Chitin degradation method

Abstract

The application discloses a chitin degradation method, which comprises the following steps: in a liquid-phase reaction system, 4-28 muM chitin degradation enzyme is added according to 0.1 g chitin, and reaction is carried out at 70-85 DEG C for 4-32 h to realize chitin degradation. The method successfully realizes efficient degradation of chitin based on a chitin degradation enzyme system composed of exochitinase and endochitinase pure enzyme or crude enzyme liquid. The preferred chitin degradation enzyme system is a crude enzyme liquid containing exochitinase mutants and endochitinase mutants, which can be used for enzymolysis without expensive protein purification steps, and the thermal stability of the two chitinases is extremely high, which greatly simplifies the enzymolysis step, reduces the production cost of enzymolysis, and has the potential for large-scale application.

Description

Technical Field

[0001] This invention relates to enzyme engineering, and more particularly to a method for chitin degradation. Background Technology

[0002] Chitin is the second largest natural biopolysaccharide after cellulose. It is composed of N-acetyl-D-glucosamine linked by β-1,4-glycosidic bonds and is widely found in the exoskeletons of crustaceans such as shrimp and crabs, insect epidermis, and fungal cell walls. Shrimp and crab shells are the primary raw material for industrial chitin extraction, and their main components include chitin, protein, and calcium carbonate. With the continuous development of the global seafood processing industry, a large amount of crustacean processing waste is generated annually. Direct landfilling or disposal not only causes environmental pollution but also wastes biomass resources such as chitin. Targeted degradation of chitin to prepare high-value-added products such as chitosan oligosaccharides, N-acetylglucosamine, and glucosamine can be widely applied in agriculture, biomedicine, food, and functional materials, representing an important direction for the resource utilization of crustacean waste.

[0003] Currently, chitin depolymerization methods mainly include chemical methods, physical-assisted methods, and bio-enzymatic methods. While traditional acid-base methods are mature and have high degradation efficiency, they typically require high temperatures and concentrated acid or alkali conditions, resulting in high energy consumption, severe equipment corrosion, significant environmental pollution, numerous byproducts, and difficulty in recovering associated resources such as proteins. Improved processes such as microwave-assisted methods, ionic liquids, and deep eutectic solvents can reduce some environmental impact, but they still generally suffer from high reagent costs, complex processes, difficulties in scale-up, or limited recycling efficiency. In contrast, enzymatic methods offer advantages such as mild reaction conditions, high substrate specificity, controllable product composition, and environmental friendliness. They can simultaneously achieve high-value utilization of chitin and associated proteins, making them an important development direction for the green biorefining of chitin.

[0004] The efficient enzymatic hydrolysis of chitin relies on the synergistic catalysis of endo- and exo-chitinases: endo-chitinases randomly cleave the glycosidic bonds within the sugar chain, generating new chain ends; exo-chitinases continuously release chitobioses or monosaccharides from these chain ends, and the synergy between the two determines the overall degradation efficiency. However, existing chitinase systems still have many limitations, such as the mismatch between the optimal reaction temperatures and thermal stability of exo- and endo-enzymes, making it difficult to maintain efficient synergistic catalysis in the same system; low enzyme efficiency for natural crystalline chitin, with insufficient catalytic activity and substrate utilization; and low expression levels, high purification costs, and poor industrial stability of some enzyme preparations, limiting continuous and large-scale applications. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for achieving efficient degradation of chitin through the synergistic action of exochitinase and endochitinase.

[0006] Technical Solution: The chitin degradation method of the present invention includes the following steps: In a liquid-phase reaction system, a chitin-degrading enzyme system with a final concentration of 4-28 μM is added per 0.1 g of chitin, and the reaction is carried out at 70-85℃ for 4-32 h to achieve chitin degradation; wherein, the chitin-degrading enzyme system contains an exochitinase and an endochitinase in a molar ratio of (1-4):1, the amino acid sequence of the exochitinase is as shown in SEQ ID NO: 1 or has at least 95% similarity to the sequence shown in SEQ ID NO: 1, and the amino acid sequence of the endochitinase is as shown in SEQ ID NO: 2 or has at least 95% similarity to the sequence shown in SEQ ID NO: 2.

[0007] Preferably, the amino acid sequence of the exochitinase is based on the sequence shown in SEQ ID NO: 1 and has any one of the following single-point mutations: W74T, S154T, S157N, T233F, T240I, T240V, and A295P; or any one of the following double-point mutations: T233F / T240I, T233F / A295P, and T240I / A295P; or any one of the following triple-point mutations: T233F / T240I / A295P; or any one of the following quadruple-point mutations: W74T / T233F / T240I / A295P, S154T / T233F / T240I / A295P, and S157N / T233F / T240I / A295P.

