Chitinase mutant d615s with high thermal stability and construction method and application thereof

Abstract

The application discloses a chitinase mutant D615S with high thermal stability and a construction method and application thereof. A wild-type chitinase Chil is subjected to thermal stability modification through a semi-rational design method, and a point mutation of aspartic acid at the 615th position is changed into serine, so that the half-life of the obtained mutant at 40 DEG C is increased by 3.6 times, and the half-life at 45 DEG C is increased by 24 times. When 100 g / L powder chitin is degraded at 45 DEG C for 60 h, 48 g / L reducing sugar is obtained, and the degradation rate is 48%; when 100 g / L lobster shells with 20% chitin content are degraded for 60 h, 9 g / L reducing sugar is obtained, and the degradation rate can reach 45%; through identification of the degradation products, it is found that the products are N acetylglucosamine and chitobiose with a mass ratio of 4:1, and have a good market prospect.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis, specifically relating to a chitinase mutant D615S with high thermal stability, its construction method, and its application. Background Technology

[0002] Chitin, also known as chitosan, is composed of... N - Acetaminophen (GlcNAc) via β A polysaccharide polymer formed by -D-(1,4)-glycosidic bonds, with the molecular formula (C8H... 13 05N) n Chitin is the second most abundant mineral on Earth after cellulose. It is mainly found in the shells of crustaceans, the exoskeletons of insects, and the cell walls of fungi. However, due to the lack of efficient refining methods, it is often disposed of as waste through landfills or dumping, taking up space and burdening the environment. Therefore, the development and efficient utilization of chitin resources is crucial. However, chitin's insolubility in water is one of the biggest obstacles to its development and utilization. [The text then abruptly shifts to a seemingly unrelated topic:] ...to extract the... β Cleavage of the -1,4-glycosidic bond to form soluble GlcNAc and low-polymerization-degree chitin oligosaccharide products is one of the main strategies for achieving efficient utilization of chitin.

[0003] Chitosan oligosaccharides and GlcNAc possess excellent biological functions, including antibacterial, antitumor, immunomodulatory, and wound-healing properties. They have significant applications in agriculture, medicine, food, and feed additives. Therefore, converting discarded chitin resources into high-value-added chitosan oligosaccharides and GlcNAc is crucial for their utilization, bringing both economic and environmental benefits.

[0004] Previous studies have shown that the optimal temperature for chitinase Chi1 is 40°C, but its half-life is 20 hours at this temperature. Crystalline chitin is difficult to degrade, requiring a long reaction time, and the resulting GlcNAc and chitosan oligosaccharides are easily contaminated by microorganisms at lower temperatures. Therefore, modifying the enzyme's thermal stability to increase its reaction temperature and enable it to catalyze for longer periods at higher temperatures is a technical challenge that needs to be addressed by those skilled in the art. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a chitinase mutant D615S with high thermal stability, its construction method, and its applications. The obtained chitinase mutant D615S has a half-life of 72 h at 40℃, which is 3.6 times that of the wild-type fungus, and a half-life of 60 h at 45℃, which is 24 times that of the wild-type fungus. m The temperature was increased by 6.6℃, which can efficiently degrade chitin-containing biomass such as powdered chitin and shrimp and crab shells.

[0006] To address the problems in the existing technology, the technical solution adopted by this invention is as follows:

[0007] A chitinase mutant D615S with high thermal stability has an amino acid sequence referenced by SEQ ID NO.1 in the sequence listing. It features a point mutation at position 615, where aspartic acid is replaced by serine. Compared to wild-type chitinase, this chitinase mutant exhibits a half-life of 72 h at 40°C (3.6 times that of the wild-type) and a half-life of 60 h at 45°C (24 times that of the wild-type). The optimal temperature for its degradation is 40°C. m The value was 56.9℃, which was 6.6℃ higher than that of the wild type. When 100 g / L of granular chitin was degraded at 45℃ for 60 h, 48 g / L of reducing sugar was obtained, with a degradation rate of 48%. Identification of the degradation products revealed that the product was... N The mass ratio of acetylglucosamine to chitobiose is 4:1. Degradation of 100 g / L lobster shells with a chitin content of 20% for 60 h yields 9 g / L of reducing sugar, achieving a degradation rate of 45%.

