Novel chitosanase and application thereof

Abstract

The invention provides novel chitosanase and application thereof, and relates to the technical field of enzyme engineering. The amino acid sequence of the chitosanase is as shown in SEQ ID NO. 1. The novel chitosanase prepared by the invention can efficiently and controllably catalyze chitosan hydrolysis to generate chitosan oligosaccharide with a specific polymerization degree. The enzyme is obtained by screening from an extreme environment through a metagenome technology, is novel in sequence, has unreported catalytic characteristics, can accurately regulate and control product composition, and meets the preparation requirements of specific high-purity chitosan oligosaccharide. Due to the characteristics, the enzyme has important application value in large-scale production of chitosan oligosaccharide, an effective way is provided for solving the problems of single product and low purity in the traditional method, and a technical foundation is laid for development of the fields of medicine, food and agriculture.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme engineering, and in particular to a novel chitosanase and application thereof. BACKGROUND

[0002] Chitosanase is an enzyme that is specifically used for catalyzing the hydrolysis of the beta-1, 4-glycosidic bond of chitosan, and can generate chitooligosaccharides with different degrees of polymerization (DP). Chitooligosaccharides have wide application potential in the fields of medicine, food, agriculture, etc., and their biological activities such as antibacterial, antioxidant and immunomodulatory activities have been widely studied. However, the traditional chitosan degradation method is difficult to realize the controllable production of chitooligosaccharides, especially for the efficient preparation of specific DP products. Chitosanase reduces the molecular weight by attacking the glycosidic chain of chitosan to generate small molecule chitooligosaccharides with biological activity. According to the classification of glycoside hydrolase (GH) family, chitosanases of different families have subtle differences in their catalytic mechanisms and substrate specificities. For example, GH46 family chitosanases usually exhibit a catalytic binary of glutamic acid (Glu) and aspartic acid (Asp), while GH75 family is considered to be a configuration inverting enzyme, which utilizes different residues to realize acid-base catalysis.

[0003] Chitooligosaccharides have attracted much attention due to their unique biological activities, but the current chitooligosaccharide preparation technology on the market generally has the problems of single product and low purity. In addition, the preparation technology of chitooligosaccharides with specific DP is not mature, and the traditional technology is difficult to realize the accurate control of the degree of polymerization of the product.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] In order to solve the above technical problems, the present application provides a novel chitosanase and application thereof. The present application provides a novel chitosanase, which can produce chitooligosaccharides with different degrees of polymerization through modification of chitosanase, and the prepared chitosanase provides a new opportunity for the application of medicine, food and agriculture. At the same time, the present application combines existing molecular biology and enzyme engineering technology, and the present application will lay a foundation for the industrial production of chitooligosaccharides.

[0006] In order to achieve the purpose of the present application, the following technical solutions are adopted: The present application provides a novel chitosanase, and the amino acid sequence of the chitosanase is shown in SEQ ID NO. 1.

[0007] SEQ ID NO. 1 MLKKICLTLLLCSTLGITGCTKQTAEKPAKTSTVSLKDPEKKEQAMALVSSAENSSLDWKAQYNYIEDIQDGRGYTAGIIGFCSGTGDMLDLVEKYTKTQPENPLAAYLPALKTVNGSDSHSGLDPDFTSAWEKASKEPEFQKAQNQIRDEQYFDPAVKQAKKDGLSVLGQFIYYDAIVMHGPGDGSVPYEECFYGIRKAALKKVKSPAEGGDETEYLTSFLDARDKVMALEDVHEDLSRTIAQRKFLKEENFTMKQPLHWVMYGEKFKL The application further provides a polynucleotide encoding the novel chitosanase.

[0008] The application further provides a recombinant vector comprising the polynucleotide.

[0009] Further, the recombinant vector comprises any one of a cloning vector or an expression vector.

[0010] The application further provides a host cell comprising the polynucleotide or the recombinant vector.

[0011] Further, the host cell is a prokaryotic cell or a eukaryotic cell.

[0012] The application further provides a recombinant strain comprising the polynucleotide or the recombinant vector.