[0008] Preferably, the amino acid sequence of the endochondrinase is based on the sequence shown in SEQ ID NO: 2, and has any one of the following single-point mutations: K22P, P23G, D149L, T184S, T184P, A186N, S231E, S234D, P263G, T266G, E268G, D278Q, W281Y; or any one of the following double-point mutations: K22P / D149L, D149L / P263G, D149L / E268G.

[0009] Preferably, the amino acid sequence of the exochitinase is based on the sequence shown in SEQ ID NO: 1, and has: a three-point mutation of T233F / T240I / A295P; or a four-point mutation of W74T / T233F / T240I / A295P, S154T / T233F / T240I / A295P, or S157N / T233F / T240I / A295P; the amino acid sequence of the endochitinase is based on the sequence shown in SEQ ID NO: 2, and has: a two-point mutation of K22P / D149L, D149L / P263G, or D149L / E268G.

[0010] Preferably, the chitin is derived from chitin-containing waste; more preferably, the chitin-containing waste includes crustacean shells, fungal biomass, and insect exoskeletons.

[0011] Preferably, the chitin undergoes mechanical activation pretreatment before degradation; more preferably, the mechanical activation pretreatment is ball milling.

[0012] Preferably, the chitin-degrading enzyme system is a mixture of crude enzyme solution obtained by thermal lysis of recombinant cells expressing exochitinase and recombinant cells expressing endochitinase; more preferably, the thermal lysis conditions are lysis at 65~75 °C for 5~30 min.

[0013] Preferably, the liquid-phase reaction system is a buffer solution with pH=5~6; more preferably, the buffer solution is a sodium acetate buffer solution.

[0014] The chitin hydrolysate prepared by the method described in this invention.

[0015] The application of the deacetylated product of the chitin hydrolysate described in this invention in promoting plant growth.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention is based on a chitin-degrading enzyme system composed of pure or crude enzyme solutions of exo-chitinase and endo-chitinase, which successfully achieves efficient degradation of chitin; 2. The preferred chitin-degrading enzyme system is a crude enzyme solution containing exo-chitinase mutants and endo-chitinase mutants, which can be used for enzymatic hydrolysis without expensive protein purification steps, and the two chitinases have extremely high thermostability matching, which greatly simplifies the enzymatic hydrolysis steps, reduces the production cost of enzymatic hydrolysis, and has the potential for large-scale application. Attached Figure Description

[0017] Picture 1 The graph shows the results of the optimization of thermal lysis conditions and thermal stability analysis of the crude chitinase solution in Example 1 of the present invention. In the graph, A is the SDS-PAGE analysis result of the V4-2 bacterial solution after lysis under different conditions, B is the SDS-PAGE analysis result of the M2-3 bacterial solution after lysis under different conditions, and C is the thermal stability curve of the V4-2 crude enzyme solution and the M2-3 crude enzyme solution during incubation at 75°C for 10 minutes. Picture 2 This is a graph showing the results of using a pure enzyme-based chitin-degrading enzyme system for the enzymatic degradation of chitin in shrimp shells in Example 2 of this invention, where A represents wild-type CD. chiB is the degradation rate curve of chitin co-hydrolyzed by TkCD enzyme at different temperatures; C is the degradation rate curve of chitin co-hydrolyzed by V4-2 and M2-3 enzyme at different temperatures; D is the XRD analysis result of chitin powder and ball-milled chitin; E is the statistical result of degradation rate of chitin powder and ball-milled chitin under the same conditions. Picture 3 The graph shows the results of the enzymatic degradation of chitin in shrimp shells using the chitinase crude enzyme solution in Example 2 of the present invention. In the graph, A is the statistical result of the degradation rate of ball-milled chitin with different amounts of crude enzyme solution, and B is the result of the degradation product component analysis based on high performance liquid chromatography. Picture 4 Figure 3 shows the results of preparing chitosan oligosaccharide after enzymatic hydrolysis of chitin in Example 3 of the present invention. Figure A is the chitosan oligosaccharide conversion rate curve under different substrate concentrations, and Figure B is the chitosan oligosaccharide conversion rate curve under different concentrations of chitin deacetylase. Picture 5 The following are the results of the foliar spraying of chitosan oligosaccharide solution on the growth of tomato seedlings in Example 3 of the present invention. A is a representative image of the growth of tomato seedlings in different treatment groups after 14 days of cultivation. B is a statistical result of the plant height of tomato seedlings in different treatment groups. C is a statistical result of the root length of tomato seedlings in different treatment groups. D is a statistical result of the aboveground fresh weight of tomato seedlings in different treatment groups. E is a statistical result of the underground fresh weight of tomato seedlings in different treatment groups. F is a result of the determination of the relative chlorophyll content of tomato seedlings in different treatment groups. G is a result of the determination of the net photosynthetic rate of tomato seedlings in different treatment groups. Picture 6 This is a diagram summarizing the overall concept and results of the present invention. Detailed Implementation

[0018] The technical solution of the present invention will be further described below.