[0008] SEQ ID NO.1:

[0009] SASFAATAWNSTTVYTGGEIVTYQGTDYKAKWWTQGNVPGAEQWGPWEAQGPVSATPVPTATTAPTATPVTTATSAPTATPKPTATPAVGTCVDAAWNSSTAYTGGQKVSYNGRTYQAQWWTSGDQPDLNTGSGKPWKDLGACGPVVATPTPTATVAPTATPVVTATPTPTATPVVTATPTATPVGPTPTPTPVVTPTVPPSTGAKQVGTYFAEWSIYGRKFFLKNVQDSGQAAKLTFLNYSFGNVYKQADGTYKCQANINKAETGNQDGGDAWALYQKGFAANESVDGVADAWGQDGKGSLKGNWNQLKKLKAKNPNMKVLISLGGWTWSKWFSAAASTDALRKTLVASCIDVWIKGNLPFDAASNAGGAGTGAGVFDGIDIDWEYPGVQGIGTNTVSPADKENNTLLMKEFREQLDAIGSQTGKRYLLTVAIGAGDEKIAATVPGEYSKYLDWINIMSYDYNGGWDAAGPTDFQSNLYQDPASPRTVDPKTGKVSKYYTDAAVKDLIARGVPAVKLHIGVPFYGRGWTGVTNVNNGLYQKATGAAKGTYESGIEDYKVLKTAPGTEYIHPVTQQTYKFDGSTFWSYDTPRDIKLKADYAKSMGMGGIFSWEADGDTANGELVEAMTHINK

[0010] Among them, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.3.

[0011] SEQ ID NO.3:

[0012]

[0013] The amino acid sequence of the chitinase mutant D615S with high thermal stability is shown in SEQ ID NO.2.

[0014] SEQ ID No. 2:

[0015] SASFAATAWNSTTVYTGGEIVTYQGTDYKAKWWTQGNVPGAEQWGPWEAQGPVSATPVPTATTAPTATPVTTATSAPTATPKPTATPAVGTCVDAAWNSSTAYTGGQKVSYNGRTYQAQWWTSGDQPDLNTGSGKPWKDLGACGPVVATPTPTATVAP TATPVVTATPTPTATPVVTATPTATPVGPTPTPTPVVTPTVPPSTGAKQVGTYFAEWSIYGRKFFLKNVQDSGQAAKLTFLNYSFGNVYKQADGTYKCQANINKAETGNQDGGDAWALYQKGFAANESVDGVADAWGQDGKGSLKGNWNQLKKLKAKN PNMKVLISLGGWTWSKWFSAAASTDALRKTLVASCIDVWIKGNLPFDAASNAGGAGTGAGVFDGIDIDWEYPGVQGIGTNTVSPADKENNTLLMKEFREQLDAIGSQTGKRYLLTVAIGAGDEKIAATVPGEYSKYLDWINIMSYDYNGGWDAAGPTD FQSNLYQDPASPRTVDPKTGKVSKYYTDAAVKDLIARGVPAVKLHIGVPFYGRGWTGVTNVNNGLYQKATGAAKGTYESGIEDYKVLKTAPGTEYIHPVTQQTYKFDGSTFWSYDTPRDIKLKADYAKSMGMGGIFSWEASGDTANGELVEAMTHINK

[0016] A DNA containing the nucleotide sequence encoding the aforementioned highly thermostable chitinase mutant D615S, as shown in SEQ ID NO.4.

[0017] SEQ ID NO.4:

[0018]

[0019] The above-mentioned method for constructing the highly thermostable chitinase mutant D615S includes the following steps:

[0020] Step 1: Extract the plasmid pET-28a(+)-Chi1;

[0021] Step 2: Design primers and perform site-directed mutagenesis using the pET28a-Chi1 plasmid as a template for full-sequence PCR. Verify the mutated strain to obtain the recombinant plasmid pET-28a(+). - D615S;

[0022] Step 3: The recombinant plasmid pET-28a(+) - D615S was introduced into E. coli BL21(DE3) to obtain a sample containing the recombinant plasmid pET-28a(+). - D615S Escherichia coli BL21 (DE3);

[0023] Step 4: Select plasmids containing pET-28a(+) - Escherichia coli BL21(DE3) D615S was cultured overnight on a shaker for 6-18 h, then induced with an inducer, and the bacterial cells were collected after centrifugation.