[0013] The application further provides a biological catalyst comprising the novel chitosanase with the amino acid sequence as shown in SEQ ID NO. 1.

[0014] The application further provides an application of the novel chitosanase in preparing chitooligosaccharide.

[0015] The application has the following technical effects: The novel chitosanase prepared by the application can efficiently and controllably catalyze chitosan hydrolysis to generate chitooligosaccharide with a specific degree of polymerization. The enzyme is obtained by screening from an extreme environment through metagenomic technology, has a novel sequence, unreported catalytic properties and can precisely regulate product composition, and meets the preparation requirements of specific high-purity chitooligosaccharide. These characteristics make the enzyme have important application value in the large-scale production of chitooligosaccharide, provide an effective way to solve the problems of single product and low purity in traditional methods, and lay a technical foundation for the development in the fields of medicine, food and agriculture. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the drawings required to be used in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and all other embodiments obtained by a person of ordinary skill in the art without creative labor on the basis of these drawings also belong to the scope of protection of the present application.

[0017] Figure 1 Sequence alignment of chitosanase EntCsn-46A and reference sequence BsCsn46A after removing signal peptide; Figure 2 Structure of chitosanase EntCsn-46A predicted by AlphaFold3; Figure 3 Nucleic acid electropherogram of chitosanase EntCsn-46A; Figure 4 Crude protein map of chitosanase EntCsn-46A; Figure 5 Pure protein map of chitosanase EntCsn-46A; Figure 6 Standard curve and fitting equation of chitosanase EntCsn-46A; Figure 7 DNS color reaction of chitosanase EntCsn-46A, wherein the left graph is a blank control and the right graph is chitosanase EntCsn-46A; Figure 8 TLC product spectrum of chitosanase EntCsn-46A; Figure 9 Effect of different pH values on the enzyme activity of chitosanase EntCsn-46A; Figure 10 Effect of different ions on the enzyme activity of chitosanase EntCsn-46A; Figure 11 Effect of different temperatures on the enzyme activity of chitosanase EntCsn-46A; Figure 12 Reaction rate of chitosanase EntCsn-46A over time. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions, and advantages of the present application clearer, the technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor also belong to the scope of protection of the present application.

[0019] In a first aspect, the present application provides a novel chitosanase, wherein the amino acid sequence of the chitosanase is shown as SEQ ID NO. 1.

[0020] SEQ ID NO. 1 MLKKICLTLLLCSTLGITGCTKQTAEKPAKTSTVSLKDPEKKEQAMALVSSAENSSLDWKAQYNYIEDIQDGRGYTAGIIGFCSGTGDMLDLVEKYTKTQPENPLAAYLPALKTVNGSDSHSGLDPDFTSAWEKASKEPEFQKAQNQIRDEQYFDPAVKQAKKDGLSVLGQFIYYDAIVMHGPGDGSVPYEECFYGIRKAALKKVKSPAEGGDETEYLTSFLDARDKVMALEDVHEDLSRTIAQRKFLKEENFTMKQPLHWVMYGEKFKL In a second aspect, the present application provides a polynucleotide encoding the novel chitosanase.

[0021] In a third aspect, the present application provides a recombinant vector comprising the polynucleotide.

[0022] In some embodiments, the recombinant vector comprises any one of a cloning vector or an expression vector.

[0023] In a fourth aspect, the present application further provides a host cell comprising the polynucleotide or the recombinant vector.

[0024] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0025] In a fifth aspect, the present application further provides a recombinant strain comprising the polynucleotide or the recombinant vector.

[0026] In a sixth aspect, the present application further provides a biocatalyst comprising the chitosanase with the amino acid sequence shown as SEQ ID NO. 1.

[0027] In a seventh aspect, the present application further provides use of the novel chitosanase in preparation of chitooligosaccharides.

