was treated in the same way as a blank control. Each treatment was performed in triplicate. The measured data were analyzed graphically, and the results are compared as follows: Figure 1 As shown in C. The enzyme exhibits the highest activity at pH 4. Compared to the wild type, the N197R mutant shows an increase of approximately 90% in enzyme activity, while the N197RCBM14 shows an increase of approximately 170%.

[0062] Example 6: Results of Mutant Product Analysis

[0063] To elucidate the catalytic properties of ThChi8 and its mutants on colloidal chitin, the hydrolysis products were analyzed using an ultra-high performance liquid chromatography-mass spectrometry imaging system. The results are as follows: Figure 5 As shown in A, B, and C, ThChi8 hydrolyzes colloidal chitin to form different polysaccharides, with chitobiose [(GlcNAc)2] being the most abundant among the hydrolysis products; in addition, chitotriose was also detected. The results indicate that the final products of ThChi8 hydrolyzing colloidal chitin are mainly oligosaccharides, especially chitobiose and trisaccharides. The enzymatic hydrolysis results of mutant N197R also showed that (GlcNAc)2 was the predominant product, and its abundance was greater than that of the wild-type enzyme under the same system, further supporting the results of this experiment. Analysis of the products of mutant N197RCBM14 showed an increased abundance of (GlcNAc)3, indicating another direction of increased enzyme activity. Based on the product signal intensity, the ThChi8N197R mutant showed an increase of approximately 60% in the total disaccharide and trisaccharide products, while the ThChi8N197RCBM14 mutant showed a 175% increase in trisaccharide signal intensity.

[0064] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

[0065] Sequence list:

[0066] Wild-type ThChi8 cDNA sequence SEQ ID NO. 1

[0067]

[0068] Wild-type ThChi8 amino acid sequence SEQ ID NO. 2

[0069] SPLATEERSVEKRANGYANSVYFTNWGIYDRNFQPADLVASDVTHVIYSFMNLQADGTVVSGDTYADFEKHYADDSWNDVGTNAYGCAKQLFKVKKANRGLKVLLSIGGWTWSTNFPSAASTDANRKNFAKTAITFMKDWGFDGIDVDWEYPADATQASNMVLLLKEVRSQLDAYAAQYAPGYHFLLTIAAPAGKDNYSKLRLADLGQVLDYINLMAYDYAGSFSPLTGHDANLFANPSNPNATPFNTDSAVKDYIKGGVPANKIVLGMPIYGRSFQNTAGIGQTYNGVGGGGGGSTGSWEAGIWDYKALPKAGATIQYDSVAKGYYSYNAGTKELISFDTPDMINTKVAYLKSLGLGGSMFWEASADKKGADSLIGTSHRALGGLDTTQNLLSYPNSKYDNIRNGLN

[0070] Single-point mutant / N197R / cDNA sequence SEQ ID NO. 3

[0071] >AGCCCACTGGCGACCGAAGAACGTAGTGTTGAAAAACGCGCGAATGGCTATGCGAATAGCGTGTATTTTACCAATTGGGGCATTTACGACCGCAATTTCCAGCCGGCGGATCTGGTGGCGAGTGATGTGACCCATGTTATTTATAGCTTTATGAACCTGCAGGCGGACGGCACCGTGGTGAGCGGTGATACCTATGCGGATTTTGAAAAACATTACGCGGACGATAGCTGGAACGATGTGGGCACCAATGCGTATGGCTGCGCGAAACAGCTGTTTAAAGTGAAAAAAGCGAATCGCGGCCTGAAAGTGCTGCTGAGCATTGGCGGTTGGACCTGGAGTACCAATTTTCCGAGCGCGGCGAGCACCGATGCGAATCGTAAAAATTTTGCGAAAACCGCGATTACCTTTATGAAGGACTGGGGCTTTGATGGCATTGACGTGGATTGGGAATATCCGGCGGATGCGACCCAGGCGAGCAATATGGTTCTGCTGCTGAAAGAAGTGCGCAGCCAGCTGGATGCGTATGCGGCGCAATATGCGCCGGGTTATCATTTTCTGCTGACCATTGCGGCGCCGGCGGGTAAAGAT CGCTATAGCAAACTGCGCCTGGCGGATCTGGGCCAGGTTTTAGATTATATTAATCTGATGGCGTACGATTACGCGGGCAGCTTTAGCCCGTTAACCGGTCATGATGCGAATCTGTTTGCGAATCCGAGCAATCCGAATGCGACCCCGTTTAATACCGATAGCGCGGTGAAAGATTATATCAAGGGCGGCGTGCCGGCGAATAAAATTGTGCTGGGCATGCCGATTTATGGCCGCAGCTTTCAGAATACCGCGGGCATTGGCCAGACCTATAATGGCGTGGGCGGCGGCGGTGGTGGTAGTACTGGTAGCTGGGAAGCGGGTATTTGGGATTATAAAGCGCTGCCGAAAGCGGGCGCGACCATTCAATATGATAGCGTGGCGAAAGGCTATTATAGCTATAATGCGGGCACCAAAGAACTGATTAGCTTTGATACCCCGGATATGATTAATACCAAAGTGGCGTATCTGAAGAGCCTGGGCCTGGGCGGTAGCATGTTTTGGGAAGCGAGCGCGGATAAAAAAGGCGCGGATAGCCTGATTGGCACCAGCCATCGTGCGTTGGGCGGTTTAGATACCACCCAGAATTTGCTGAGCTATCCGAATAGCAAATATGACAATATCCGCAACGGCCTGAACCTCGAG