[0024] Step 5: Add buffer solution to the bacterial cells for resuspending, then sonicate to lyse, centrifuge to collect the supernatant, and refrigerate for later use.

[0025] As an improvement, the upstream primer is D615S-F: AGCTGGGAAGCAAGTGGCGACACGGCCAACGGCGAGCTGGTT, and the downstream primer is D615S-R: CGTGTCGCCACTTGCTTCCCAGCTGAAGATACCGCCCATGCCCAT.

[0026] The above-mentioned chitinase mutant D615S with high thermal stability is used in the degradation of biomass containing chitin.

[0027] As an improvement, the highly thermostable chitinase mutant D615S degraded 100 g / L of powdered chitin at 45°C for 60 h, yielding 48 g / L of reducing sugar, with a degradation rate of 48%. The degradation products were identified as follows: N - Acetaminoglycine and chitobiose, in a mass ratio of 4:1.

[0028] As an improvement, when the chitinous substance is 100 g / L of lobster shell powder with a chitin content of 20%, it produces 9 g / L of reducing sugar after 60 h of degradation, and the degradation rate reaches 45%.

[0029] Beneficial effects:

[0030] Compared with existing technologies, the present invention provides a chitinase mutant D615S with high thermal stability, its construction method, and its applications, which have the following advantages:

[0031] 1. This invention uses SEQ ID NO.1 in the sequence listing as a reference sequence, which has a point mutation at position 615 of aspartic acid, which is mutated to serine. Compared with wild-type chitinase, the chitinase mutant has a half-life of 72 h at 40°C, which is 3.6 times that of the wild-type, and a half-life of 60 h at 45°C, which is 24 times that of the wild-type. The optimal temperature is 40°C. m The temperature was 56.9℃, 6.6℃ higher than the wild type, which allows for better degradation of the chitin in the powder particles. N Acetaminoglycine and chitosan oligosaccharides have promising prospects for industrial applications.

[0032] 2. The chitinase Chi1 mutant D615S of this invention exhibits good degradation effects on natural particulate chitin. When 100 g / L of particulate chitin is degraded at 45°C for 60 h, 48 g / L of reducing sugar is obtained, representing a degradation rate of 48%. When 100 g / L of lobster shell containing 20% ​​chitin is degraded for 60 h, 9 g / L of reducing sugar is obtained, achieving a degradation rate of 45%. Identification of the degradation products revealed that the product is... N The mass ratio of acetylglucosamine to chitobiose is 4:1. Attached Figure Description

[0033] Figure 1 The half-lives of wild-type chitinase Chi1 and chitinase mutant D615S in this invention are at 40°C and 45°C, respectively. (A) is the half-life of Chi1 and D615S at 40°C, and (B) is the half-life of Chi1 and D615S at 45°C.

[0034] Figure 2 Determination of the optimal temperature for wild-type chitinase Chi1 and chitinase mutant D615S in this invention.

[0035] Figure 3 This is the time curve of the degradation of powdered chitin to reducing sugar by the chitinase mutant D615S at 45°C in this invention.

[0036] Figure 4 This refers to the reducing sugar produced by the degradation of shrimp and crab shells by the chitinase mutant D615S at 45°C for 60 h in this invention. Detailed Implementation

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention.

[0038] The present invention will be further described below through embodiments, but these are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] In the quantitative experiments in the following examples, three replicate experiments were set up, and the results were averaged.

[0041] The plasmids used in this invention E. coli DH5α and E. coli Materials such as DH5αBL21 (DE3) are all currently available commercially available; any technical means not mentioned are conventional in this field and will not be elaborated here. Example 1

[0042] A method for constructing the chitinase mutant D615S with high thermal stability includes the following steps:

[0043] Step 1: Extract the pET-28a(+)-Chi1 plasmid according to the standard procedure. The sequence is shown in SEN ID NO.3.

[0044] Step 2: Design primers to perform site-directed mutagenesis using the pET28a-Chi1 plasmid as a template for full-sequence PCR.