[0028] The present application will be further described in conjunction with specific embodiments as follows: Embodiment 1 SRR14318403 from acid mine extreme environment was downloaded from NCBI public database. Quality control was performed using TrimGalore to remove adapter sequences and low-quality reads (quality score <20 points) to obtain clean reads. MEGAHIT was used to assemble clean reads. The protein sequence was obtained by predicting open reading frame using the prodigal tool. Protein sequence: MLKKICLTLLLCSTLGITGCTKQTAEKPAKTSTVSLKDPEKKEQAMALVSSAENSSLDWKAQYNYIEDIQDGRGYTAGIIGFCSGTGDMLDLVEKYTKTQPENPLAAYLPALKTVNGSDSHSGLDPDFTSAWEKASKEPEFQKAQNQIRDEQYFDPAVKQAKKDGLSVLGQFIYYDAIVMHGPGDGSVPYEECFYGIRKAALKKVKSPAEGGDETEYLTSFLDARDKVMALEDVHEDLSRTIAQRKFLKEENFTMKQPLHWVMYGEKFKL A sequence with a similarity of 47.7% was obtained by diamond alignment, and the most similar sequence compared with the NCBI public database was chitosanase [Enterococcus hulanensis], Sequence ID: WP_137665303.1, and its structure was predicted by AlphaFold3. The related enzyme activity and catalytic characteristics have not been reported before, wherein, Figure 1 is a sequence alignment chart of chitosanase EntCsn-46A and reference sequence BsCsn46A. The structure of chitosanase EntCsn-46A is as Figure 2 indicated. The lower sequence similarity indicates that EntCsn-46A is a novel chitosanase. Since it is derived from an extreme environment, the enzyme may have good stability.

[0029] Example 2 After macrogenomic comparison and screening, a chitosanase with chitosan degradation was screened and named EntCsn-46A. The chitosanase gene sequence was synthesized after removing the signal peptide using SignalP software, which was used to construct a plasmid. The constructed plasmid was subjected to experiments to determine whether the plasmid was successfully constructed, and the experimental results are as Figure 3 indicated.

[0030] I. Plasmid transformation 1. Plasmid pretreatment The recombinant plasmid solution was centrifuged at 12,000xg at 4°C for 1 min, and the supernatant was discarded.

[0031] Add 20 μL sterile ultrapure water (DNase / RNase-free) to fully resuspend the plasmid pellet.

[0032] 2. Heat shock transformation Take 2 μL plasmid solution and add to 100 μL pre-cooled E. coli BL21(DE3) competent cells, and let stand on ice for 30 min. Heat shock at 42°C for 45 s (avoid excessive cell damage), and quickly place in ice bath for 2 min.

[0033] Add 900 μL sterile LB medium (without antibiotics), and incubate at 37°C with shaking at 220 rpm for 1 h.

[0034] 3. Plating for screening After centrifugation (5,000 x g, 5 min), discard 900 μL supernatant, and resuspend the bacterial pellet in about 100 μL.

[0035] Take all the bacterial solution and plate on LB agar plates containing 50 μg / mL kanamycin, and incubate at 37°C for 12-16 h.

[0036] II. Positive clone verification 1. Colony PCR (1) Template preparation: pick a single colony and suspend in 10 μL sterile water, and spot on a resistance plate, and incubate at 37°C. The remaining bacterial suspension is used for PCR.

[0037] (2) PCR system (20 μL): 2 x Taq Master Mix: 10 μL, forward / reverse primers (10 μM): 1 μL each, template: 1 μL, sterile water: 7 μL (3) Reaction program: pre-denaturation: 95°C for 15 min, cycle: 95°C for 30 s→60°C for 30 s→72°C for 1 min / kb, for 30 cycles, final extension: 72°C for 5 min (4) Electrophoresis verification: Take 5 μL PCR product and perform 1.5% agarose gel electrophoresis (120 V, 20 min), and confirm the target band under UV imaging.

[0038] (5) After sequencing verification, pick the positive clone in step (1), inoculate into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and incubate at 37°C with shaking at 200 rpm for 12-16 h. Store in 25% glycerol at -80°C.

[0039] III. Fermentation and induced expression (1) Take 50 μL competent cells to 5 mL LB liquid medium containing 50 μg / mL kanamycin, and incubate at 37°C, 200 rpm for 12-16 h. At the same time, set up a negative control (optional E. coli BL21 competent cells), take 50 μL competent cell solution to 5 mL LB liquid medium without 50 μg / mL kanamycin, and incubate at 37°C, 200 rpm for 12-16 h.