[0019] Example 1: Preparation of chitin-degrading enzyme system 1. Preparation of purified exochitinase Based on the wild-type exochitinase amino acid sequence shown in SEQ ID NO: 1, a wild-type exochitinase expression sequence shown in SEQ ID NO: 2 was designed. The pET-28a(+) expression vector containing this expression sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., resulting in the recombinant plasmid pET-28a-CD. chi .

[0020] pET-28a-CD chiUsing the W74T mutant primers shown in Table 1 as templates, single-point mutant plasmids were constructed using the Quick Change site-directed mutagenesis kit (purchased from Agilent Technologies, Inc., catalog number 200523). The amplified PCR fragments were digested with DpnI enzyme to remove the template before transformation into... E. coli DH5α competent cells were cultured, and single colonies were picked after culture. Beijing Qingke Biotechnology Co., Ltd. was commissioned to sequence and verify the correctness of the mutation site. After successful verification, the single-point mutant plasmid pET-28a-CD was extracted. chi -W74T and transfer E. coli BL21(DE3) competent cells were used to obtain a recombinant single-point mutant expression strain. E. coli BL21(DE3) / pET-28a-CD chi -W74T.

[0021] According to the above method, pET-28a-CD chi Using the templates, single-point mutant plasmids were constructed and transformed into the following primers: S154T, S157N, T233F, T240I, T240V, and A295P, as shown in Table 1. E. coli Recombinant mutant expression strains were obtained from BL21(DE3) competent cells. E. coli BL21(DE3) / pET-28a-CD chi -S154T, E. coli BL21(DE3) / pET-28a-CD chi -S157N、 E. coli BL21 (DE3) / pET-28a-CD chi -T233F、 E. coli BL21(DE3) / pET-28a-CD chi -T240I, E. coli BL21 (DE3) / pET-28a-CD chi -T240V, E. coli BL21(DE3) / pET-28a-CD chi -A295P.

[0022] According to the above method, pET-28a-CD chi Using -T233F as a template, double-point mutant plasmids were constructed based on the T240I and A295P mutant primers shown in Table 1, respectively; pET-28a-CD was used as the template. chi Using -T240I as a template, a double-point mutant plasmid was constructed based on the A295P mutation primers shown in Table 2; the resulting plasmids were then transformed into... E. coliBL21(DE3) competent cells were used to obtain a recombinant mutant expression strain. E. coli BL21(DE3) / pET-28a-CD chi -T233F / T240I, E. coli BL21(DE3) / pET-28a-CD chi -T233F / A295P, E. coli BL21(DE3) / pET-28a-CD chi -T240I / A295P.

[0023] According to the above method, pET-28a-CD chi Using T233F / T240I as a template, a three-point mutant plasmid was constructed based on the A295P mutation primers shown in Table 1; further, using the obtained three-point mutant plasmid as a template, a four-point mutant plasmid was constructed based on the W74T, S154T, and S157N mutation primers shown in Table 2, respectively; the obtained three-point mutant plasmid and four-point mutant plasmid were then transformed into... E. coli BL21(DE3) competent cells were used to obtain a recombinant mutant expression strain. E. coli BL21(DE3) / pET-28a-CD chi -T233F / T240I / A295P, E. coli BL21(DE3) / pET-28a-CD chi -W74T / T233F / T240I / A295P E. coli BL21 (DE3) / pET-28a-CD chi -S154T / T233F / T240I / A295、 E. coli BL21 (DE3) / pET-28a-CD chi -S157N / T233F / T240I / A295P.