[0045] The primers were designed as follows: upstream primer D615S-F is AGCTGGGAAGCAAGTGGCGACACGGCCAACGGCGAGCTGGTT, and downstream primer D615S-R is CGTGTCGCCACTTGCTTCCCAGCTGAAGATACCGCCCATGCCCAT.

[0046] Using pET28a-Chi1 plasmid as a template, site-directed mutagenesis was performed by full-sequence PCR (sequence shown as SEN ID NO.3).

[0047] The PCR system is shown in Table 1.

[0048] Table 1. PCR reaction system for whole plasmids

[0049] Components Volume (μL) pET28a-Chi1 plasmid 1 D615S-F 1 D615S-R 1 2×Phanta Flash Master Mix (Dye Plus) 25 <![CDATA[ddH2O]]> 23

[0050] The reaction conditions for the above PCR are:

[0051] Pre-denaturation at 98℃ for 3 min; denaturation at 98℃ for 10 s, annealing at 60℃ for 5 s, extension at 72℃ for 10 s, for 30 cycles; final extension at 72℃ for 10 min. The amplified product was recovered after amplification.

[0052] Step 3: The PCR product was digested with DpnI at 37°C for 2 h and then inactivated at 70°C for 10 min.

[0053] Step 4, convert the product obtained in step 3 to... E. coli DH5α competent cells.

[0054] Step 5: Select positive transformants from the mutants obtained in Step 4, extract plasmids, and send them to Universal Biotech for sequencing verification.

[0055] Step 6: The recombinant plasmid pET-28a(+)-D615S was introduced into Escherichia coli BL21(DE3) to obtain Escherichia coli BL21(DE3) containing the recombinant plasmid pET-28a(+)-D615S.

[0056] Step 7: Select recombinant Escherichia coli BL21 (DE3) pET-28a(+)-D615S and inoculate it into 5 mL LB medium containing 100 μg / mL kanamycin. Incubate overnight at 37°C and 200 rpm in a shaking incubator.

[0057] Step 8: Inoculate the above bacterial suspension into 100 mL of LB medium containing 100 μg / mL kanapenem at a volume ratio of 1:100, and incubate at 37°C with shaking at 200 rpm until OD. 600 Reaching 0.6-0.8;

[0058] Step 9: Add IPTG (inducer) to a final concentration of 0.5 mmol / L, and incubate at 18°C ​​and 200 rpm with shaking for 16 h.

[0059] Step 10: Collect the above bacterial culture into a centrifuge tube, centrifuge at 4°C and 6000 rpm for 10 min, discard the supernatant and collect the precipitate;

[0060] Step 11: Add 10 mL of PBS buffer (pH 7.0, 50 mM) to the precipitate to resuspend the bacterial cells;

[0061] Step 12: Bacterial lysis by ultrasound on ice. The ultrasound power is 300 W. Each ultrasound treatment lasts for 2 seconds, with a 3-second interval, for a total of 10 minutes.

[0062] Step 13: Centrifuge at 7000 rpm for 15 min at 4℃, collect the supernatant of the lysate, and store at 4℃ for later use. Comparative Example 1

[0063] Wild-type chitinase Chi1 was expressed using steps 7-13 as described in Example 1. Example 2

[0064] Chitinase activity assay

[0065] 1. DNS reagent preparation

[0066] Weigh 182.0 g of potassium sodium tartrate, add 0.5 L of pure water, heat to dissolve at 50 ℃, then add 6.3 g of 3,5-dinitrosalicylic acid (DNS), 21.0 g of sodium hydroxide, and 5.0 g of phenol, stir until completely dissolved, cool, add pure water to make up to 1.0 L, store in the dark, and let stand for 7 days before use.

[0067] 2. Establishment of the standard curve for enzyme activity assay

[0068] The activity of chitinase was determined using the DNS (3,5-dinitrosalicylic acid) method. Separately, samples were taken... N - Acetaminoglycine standard solution (1 mg / mL): 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL were dispensed into 4 mL tubes, and diluted to 1.0 mL with pure water. 1 mL of DNS reagent was accurately added to each tube. The tubes were heated in a boiling water bath for 5 min, cooled under running water, and the absorbance was measured at 540 nm. Establishment N - Standard curve for acetylglucosamine.