[0040] (2) Transfer 5 mL of the bacterial solution to 50 mL of LB medium (containing 50 μg / mL kanamycin) at a 1:50 inoculation ratio, and incubate at 37°C, 200 rpm until OD600=0.6-0.8 (about 2-3 hours).

[0041] Add IPTG to a final concentration of 0.3 mM (0.1-1 mM), and transfer to 16-17°C, 160 rpm for 16-18 h of induction.

[0042] Four, protein extraction and detection 1. Bacterial collection and lysis Weigh the empty centrifuge tube, collect the fermentation broth, centrifuge (105,000 x g, 10 min) to collect the bacterial cells, reserve 1 mL of the fermentation supernatant, weigh the centrifuge tube containing the bacterial cells, and calculate the wet weight of the bacterial cells.

[0043] Use the iron hammer kit (refer to the instruction manual) for cell disruption. The supernatant of the iron hammer broken solution is obtained.

[0044] 2. SDS-PAGE analysis (1) Collect the supernatant and precipitate of the iron hammer broken solution, resuspend the precipitate in 1 mL of buffer solution, and analyze the protein composition in the fermentation supernatant, broken supernatant, and broken precipitate using SDS-PAGE technology. The specific method is as follows: mix 20 μL of sample with 5 μL of 4x loading buffer, and dye with protein dyeing agent at a ratio of sample:dye = 4:1, and boil for 10 min.

[0045] (2) Centrifuge at 12000 rpm for 10 min, and use the supernatant for loading. The loading order is: protein marker-fermentation supernatant-broken supernatant-broken precipitate (3) Gel preparation method: refer to 12.5% SDS-PAGE denatured acrylamide color gel rapid preparation kit (4) The electrophoresis conditions are low voltage gel pressing (80V constant voltage until bromophenol blue enters the separation gel): 12.5% separation gel, then run the separation gel at 120V, and stop running the gel when the blue band reaches the bottom, about 90 min of electrophoresis.

[0046] Staining and destaining: Coomassie brilliant blue R-250 staining for 30 min, destaining with destaining solution (10% acetic acid + 40% ethanol) until the background was transparent, and then the protein gel map of the chitosanase EntCsn-46A crude protein was obtained. The experimental results are shown in Figure 4 We can see that the protein molecular weight of the chitosanase EntCsn-46A crude protein is close to 29 between 33-25, which is consistent with the expected protein molecular weight (28.95), proving that the chitosanase EntCsn-46A has been correctly expressed.

[0047] 4. Protein purification 1. Sample preparation Collect the supernatant and the precipitate of the iron hammer broken liquid, and resuspend the precipitate in 1 mL of buffer solution.

[0048] 2. Magnetic bead pretreatment Balance the magnetic beads: Take an appropriate amount of Ni-NTA magnetic beads (according to the recommended ratio of the manufacturer), place them on the magnetic stand for adsorption for 1 min, and discard the storage solution.

[0049] Wash 3 times with 5 times the volume of the magnetic beads with binding buffer, and discard the supernatant after adsorption for 1 min each time.

[0050] 3. Protein binding Mix the lysate supernatant and the resuspended precipitate with the balanced magnetic beads, and incubate at room temperature for 30-60 min (or 4°C for 1-2 h) with gentle shaking. Adsorb for 1 min on the magnetic stand, and discard the unbound liquid.

[0051] 4. Wash the impurities Wash 3 times with 5 times the volume of the magnetic beads with washing buffer: Add buffer, and resuspend the magnetic beads gently. Adsorb for 1 min on the magnetic stand, and discard the supernatant.

[0052] 5. Elution of target protein Add 1 times the volume of the magnetic beads with elution buffer, and incubate at room temperature for 10-15 min with shaking.

[0053] Adsorb for 1 min on the magnetic stand, and collect the eluate (containing His-tagged protein). Repeat the elution 1-2 times, and combine the eluates. Then obtain the pure enzyme solution.