[0024] Table 1 CD chi Single-point mutation primers

[0025] Single colonies of the 14 recombinant mutant expression strains obtained above and E. coli BL21(DE3) / pET-28a-CD chi Single colonies were inoculated into 50 mL LB liquid medium containing a final concentration of 50 μg / mL kanamycin and incubated at 37°C until OD500. 600=0.6, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, and induce expression at 20℃ for 24 h; after induction, collect bacterial cells by centrifugation at 6500× g for 10 min, add 15 mL of lysis buffer (pH=8.0, formulation: 50 mM Tris-HCl, 400 mM NaCl, 5% (v / v) glycerol, and the remainder water) to resuspend the bacterial cells, and sonicate them at 300W and 4℃ for 10 min. After centrifugation at 14000× g at 4℃, collect the supernatant and purify it using immobilized metal ion affinity chromatography (IMAC) using a protein purifier. Specifically: The Ni affinity chromatography column (chromatographic media purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number HP202-01) was pre-equilibrated with approximately 5 column volumes of equilibration buffer, and then the aforementioned supernatant was added. The column was washed with washing buffer to remove contaminating proteins; the UV detection signal showed no peak at A280. The target protein was eluted with 50 mL of elution buffer and collected. The protein concentrations of the purified wild-type chitinase and 26 CDchi single-point mutants were determined using the Bradford method.

[0026] The equilibration buffer consisted of 50 mM Tris-HCl, 400 mM NaCl, and 10 mM imidazole, with the remainder being water, pH=8.0; the washing buffer consisted of 50 mM Tris-HCl, 400 mM NaCl, and 20 mM imidazole, with the remainder being water, pH=8.0; the elution buffer consisted of 50 mM Tris-HCl, 400 mM NaCl, and 200 mM imidazole, with the remainder being water, pH=8.0. 2. Preparation of purified endochondral chitinase. Based on the wild-type endochondrinase amino acid sequence shown in SEQ ID NO: 2, the wild-type endochondrinase expression sequence shown in SEQ ID NO: 4 was designed, and the pET-28a(+) expression vector containing the expression sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., to obtain the recombinant plasmid pET-28a-TkCD.

[0027] Using pET-28a-TkCD as a template, and based on the K22P mutation primers shown in Table 2, a single-point mutant plasmid was constructed using the Quick Change site-directed mutagenesis kit. The amplified PCR fragment was then digested with DpnI enzyme to remove the template before transformation into... E. coli DH5α competent cells were cultured, and single colonies were picked after culture. Beijing Qingke Biotechnology Co., Ltd. was commissioned to sequence and verify the correctness of the mutation site. After successful verification, the single-point mutant plasmid pET-28a-TkCD-W74T was extracted and transformed into... E. coliBL21(DE3) competent cells were used to obtain a recombinant single-point mutant expression strain. E. coli BL21(DE3) / pET-28a-TkCD-K22P.

[0028] Following the method described above, using pET-28a-TkCD as a template, single-point mutant plasmids were constructed and transformed into the following primers: P23G, D149L, T184P, T184S, A186N, S231E, S234D, P263G, T266G, E268G, D278P, and W281Y, as shown in Table 2. E. coli Recombinant mutant expression strains were obtained from BL21(DE3) competent cells. E. coli BL21(DE3) / pET-28a-TkCD-P23G、 E. coli BL21 (DE3) / pET-28a-TkCD-D149L, E. coli BL21 (DE3) / pET-28a-TkCD-T184P, E. coli BL21 (DE3) / pET-28a-TkCD-T184S, E. coli BL21 (DE3) / pET-28a-TkCD-A186N, E. coli BL21 (DE3) / pET-28a-TkCD-S231E, E. coli BL21 (DE3) / pET-28a-TkCD-S234D, E. coli BL21 (DE3) / pET-28a-TkCD-P263G, E. coli BL21 (DE3) / pET-28a-TkCD-T266G, E. coli BL21 (DE3) / pET-28a-TkCD-E268G, E. coli BL21 (DE3) / pET-28a-TkCD-D278P, E. coli BL21(DE3) / pET-28a-TkCD-W281Y.

[0029] Following the above method, using pET-28a-TkCD-D149L as a template, double-point mutant plasmids were constructed based on the K22P, P263G, and E268G mutant primers shown in Table 2, respectively; the resulting plasmids were then transformed into... E. coli BL21(DE3) competent cells were used to obtain a recombinant mutant expression strain. E. coli BL21(DE3) / pET-28a-TkCD-K22P / D149L、 E. coliBL21(DE3) / pET-28a-TkCD-D149L / P263G、 E. coli BL21(DE3) / pET-28a-TkCD-D149L / E268G.

[0030] Table 2 Primers for TkCD single-point mutation

[0031] Single colonies of the 16 recombinant mutant expression strains obtained above and E. coli BL21(DE3) / pET-28a-TkCD single colonies were inoculated into 50 mL LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured, IPTG induced, lysed, and purified to obtain TkCD mutant protein and wild-type TkCD protein; the protein concentration of the purified enzyme was determined using the Bradford method.