[0072] Single point mutant / N197R / Amino acid sequence SEQ ID NO. 4

[0073] SPLATEERSVEKRANGYANSVYFTNWGIYDRNFQPADLVASDVTHVIYSFMNLQADGTVVSGDTYADFEKHYADDSWNDVGTNAYGCAKQLFKVKKANRGLKVLLSIGGWTWSTNFPSAASTDANRKNFAKTAITFMKDWGFDGIDVDWEYPADATQASNMVLLLKEVRSQLDAYAAQYAPGYHFLLTIAAPAGKD RYSKLRLADLGQVLDYINLMAYDYAGSFSPLTGHDANLFANPSNPNATPFNTDSAVKDYIKGGVPANKIVLGMPIYGRSFQNTAGIGQTYNGVGGGGGGSTGSWEAGIWDYKALPKAGATIQYDSVAKGYYSYNAGTKELISFDTPDMINTKVAYLKSLGLGGSMFWEASADKKGADSLIGTSHRALGGLDTTQNLLSYPNSKYDNIRNGLN

[0074] N197RCBM14 cDNA sequence SEQ ID NO. 5

[0075] >GGATCCAAGCCCGTTAGCGACCGAAGAACGCAGCGTTGAAAAACGCGCGAATGGCTATGCGAATAGCGTGTATTTTACCAATTGGGGCATTTACGATCGCAATTTTCAGCCGGCGGATCTGGTGGCGAGCGATGTTACCCATGTGATTTATAGCTTTATGAATCTGCAGGCGGATGGCACCGTGGTGAGCGGCGATACCTATGCGGATTTTGAAAAACATTATGCGGACGACAGCTGGAACGATGTGGGCACCAATGCGTATGGCTGCGCGAAACAGCTGTTTAAAGTGAAAAAAGCGAATCGCGGCCTGAAAGTGCTGCTGAGCATTGGCGGTTGGACCTGGAGCACCAATTTTCCGAGCGCGGCGAGCACCGATGCGAATCGTAAAAATTTTGCGAAAACCGCGATTACCTTTATGAAGGATTGGGGCTTTGATGGCATTGATGTGGATTGGGAATATCCGGCGGATGCGACCCAGGCGAGCAATATGGTTCTGCTGCTGAAAGAAGTGCGCAGCCAGCTGGATGCGTATGCGGCACAATATGCGCCGGGTTATCATTTTCTGCTGACCATTGCGGCGCCGGCGGGTAAAGAT CGCTATAGCAAACTGCGCCTGGCGGATCTGGGCCAGGTTTTAGATTATATTAATCTGATGGCGTACGACTACGCGGGCAGCTTTAGCCCGTTAACCGGTCATGATGCGAATCTGTTTGCGAATCCGAGCAATCCGAATGCGACCCCGTTTAATACCGATAGCGCGGTGAAAGATTATATCAAGGGCGGCGTGCCGGCGAATAAAATTGTGCTGGGCATGCCGATTTATGGCCGCAGCTTTCAGAATACCGCGGGCATTGGCCAGACCTATAATGGCGTGGGCGGCGGCGGTGGTGGTAGTACTGGTAGCTGGGAAGCGGGTATTTGGGATTATAAAGCGCTGCCGAAAGCGGGCGCGACCATTCAATATGATAGCGTGGCGAAAGGCTATTATAGCTATAATGCGGGCACCAAAGAACTGATTAGCTTTGATACCCCGGATATGATTAATACCAAAGTGGCGTATCTGAAGAGCCTGGGCCTGGGCGGTAGCATGTTTTGGGAAGCGAGTGCGGATAAAAAAGGCGCGGATAGCCTGATTGGCACCAGCCATCGTGCGTTGGGTGGTTTAGATACCACCCAAAATCTGCTGAGCTATCCGAATAGCAAATATGACAATATCCGCAACGGCCTGAATGACTTCGCGGGCTTTAGCTGCAATCAGGGCCGCTATCCGCTGATTCAGACCCTGCGCCAAGAACTGAGCCTGCCGTATTTACCGAGCGGCACCCCAGAATTGGAAGTGCCGAAACCAGGTCAACCAAGCGAACCAGAACATGGCCCAAGCCCGGGCCAAGATACCTTTTGTCAAGGCAAAGCGGATGGCCTGTATCCGAATCCGCGCGAACGTAGCAGTTTTTATAGCTGCGCGGCGGGCCGTTTGTTTCAGCAAAGTTGTCCAACCGGCCTGGTGTTTAGCAATAGCTGCAAATGCTGCACCTGGAATTAACTCGAG