[0054] 5. SDS-PAGE analysis (1) Mix 20 μL of sample with 5 μL of 4x loading buffer, and dye with protein dyeing agent according to the ratio of sample:dyeing agent = 4:1, and boil for 5 min (2) Centrifuge at 12000 rpm for 10 min, and use the supernatant for loading. The loading order is protein marker—broken liquid supernatant—broken liquid precipitate (3) Glue method: reference 12.5% SDS-PAGE denatured acrylamide color gel rapid preparation kit (4) Electrophoresis conditions are low voltage glue (80V constant voltage to bromophenol blue into the separation glue), and then use 120V to run the separation glue, until the blue band to the bottom, about 90min electrophoresis, stop running glue.

[0055] Staining and decolorization: Coomassie brilliant blue R-250 staining for 30 min, and decolorizing to the background transparent after decolorizing solution (10% acetic acid + 40% ethanol) to obtain chitosanase EntCsn-46A pure protein protein gel map, the experimental results are shown in Figure 5 We can see that the protein molecular weight of chitosanase EntCsn-46A is close to 29 between 33-25, which is consistent with the expected protein molecular weight (28.95), which proves that chitosanase EntCsn-46A has been correctly expressed, and the experiment proves that the gene can be effectively expressed and the active chitosanase is purified, which provides a material basis for the follow-up functional research.

[0056] Example 3 Chitosanase enzyme activity detection Determination of reducing sugar content in hydrolysate Prepare standard curve: prepare 1 mg / mL glucosamine hydrochloride standard solution (accurately weigh 100 mg standard, constant volume to 100 mL), respectively, 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL standard solution into 25 mL stoppered colorimetric tube, and make up to 3 mL with distilled water. Then mix with 2 mL DNS reagent and shake well, color for 10 min in boiling water bath, cool with running water, constant volume to 5 mL with distilled water, and measure the absorbance at 540 nm, with distilled water and DNS reagent as blank.

[0057] Fitting equation: with concentration as abscissa (x, mg / mL) and OD value as ordinate (y), fitting linear regression equation y=ax+b, R 2 ≥0.99, calculate enzyme activity according to standard curve and fitting equation, fitting curve and fitting equation are shown in Figure 6

[0058] ​Sample enzyme activity determination (DNS colorimetric method): 1 g of chitosan was dissolved in 200 mM of pH 6.5 acetic acid-sodium acetate buffer solution to obtain a 1% chitosan solution. The crude enzyme solution or the purified enzyme solution was diluted with distilled water to an appropriate multiple, 100 μL of the diluted enzyme solution was added to 200 μL of 1% chitosan solution, and the volume was made to 1 mL with acetic acid-sodium acetate buffer solution, and mixed uniformly, and placed in a 45°C water bath for reaction for 20 min, 2 mL of DNS solution was added to terminate the reaction, boiled for 5 min for color development, and then made to 5 mL with distilled water, centrifuged at 12000 rpm for 5 min, and the absorbance value was measured at 540 nm, with three parallel samples in each group. The blank control was inactivated enzyme, and the experimental process was as shown in Figure 7 The left graph is the blank control, and the right graph is chitosanase EntCsn-46A. The unit of enzyme activity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute per mL of enzyme solution at 40°C. The final enzyme activity of chitosanase EntCsn-46A was measured to be 6.02 U / g, which provided a benchmark for quantifying the catalytic efficiency of the enzyme and proved that the enzyme indeed has the ability to catalyze the hydrolysis of chitosan.