[0032] 3. Preparation of crude chitinase solution The above results E. coli BL21 (DE3) / pET-28a-CD chi -S154T / T233F / T240I / A295P, E. coli Single colonies of BL21(DE3) / pET-28a-TkCD-D149L / E268G were inoculated into 1 L LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C until OD500. 600 =0.6, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, and induce expression at 20℃ for 24 h; after induction, collect bacterial cells by centrifugation at 6500× g for 10 min, add 50 mL of lysis buffer (pH=8.0, formulation: 50 mM Tris-HCl, 400 mM NaCl, 5% (v / v) glycerol, and the remainder being water) to resuspend the bacterial cells, and thermally lyse them at 65℃, 70℃, and 75℃ for 5, 10, 15, 20, 25, or 30 min respectively, centrifuge and collect the supernatant to obtain CD-containing bacteria prepared under different thermal lysis conditions. chi The supernatant of S154T / T233F / T240I / A295P (V4-2 crude enzyme solution) and the supernatant containing TkCD-D149L / E268G prepared under different thermal lysis conditions (M2-3 crude enzyme solution) were analyzed. The protein concentration in the crude enzyme solution was determined using the Bradford method. The lysis effect of the above crude enzyme solutions was analyzed by SDS-PAGE, with the unlysolved enzyme-containing bacterial solution used as a control.

[0033] The results are as follows Picture 1As shown in A and B, complete cell disruption can be achieved by thermal lysis at 75℃ for 5 minutes, resulting in high purity of the target protein and no loss of enzyme activity. 1 L of bacterial culture can yield 156 mg V4-2 and 80 mg M2-3 through thermal lysis, which can significantly reduce production costs.

[0034] Crude enzyme solutions of V4-2 or M2-3 prepared under 75℃ thermal lysis for 5 min were incubated at 75℃ for 0, 5, or 10 min, then cooled on ice for 5 min. Enzyme activity was then determined using the 3,5-dinitrosalicylic acid (DNS) method. N-acetylglucosamine standard solutions at concentrations of 0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, and 0.4 mg / mL were prepared and added to 10 mL centrifuge tubes. Each tube was then mixed with 1 mL of DNS solution and boiled in a 100℃ water bath for 15 min for color development. After the reaction, the solutions were cooled to room temperature, and the absorbance (OD) of the N-acetylglucosamine solutions at different concentrations after the reaction was measured at 540 nm. 540 Plotting N-acetylglucosamine concentration on the x-axis, OD 540 A standard curve was plotted on the ordinate (y). The standard curve was: y = 1.41x - 0.308. A 2% (w / v) chitin substrate solution (chitin, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number C104157) prepared in 5 mL of 50 mM sodium acetate buffer (pH 5.8) was added, along with 0.05 mL of the previously obtained crude enzyme solutions V4-2 and M2-3. The reaction was carried out at 85 °C with shaking for 5 min. The reaction was terminated by adding 1 mL of DNS reagent, followed by color development in a boiling water bath at 100 °C for 15 min. After cooling, the OD was measured. 540 Enzyme activity was calculated based on the standard curve.

[0035] Enzyme activity unit U is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under the above conditions.

[0036] The results are as follows Picture 1 As shown in C, crude enzyme solution V4-2 or crude enzyme solution M2-3 exhibit good thermal stability.

[0037] 4. Preparation of chitin-degrading enzyme system The purified exochitinase obtained above was mixed with the purified endochitinase at molar ratios of 1:1, 2:1, 3:1, and 4:1 (total enzyme amount of 60 nmol) to obtain chitin-degrading enzyme systems for chitin degradation.

[0038] The CD prepared under the aforementioned 75℃ pyrolysis 5 min condition was... chiThe crude enzyme solution of S154T / T233F / T240I / A295P and the crude enzyme solution of TkCD-D149L / E268G were mixed in a molar ratio of exochitinase to endochitinase of 3:1 to obtain a chitin-degrading enzyme system for chitin degradation.