[0076] N197RCBM14 Amino Acid Sequence SEQ ID NO. 6

[0077] SPLATEERSVEKRANGYANSVYFTNWGIYDRNFQPADLVASDVTHVIYSFMNLQADGTVVSGDTYADFEKHYADDSWNDVGTNAYGCAKQLFKVKKANRGLKVLLSIGGWTWSTNFPSAASTDANRKNFAKTAITFMKDWGFDGIDVDWEYPADATQASNMVLLLKEVRSQLDAYAAQYAPGYHFLLTIAAPAGKD R YSKLRLADLGQVLDYINLMAYDYAGSFSPLTGHDANLFANPSNPNATPFNTDSAVKDYIKGGVPANKIVLGMPIYGRSFQNTAGIGQTYNGVGGGGGGSTGSWEAGIWDYKALPKAGATIQYDSVAKGYYSYNAGTKELISFDTPDMINTKVAYLKSLGLGGSMFWEASADKKGADSLIGTSHRALGGLDTTQNLLSYPNSKYDNIRNGLNDFAGFSCNQGRYPLIQTLRQELSLPYLPSGTPELEVPKPGQPSEPEHGPSPGQDTFCQGKADGLYPNPRERSSFYSCAAGRLFQQSCPTGLVFSNSCKCCTWN

[0078] pET-29a(+)-ThChi8N197R cDNA Sequence SEQ ID NO. 7

[0079]

[0080]

Claims

1. An endochondrinase mutant, characterized in that, The amino acid sequence of the endochondrinase mutant is shown in SEQ ID NO.4 or SEQ ID NO.

6.

2. The gene encoding the endochondrinase mutant of claim 1.

3. The gene according to claim 2, characterized in that, The gene sequence is shown in SEQ ID NO.3 or SEQ ID NO.

5.

4. A recombinant vector, characterized in that, The recombinant vector contains the endogenous chitinase mutant gene as described in claim 2 or 3, and the expression vector of the recombinant vector is pET-29a(+).

5. A recombinant bacterial strain, characterized in that, The recombinant strain contains the endogenous chitinase mutant gene as described in claim 2 or 3 or the recombinant vector as described in claim 4, and the recombinant strain is Escherichia coli Rosetta (DE3).

6. A method for producing the endochondrinase mutant according to claim 1, characterized in that, The production method includes the following steps: constructing the gene encoding the endochondrinase mutant into an expression vector, transferring the expression vector into a host bacterium for expression, and obtaining the endochondrinase mutant from the host bacterium.

7. The application of the endochondrinase mutant according to claim 1 in chitin degradation and biomass conversion.

8. The application according to claim 7, characterized in that, The endochitinase mutant was used for chitin degradation and biomass conversion under conditions of 40–60°C and pH 4.0–6.

0.

9. The application according to claim 8, characterized in that, The temperature is 40°C; the pH is 4.

0.

10. The application according to any one of claims 7 to 9, characterized in that, The endochitinase mutant is used to degrade colloidal chitin to generate chitin oligosaccharides.

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