[0059] Example 4: Catalytic characteristics of chitosanase Thin layer chromatography (TLC): 50 μL of pure enzyme solution was added to 950 μL of 1% chitosan solution, and reacted for 1 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 6 h, 12 h, 24 h, 48 h, respectively, and then centrifuged at 8000 rpm for 10 min, and the supernatant was taken and directly spotted on a silica gel plate with a capillary (the distance between each spot was 0.50 cm, and the distance from the bottom end was 1.0 cm), and 5% glucosamine and chitooligosaccharide aqueous solution were used as controls. The plate was placed in a chromatography tank and developed upward in a developing agent (isopropanol:ammonia water:water = 15:7.5:4 V / V / V), and after development, it was blown dry, and then 1% (w / v) ninhydrin ethanol solution was sprayed as a color developing agent, and finally it was colored in a 105°C drying oven for 20 min. The TLC product spectrum of chitosanase EntCsn-46A is shown in Figure 8 As can be seen, within 48 h, the enzymatic hydrolysis products of chitosanase EntCsn-46A were always dominated by (GlcN)2, with high purity, and the enzyme was most skilled at cutting one disaccharide unit in each hydrolysis reaction, and its active site could most effectively bind and cut the substrate sequence that produced disaccharide as a product. This experiment showed that chitosanase EntCsn-46A might be a strict "disaccharide generator".

[0060] Example 5: pH tolerance of chitosanase To determine the optimum temperature of chitosanase, 50 μL of crude enzyme solution of E. coli BL21 engineering bacteria (10 9 CFU / mL) and 450 μL of 1% colloidal chitosan were taken, and 500 μL of acetic acid-sodium acetate buffer (pH=4.5) was added. The reaction was carried out at pH=2, 3, 4, 5, and 6 for 15 min, respectively. The corresponding enzyme activity was measured by the DNS method. The enzyme activity value measured under the best temperature condition was used as the reference ratio to calculate the relative enzyme activity under other conditions. The experimental results are shown in Table 1. Figure 9 As can be seen from Table 1, Figure 9 the enzyme activity of chitosanase EntCsn-46A is low at pH=2 and 3, and is high at pH=4, 5, and 6. The enzyme activity gradually increases with increasing pH, and reaches the highest value at pH=6. The optimum pH of chitosanase EntCsn-46A is 6, indicating that the catalytic efficiency of the enzyme is highest in a slightly acidic environment, and the adaptability is good in a neutral to weakly acidic environment (pH=4-6). This experiment shows that EntCsn-46A is an enzyme that adapts to weakly acidic environment, which provides convenience for its application in various industrial conditions.

[0061] Example 6: Ion tolerance of chitosanase To determine the effect of different ions on chitosanase activity, the ion concentration in the reaction solution was 0.5 mmol / L. 50 μL of crude enzyme solution of E. coli BL21 engineering bacteria (10 9 CFU / mL) and 450 μL of 1% colloidal chitosan were taken, and 500 μL of acetic acid-sodium acetate buffer (pH=4.5) was added. The reaction was carried out at 45°C and for 15 min (the optimum temperature and pH measured) to detect the effect of different ions on chitosanase activity. 2+ (MnSO4•H2O), Zn 2+ (ZnSO4•7H2O), Fe 2+ (FeSO4•7H2O), Mg 2+ (MgSO4•7H2O), Cu 2+ (Anhydrous CuSO4), Fe 3+ (FeCl3•6H2O), Ca 2+ (CaCI2•2H2O), Al 3+ (Al2(SO4)3•16H2O), Cr 3+ (CrCl3•6H2O), Co 2+ (CoSO4•7H2O), Ni 2+ (NiSO4·6H2O), K + (K2SO4), NH4 +The effect of ((NH4)2SO4) plasma on surface-displayed chitosanase was investigated. The corresponding enzyme activity was measured using the DNS method. The enzyme activity measured under conditions without added ions was used as the highest percentage baseline, and the relative enzyme activity under other conditions was calculated. Experimental results are as follows: Figure 10 As shown, from Figure 10 As can be seen, the ion that promotes the activity of chitosanase EntCsn-46A is Mn. 2+ Co 2+ Zn 2+ Enhancing the relative enzyme activity to over 100% (up to approximately 140%) can improve the enzyme's catalytic efficiency.

[0062] Ions that inhibit the activity of chitosanase EntCsn-46A include: Cu. 2+ NH4 + Cr 3+ When the relative enzyme activity is reduced to below 100% (minimum approximately 85%), enzyme activity may be inhibited by disrupting the enzyme structure or competing for active sites; ions that have no significant effect on the enzyme activity of chitosanase EntCsn-46A include: Ca... 2+ Fe 3+ When the relative enzyme activity is close to 100%, the enzyme exhibits strong tolerance. This experiment investigated the effects of different metal ions on the enzyme activity, providing an important reference for selecting appropriate reaction conditions (such as buffer composition) in practical applications.