[0039] Example 2: Chitin-degrading enzyme system for chitin degradation 1. Preparation of chitin from shrimp shells The shells of Litopenaeus vannamei were washed with deionized water, dried at 60℃, and pulverized through a 150-mesh sieve to obtain shell powder. 1 g of shell powder was added to 10 mL of deionized water, and 1000 U of alkaline protease (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number P750258) was added. The protein was degraded at 55℃ and pH=10.0 for 10 h. The precipitate was collected by centrifugation at 10000× g, washed with deionized water, and dried at 60℃ for 10 h to obtain deproteinized powder. 1 M acetic acid with a solid-liquid ratio of 1:20 (w / v) was added, and the mixture was demineralized at room temperature for 30 min. The precipitate was collected by centrifugation at 10000 g, washed with deionized water, and dried at 60℃ for 10 h to obtain shell chitin powder.

[0040] 2. Chitin-degrading enzyme systems based on pure enzymes for chitin degradation The enzyme degradation reaction system was prepared as follows: 1 g of shrimp shell chitin powder was added to 10 mL of 0.1 M sodium acetate buffer (pH=5.8); a chitin-degrading enzyme system consisting of V4-2 and M2-3 in a 1:1 molar ratio (total enzyme content 60 nmol); and wild-type CD4+ in a 1:1 molar ratio (total enzyme content 60 nmol). chi Chitin-degrading enzyme system composed of TkCD; degradation of chitin by the chitin-degrading enzyme system composed of V4-2 and M2-3, at 75℃ or 80℃ for 0, 4, 8, 12, 16, 20, 24, 28 and 32 h; degradation of wild-type CD chi The degradation of chitin by the chitin-degrading enzyme system composed of TkCD was carried out at 70℃ or 75℃ for 0, 4, 8, 12, 16, 20, 24, 28, and 32 h. After the reaction, the precipitate was collected by centrifugation at 10000 × g and dried at 80℃ for 10 h. The weight loss was then measured. The degradation rate was calculated by the weight loss method using the formula: Degradation rate = Weight loss / Original weight × 100%.

[0041] The results are as follows Picture 2 As shown in A and B, the chitin-degrading enzyme system composed of V4-2 and M2-3 achieved a degradation rate of up to 29.1% after reacting at 75℃ for 32 h, which was significantly higher than that of wild-type CD4+. chi The degradation rate of the / TkCD chitin-degrading enzyme system was 23.5%.

[0042] Further optimization of the molar ratio of V4-2 to M2-3 was carried out while maintaining a total enzyme content of 60 nmol. Four ratios were set: 1:1, 2:1, 3:1, and 4:1. At the same time, a single enzyme digestion system of V4-2 with a total enzyme content of 60 nmol (1:0) was set up, and chitin degradation was carried out at 75℃ for 32 h.

[0043] The results are as follows Picture 2 As shown in Figure C, the degradation rate was highest at a molar ratio of V4-2 to M2-3 of 3:1, reaching 34.8%.

[0044] 3. Chitin-degrading enzyme systems based on purified enzymes are used for pretreatment of chitin degradation. The aforementioned shrimp shell chitin powder was pretreated using a low-temperature ball mill (purchased from Fuld (Shanghai) Instrument Equipment Co., Ltd., model MM400), specifically by ball milling at 30 Hz for 30 min. X-ray powder diffraction was then performed using an X-ray powder diffractometer (purchased from Rigaku Corporation, model MiniFlex 600) within the range of 5° to 30° at a scanning rate of 4° / min. The crystallinity index (CI) was calculated using the following formula: Crystallinity index (%) = (I 110 -I am ) / I 110 ×100% Among them, I 110 For the maximum intensity of the (110) peak at 2θ = 19°, I am The X-ray diffraction (XRD) results for the amorphous diffraction intensity at 2θ = 12.6° (021) are as follows: Picture 2 As shown in D, the results indicate that the chitin crystallinity of the shrimp shell chitin powder decreased from 79.2% to 66.9% after ball milling.

[0045] The enzyme degradation reaction system was prepared as follows: 1 g of ball-milled shrimp shell chitin powder and a chitin-degrading enzyme system consisting of V4-2 and M2-3 in a molar ratio of 3:1 with a total enzyme amount of 60 nmol were added to 10 mL of 0.1 M sodium acetate buffer at pH=5.8. After reacting at 75℃ for 32 h, the degradation rate was calculated by the aforementioned method.

[0046] The results are as follows Picture 2 As shown in E, after pretreatment of chitin, the degradation rate of the chitin-degrading enzyme system composed of V4-2 and M2-3 can reach 56.3%.