[0069] 3. Chitinase activity assay

[0070] The enzyme activities of Chi1 and its mutant D615S were determined using 10 g / L colloidal chitin as a substrate. 100 μL of the substrate was added to 850 μL of PBS (pH 7.0, 50 mM), preheated for 10 min, and then 50 μL of the purified enzyme solution obtained in step 9 of Example 3 was added. After reacting for 30 min, the mixture was transferred to a boiling water bath and boiled for 5 min to inactivate the enzyme. Then, 1 mL of DNS solution was added, and the mixture was boiled at 100 °C for 5 min. After cooling to room temperature, the mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was measured at 540 nm. An equal volume of inactivated enzyme solution was used as a blank control. Enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol of reducing sugar per minute at 37 °C.

[0071] The preparation method of the above colloidal chitin is as follows:

[0072] Weigh 10 g of granular chitin (CP), add 40 mL of acetone and mix thoroughly. Then add 100 mL of 36% concentrated hydrochloric acid and stir until transparent. Slowly add the mixture to a beaker containing 2 L of 95% ethanol aqueous solution and stir quickly until fully mixed. Place the beaker in a refrigerator at 4°C and let it stand overnight to obtain precipitated colloidal chitin. Rinse repeatedly with sterile water and adjust the pH to 7. Then make up to volume with pH 7.0 (PBS buffer, 50 mM). Example 3

[0073] Determination of the optimal temperature for the highly thermally stable chitinase mutant D615S

[0074] Using 10 g / L colloidal chitin as a substrate, the reaction system contained 100 μL colloidal chitin, 850 μL PBS buffer (pH 7.0, 50 mM), and 50 μL crude enzyme solution. The reaction was carried out at temperature gradients of 25℃, 30℃, 37℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃ for 30 min. After the reaction, 1 mL of DNS reagent was immediately added to each reaction system, and the mixture was boiled for 5 min. The absorbance was then measured using a 540 nm UV-Vis spectrophotometer to determine the enzyme activities of Chi1 and D615S at different temperatures. Curves were plotted based on the relative enzyme activities at different temperatures, as shown below. Figure 2 As shown in the figure, the chitinase mutant D615S is the same as the wild-type chitinase Chi1, and the optimal reaction temperature is still 40℃. Example 4

[0075] (1) Determination of half-life

[0076] Enzyme solutions with a concentration of 0.1 g / L were incubated in water baths at different temperatures (35°C, 40°C, 45°C, 50°C) for different durations (0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 16 h, 20 h, 24 h, 36 h, 48 h, 60 h, 72 h). After cooling on ice for 10 min, enzyme activity was measured at the optimal temperature according to the method described in Example 3. The unincubated enzyme activity was taken as 100%, and the residual enzyme activity at different incubation times was calculated. Curves were plotted, and the residual enzyme activity and treatment time were used to create enzyme activity decay curves. The half-life was calculated from the curves to obtain the half-life (t) of the chitinase mutant. 1 / 2 Wild-type chitinase Chi1 was tested following the same steps as above for comparison. The results are as follows: Figure 1As shown, the wild-type chitinase Chi1 has a half-life of 20 h at 40 °C and 2.5 h at 45 °C. The chitinase mutant D615S has a half-life of 72 h at 40 °C, which is 3.6 times that of the wild-type chitinase Chi1, and a half-life of 60 h at 45 °C, which is 24 times that of the wild-type chitinase Chi1.

[0077] (2) T m Value determination

[0078] Surface unwinding T m It can also characterize the thermodynamic stability of enzyme proteins, which can be determined by the DSF method. For each mutated enzyme protein sample, 2 μL of 10×SYPRO® Orange dye and 20 μL of purified mutated protein sample at a concentration of 200 mg / L were mixed and incubated on ice for 30 min.

[0079] The assay was performed using a real-time quantitative PCR system, with the temperature increased from 25°C to 90°C at a rate of 1°C / min, and three parallel experiments were conducted. The Tg of wild-type chitinase Chi1 was determined by measurement. m The value was 50.3℃, and the T value of the chitinase mutant D615S was... m The value was 56.9℃, which is 6.6℃ higher than that of the wild type. Example 5

[0080] The highly thermally stable chitinase mutant D615S degrades particulate chitin.