[0063] Example 7: Temperature tolerance of chitosanase To determine the optimal temperature for chitosanase, 50 μL of engineered LE.coli BL21 bacteria (10 9 A crude enzyme solution (CFU / mL) and 450 μL of 1% colloidal chitosan were added, followed by 500 μL of acetate-sodium acetate buffer (pH=4.5). The mixture was reacted at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 10 min each. The corresponding enzyme activities were measured using the DNS method. The enzyme activity values ​​measured under the optimal temperature conditions were taken as the highest quotient standard, and the relative enzyme activities under other conditions were calculated. Experimental results are as follows: Figure 11 As shown, from Figure 11 As can be seen, the activity of chitosanase EntCsn-46A increases with increasing temperature from 20℃ to 40℃, reaching its peak at 40℃. From 40℃ to 80℃, the activity of chitosanase EntCsn-46A gradually decreases with increasing temperature. The optimal temperature for chitosanase EntCsn-46A is 40℃. The enzyme activity exhibits a single-peak curve with temperature change, slowly decreasing after reaching the optimal temperature. This confirms that EntCsn-46A is a room-temperature enzyme, with its optimal temperature close to, and slightly higher than, the optimal temperature of most enzymes. This is beneficial for accelerating the reaction rate at around 40℃ and may reduce contamination by other microorganisms.

[0064] Example 8: Change of reaction rate of chitosanase with time To determine the change of reaction rate of chitosanase with time, 50 μL of crude enzyme solution of E. coli BL21 engineering bacteria (10 9 CFU / mL) and 450 μL of 1% colloidal chitosan were taken, and 500 μL of acetic acid-sodium acetate buffer (pH=4.5) was added, and the corresponding enzyme activity was measured by the DNS method at 1 min, 5 min, 10 min, 15 min, and 25 min, and the experimental results are shown in Figure 12 As can be seen from the above experiment, the final enzyme activity and the slope of time are converted into the conversion rate of enzyme, and it can be seen that the reaction rate of the enzyme reaches a peak at the initial stage of the reaction of chitosanase EntCsn-46A (1 min); in the late stage of the reaction of the enzyme (5 min-25 min): the rate of the enzyme gradually slows down, and the curve slope decreases.

[0065] As can be seen from the above experiment, the present application successfully screens a novel chitosanase EntCsn-46A from an extreme environment, and through recombinant expression, purification and functional verification, it can be seen that the chitosanase has the advantages of novel sequence, high catalytic efficiency, controllable product, etc., and can solve the problems of single product and low purity in the traditional chitosan degradation method, and provides a reliable tool for realizing the large-scale production of chitooligosaccharides with specific degree of polymerization, and has a broad application prospect in the fields of medicine, food and agriculture.

[0066] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present application.

Claims

1. A novel chitosanase, characterized in that, The amino acid sequence of the chitosanase is shown as SEQ ID NO.

1.

2. A polynucleotide, comprising, The novel chitosanase as claimed in claim 1.

3. A recombinant vector, characterized in that, The polynucleotide as claimed in claim 2.

4. The recombinant vector of claim 3, wherein, The recombinant vector includes any one of a cloning vector or an expression vector.

5. A host cell, characterized in that, The polynucleotide as claimed in claim 2 or the recombinant vector as claimed in any one of claims 3-4.

6. The host cell of claim 5, wherein, The host cell is a prokaryotic cell or a eukaryotic cell.

7. A recombinant bacterial strain, characterized in that, The polynucleotide as claimed in claim 2 or the recombinant vector as claimed in any one of claims 3-4.

8. A biocatalyst characterized in that, The novel chitosanase comprising the amino acid sequence shown as SEQ ID NO.

1.

9. Use of the chitosanase as claimed in claim 1 in the preparation of chitooligosaccharides.

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