[0047] 4. Chitin-degrading enzyme system based on crude enzyme solution for pretreatment of chitin degradation The enzyme degradation reaction system was prepared as follows: 1 g of the pretreated shrimp shell chitin powder was added to 10 mL of 0.1 M sodium acetate buffer (pH=5.8), along with a chitin degradation enzyme system consisting of V4-2 crude enzyme solution and M2-3 crude enzyme solution in a molar ratio of 3:1 (total enzyme volume 67 nmol, 1 mL of crude enzyme solution), 134 nmol, or 201 nmol, 3 mL of crude enzyme solution). After reacting at 75℃ for 32 h, the amount of reducing sugar released was measured, and the degradation rate was calculated using the aforementioned formula.

[0048] The results show that... Picture 3 As shown in Figure A, when the total enzyme content is 201 nmol, the chitin degradation rate reaches 67.6%, which is 20.1% higher than that of the purified enzyme degradation system with a total enzyme content of 201 nmol.

[0049] A chitin-degrading enzyme system was prepared based on crude enzyme solutions V4-2 and M2-3 in a molar ratio of 3:1 (total enzyme content 268 nmol, total crude enzyme solution volume 4 mL). After reacting at 75℃ for 32 h, the product components were further analyzed by high-performance liquid chromatography (HPLC). The specific steps were as follows: A Shimadzu LC20A HPLC system equipped with an evaporative light scattering detector was used. An amino column (purchased from Lisennoco Scientific Instruments (Shanghai) Co., Ltd., catalog number NH2P-50 4E) was employed at 30℃. The mobile phase was a mixture of 70% (v / v) acetonitrile and 30% (v / v) water, with isocratic elution and a flow rate of 1 mL / min. A standard curve was established using standard chitosan oligosaccharides with a degree of polymerization (DP) of 1–6 (purchased from Qingdao Bozhi Huili Biotechnology Co., Ltd., catalog number MCN1-6). The hydrolysis products were quantitatively analyzed using the external standard method.

[0050] The results are as follows Picture 3 As shown in B, in the product of chitin hydrolysis of ball-milled chitin by a chitin-degrading enzyme system with a total enzyme content of 268 nmol, 86.5% is N-acetylglucosamine and 13.5% is N-acetylchitobiose.

[0051] Example 3: Verification of the growth-promoting effect of chitin hydrolysate on tomato seedlings The recombinant plasmid pET-28a-TsDac, containing the chitin deacetylase TsDac (GenBank: WP_058946334.1), was transformed into... E. coli BL21(DE3) was cultured, IPTG induced, lysed, and purified to obtain chitin deacetylase TsDac. The concentration of the purified protein was determined using the Bradford method.

[0052] A chitin-degrading enzyme system was prepared based on a 3:1 molar ratio of crude enzyme solution V4-2 (total enzyme content 268 nmol, total crude enzyme solution volume 4 mL) and crude enzyme solution M2-3, along with 1 g of pre-treated shrimp shell chitin powder by ball milling. After reacting at 75℃ for 32 h, the hydrolysis products were quantitatively analyzed using the aforementioned method. Based on the quantitative analysis results, the hydrolysis products were diluted to a final chitin oligosaccharide concentration of 25, 50, 75, or 100 mg / mL. Chitin deacetase TsDac was added to a final concentration of 250 mg / L, and the reaction was carried out at 37℃ for 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 h, respectively. The conversion efficiency of chitin oligosaccharides was determined using the o-phthalaldehyde (OPA) method.

[0053] Meanwhile, the hydrolysate of chitosan oligosaccharide diluted to a final concentration of 25 mg / mL was added to chitin deacetylase TsDac at final concentrations of 50, 100, 125, 250, and 500 mg / L, respectively, and reacted at 37°C for 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, or 3 h. The conversion efficiency of chitosan oligosaccharide was determined using the OPA method.

[0054] The results are as follows Picture 4 As shown, the conversion efficiency of chitosan oligosaccharide was the highest, reaching 77.6%, when the final concentration of chitin deacetylase TsDac was 250 mg / L and the final concentration of chitosan oligosaccharide was 25 mg / mL. Hydrolysate with chitosan oligosaccharide as the main component was obtained and used for subsequent crop growth promotion experiments.