[0081] 100 µL of 10 g / L chitinase mutant D615S, 100 g / L of powdered chitin (purchased from Aladdin), and 900 µL of PBS (pH 7.0, 50 mM) buffer were added to the system. The reaction was carried out at 45 °C for 60 h. Samples were taken every 12 h to detect the amount of reducing sugar produced, and a progress curve was plotted. The distribution of the products was also detected. Figure 3 As shown, the main products generated are GlcNAc and chitobiose. The degradation rate is 48% at 60 h. The proportion of GlcNAc increases with time, and the ratio of GlcNAc to chitobiose is 4:1 at 60 h. Example 6

[0082] The highly thermally stable chitinase mutant D615S degrades chitin-like biomass.

[0083] Take 100 µL of Chi1 enzyme solution with a concentration of 10 g / L, and add 100 g / L of lobster shell powder (chitin content 20-25 g / L), mitten crab shell powder (chitin content 15-20 g / L), and swimming crab shell powder (chitin content approximately 15 g / L), respectively. Add 900 µL of PBS buffer (pH 7.0, 50 mM) and react at 45℃ for 60 h. Measure the amount of reducing sugar produced using DNS. The results are as follows: Figure 4 As shown, the chitinase mutant with high thermal stability can effectively degrade both lobster and crab shells, with the most significant degradation on lobster shells, producing 9 g / L of chitin oligosaccharides and achieving a degradation rate of 45%.

[0084] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A chitinase mutant D615S having high thermal stability, characterized in that, The amino acid sequence thereof is taken as the reference sequence in the sequence listing, and the aspartic acid at position 615 is mutated to serine. The amino acid sequence of the chitinase mutant D615S is shown as SEQ ID NO.

2. Compared with the wild-type chitinase, the half-life of the chitinase mutant at 40℃ is 72 h, which is 3.6 times that of the wild-type strain, and the half-life at 45℃ is 60 h, which is 24 times that of the wild-type strain. The optimal temperature is 40℃, and the T m value is 56.9℃, which is 6.6℃ higher than that of the wild-type strain.

2. A DNA molecule encoding the chitinase mutant D615S of claim 1, the nucleotide sequence of which is shown as SEQ ID NO.

4.

3. A method for constructing chitinase mutant D615S having high thermal stability based on claim 1, characterized in that, The method comprises the following steps: Step 1, extracting the plasmid of pET-28a(+)-Chil; Step 2, design primers, take pET28a-Chil plasmid as template to carry out full sequence PCR to carry out site-directed mutation, and verify the mutant strain, obtain recombinant plasmid pET-28a(+) - D615S; Step 3, the recombinant plasmid pET-28a(+) - D615S was introduced into E. coli BL21(DE3) to obtain E. coli BL21(DE3) containing the recombinant plasmid pET-28a(+) - D615S was introduced into E. coli BL21(DE3) to obtain E. coli BL21(DE3) containing the recombinant plasmid pET-28a(+) - Step 4, select the recombinant plasmid pET-28a(+) - E. coli BL21 (DE3) of D615S was cultured for 6-18 h in a shaker, and then an inducer was added to induce the culture. The bacterial cells were collected after centrifugation. Step 5, adding buffer to the bacteria for resuspension, then ultrasonic lysis, centrifugal collection of supernatant, and refrigeration for standby.

4. The method for constructing chitinase mutant D615S having high thermal stability according to claim 3, characterized in that, The upstream primer in the primer is D615S-F: AGCTGGGAAGCAAGTGGCGACACGGCCAACGGCGAGCTGGTT, and the downstream primer is D615S-R: CGTGTCGCCACTTGCTTCCCAGCTGAAGATACCGCCCATGCCCAT.

5. Application of the chitinase mutant D615S with high thermal stability of claim 1 in degrading biomass containing chitin.

6. Use according to claim 5, characterized in that, The chitinase mutant D615S with high thermal stability degrades 100 g / L of powder chitin for 60 h at 45℃, and 48 g / L of reducing sugar is obtained, with a degradation rate of 48%. Through identification of the degradation products, the products are N -N-acetylglucosamine and chitobiose, and the mass ratio is 4:1.

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