[0055] Dwarf red tomato seeds (provided by Jiangsu Xingnong Matrix Technology Co., Ltd.) were surface-sterilized with 5% sodium hypochlorite for 15 minutes, germinated at 30℃ for 48 hours, and sown into seedling substrate. When the seeds had 4 true leaves, they were transplanted into flowerpots containing 80 g of culture medium (purchased from Jiangsu Xingnong Matrix Technology Co., Ltd.). Treatment groups were set up: each plant was sprayed with 5 mL of the aforementioned hydrolysate diluted to 100, 200, 400, 600, and 800 mg / L of chitosan oligosaccharide, once every 3 days for a total of 3 times; the control group was sprayed with an equal volume of deionized water. After 14 days of cultivation, growth indicators and photosynthetic parameters were measured, including: measuring the fresh weight of the aboveground and underground parts; measuring plant height and root length; measuring the relative chlorophyll content using a chlorophyll meter (purchased from Konica Minolta (China) Investment Co., Ltd., model SPAD-502); and measuring the net photosynthetic rate (Pn) using a portable photosynthetic measurement system (purchased from Beijing Laikuo Biotechnology Co., Ltd., model LI-6800). The measurement conditions were: sunny mornings from 9:00 to 11:00, with a light intensity of 1000 μmol / m² maintained in an artificial climate chamber. -2 s -1 Temperature 25℃, CO2 concentration 400 μmol -1 . The results are as follows Picture 5 As shown, the treatment group with a final concentration of 600 mg / L of chitosan oligosaccharide hydrolysate showed the best effect. Compared with the control group, the tomato seedling height increased by 34.2%, root length increased by 28.8%, aboveground fresh weight increased by 33.2%, and underground fresh weight increased by 46.3%. The relative chlorophyll content increased by 17.3%, the net photosynthetic rate increased by 67.9%, and the intercellular CO2 concentration decreased significantly. This indicates that the prepared chitin hydrolysate can significantly improve the photosynthetic efficiency of tomatoes, promote plant growth, and has excellent potential as a crop growth stimulant.

[0056] The overall concept and results of this invention can be summarized as follows: Picture 6 As shown.

Claims

1. A method for chitin degradation, characterized in that, The method includes the following steps: In a liquid-phase reaction system, a chitin-degrading enzyme system with a final concentration of 4-28 μM is added per 0.1 g of chitin, and the reaction is carried out at 70-85℃ for 4-32 h to achieve chitin degradation; wherein, the chitin-degrading enzyme system contains an exochitinase and an endochitinase in a molar ratio of (1-4):1, the amino acid sequence of the exochitinase is as shown in SEQ ID NO: 1 or has at least 95% similarity to the sequence shown in SEQ ID NO: 1, and the amino acid sequence of the endochitinase is as shown in SEQ ID NO: 2 or has at least 95% similarity to the sequence shown in SEQ ID NO:

2.

2. The chitin degradation method according to claim 1, characterized in that, The amino acid sequence of the exochitinase is based on the sequence shown in SEQ ID NO: 1, and has any one of the following single-point mutations: W74T, S154T, S157N, T233F, T240I, T240V, A295P; or any one of the following double-point mutations: T233F / T240I, T233F / A295P, T240I / A295P; or any one of the following triple-point mutations: T233F / T240I / A295P; or any one of the following quadruple-point mutations: W74T / T233F / T240I / A295P, S154T / T233F / T240I / A295P, S157N / T233F / T240I / A295P.

3. The chitin degradation method according to claim 1, characterized in that, The amino acid sequence of the endochondrinase is based on the sequence shown in SEQ ID NO: 2, and has any one of the following single-point mutations: K22P, P23G, D149L, T184S, T184P, A186N, S231E, S234D, P263G, T266G, E268G, D278Q, W281Y; or any one of the following double-point mutations: K22P / D149L, D149L / P263G, D149L / E268G.

4. The chitin degradation method according to claim 2 or 3, characterized in that, The amino acid sequence of the exochitinase is based on the sequence shown in SEQ ID NO: 1, and has: a three-point mutation of T233F / T240I / A295P; or a four-point mutation of W74T / T233F / T240I / A295P, S154T / T233F / T240I / A295P, or S157N / T233F / T240I / A295P; the amino acid sequence of the endochitinase is based on the sequence shown in SEQ ID NO: 2, and has: a two-point mutation of K22P / D149L, D149L / P263G, or D149L / E268G.

5. The chitin degradation method according to claim 1, characterized in that, The chitin is derived from chitin-containing waste.

6. The chitin degradation method according to claim 5, characterized in that, The chitin undergoes mechanical activation pretreatment before degradation.

7. The chitin degradation method according to claim 1, characterized in that, The chitin-degrading enzyme system is a mixture of crude enzyme solution obtained by thermal lysis of recombinant cells expressing exochitinase and recombinant cells expressing endochitinase.

8. The chitin degradation method according to claim 1, characterized in that, The liquid-phase reaction system is a buffer solution with a pH of 5 to 6.

9. A chitin hydrolysate prepared by the method according to any one of claims 1 to 8.

10. The use of the deacetylated product of the chitin hydrolysate of claim 9 in promoting plant growth.

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