A novel chitosanase with high catalytic performance and application thereof

Abstract

The application discloses a novel chitosanase with high catalytic performance and application thereof, and belongs to the technical field of enzyme engineering. The amino acid sequence of the chitosanase is shown as SEQ ID NO. 1. The chitosanase is obtained by modification of a macrogenomic sequence in an extreme environment, and has novel and unique catalytic characteristics. The modified sequence can efficiently and controllably catalyze chitosan hydrolysis, and precisely regulate the polymerization degree of a product, so that chitooligosaccharide with a specific polymerization degree and high purity is obtained. The problems of strong randomness of chitosanase hydrolysis, complex product composition and low yield of chitooligosaccharide with a target polymerization degree in the prior art are solved.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a novel chitosanase with high catalytic performance and its applications. Background Technology

[0002] Chitosanase is an enzyme specifically designed to catalyze the hydrolysis of the β-1,4-glycosidic bonds in chitosan, generating chitosan oligosaccharides with varying degrees of polymerization (DP). Chitosan oligosaccharides have broad application potential in medicine, food, and agriculture, and their antibacterial, antioxidant, and immunomodulatory activities have been extensively studied. However, traditional chitosan degradation methods struggle to achieve controlled production of chitosan oligosaccharides, especially for the efficient preparation of specific DP products. Chitosanase reduces the molecular weight of chitosan by attacking its glycosidic chains, generating bioactive small-molecule chitosan oligosaccharides. According to the classification of glycoside hydrolases (GH) families, different families of chitosanases exhibit subtle differences in their catalytic mechanisms and substrate specificity. For example, GH46 family chitosanases typically exhibit a catalytic binary of glutamate (Glu) and aspartic acid (Asp), while the GH75 family is considered a configuration-reversing enzyme, utilizing different residues to achieve acid-base catalysis.

[0003] Chitosan oligosaccharides have attracted much attention due to their unique biological activities. However, current chitosan oligosaccharide preparation technologies on the market generally suffer from problems such as strong randomness of substrate hydrolysis, complex product composition, and low product purity. Traditional technologies make it difficult to achieve precise control of the degree of polymerization of the product. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a novel chitosanase with high catalytic performance and its applications. By modifying the chitosanase, it is possible to produce chitosan oligosaccharides with specific degrees of polymerization, solving the problems of high randomness in hydrolysis, complex product composition, and low yield of chitosan oligosaccharides with the target degree of polymerization in existing chitosanases. This provides technical support for the large-scale preparation of specific active chitosan oligosaccharides.

[0005] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides a novel chitosanase with high catalytic performance, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] SEQ ID NO.1 GLIGSDLPQDGDHVSEPDQGNSSAEISEGLRNPTTKDRAARIISSFENSTTEIQYGFAKDIGDGRGITAGRAGFTSRTHDLLLVVERYREKSPGNTLEKFIAPLRAVDGTDSTAGLSGFIETWQTAALTDPALR EVQDAVVDELYFNPAVQAAEKLHIRSPLGQLIFWDSITQHGGGTDPDGLPAMTTEVESEYGKAGGQYTEREWLQRFLEVRREHLSYAADPTTREAWKRSVPRVDALESLVLNNNLALAPPIRWRVYGDEFSLDQ The present invention also provides a polynucleotide encoding the above-mentioned novel chitosanase.

[0007] The present invention also provides a recombinant vector comprising the above-mentioned polynucleotides.

[0008] Furthermore, the recombinant vector includes either a cloning vector or an expression vector.

[0009] The present invention also provides a host cell comprising the above-described polynucleotide or recombinant vector.

[0010] Furthermore, the host cell is a prokaryotic cell or a eukaryotic cell.

[0011] The present invention also provides a recombinant strain comprising the above-mentioned polynucleotide or recombinant vector.

[0012] The present invention also provides a novel chitosanase biocatalyst comprising the above-described amino acid sequence as shown in SEQ ID NO.1.

[0013] The present invention also provides the application of a novel chitosanase with high catalytic performance in the preparation of chitosan oligosaccharides, the amino acid sequence of which is shown in SEQ ID NO.1.

[0014] Furthermore, the chitosan oligosaccharide is a disaccharide, trisaccharide, or tetrasaccharide.

[0015] The present invention has the following technical effects: The novel chitosanase prepared in this invention can efficiently and controllably catalyze the hydrolysis of chitosan to generate chitosan oligosaccharides with a specific degree of polymerization. This enzyme, obtained through metagenomic modification, possesses unique catalytic properties, enabling precise control of product composition and accurate catalysis of chitosan hydrolysis. It specifically enriches highly active di- to tetrameric chitosan oligosaccharides, meeting the requirements for preparing chitosan oligosaccharides with specific degrees of polymerization. These characteristics make this enzyme of significant application value in the large-scale production of chitosan oligosaccharides, solving the problems of high randomness in hydrolysis, complex product composition, and low yield of chitosan oligosaccharides with the target degree of polymerization found in existing chitosanases. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 The structure of chitosanase GEEGC-CNS-263 predicted by AlphaFold3; Figure 2 Electrophoresis image of chitosanase GEEGC-CNS-263; Figure 3 Crude protein diagram of chitosanase GEEGC-CNS-263; Figure 4 Image of purified protein of chitosanase GEEGC-CNS-263; Figure 5 Standard curve and fitting equation of chitosanase GEEGC-CNS-263; Figure 6 The DNS colorimetric reaction of chitosanase GEEGC-CNS-263, where the left figure is the blank control and the right figure is chitosanase GEEGC-CNS-263; Figure 7 TLC product spectrum of chitosanase GEEGC-CNS-263; Figure 8 Effects of different pH values ​​on the activity of chitosanase GEEGC-CNS-263; Figure 9 Effects of different ions on the activity of chitosanase GEEGC-CNS-263; Figure 10 Effects of different temperatures on the activity of chitosanase GEEGC-CNS-263; Figure 11: Graph showing the change in reaction rate of chitosanase GEEGC-CNS-263 over time. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] In a first aspect, the present invention provides a novel chitosanase with high catalytic performance, the amino acid sequence of which is shown in SEQ ID NO.1.

[0020] SEQ ID NO.1 GLIGSDLPQDGDHVSEPDQGNSSAEISEGLRNPTTKDRAARIISSFENSTTEIQYGFAKDIGDGRGITAGRAGFTSRTHDLLLVVERYREKSPGNTLEKFIAPLRAVDGTDSTAGLSGFIETWQTAALTDPALR EVQDAVVDELYFNPAVQAAEKLHIRSPLGQLIFWDSITQHGGGTDPDGLPAMTTEVESEYGKAGGQYTEREWLQRFLEVRREHLSYAADPTTREAWKRSVPRVDALESLVLNNNLALAPPIRWRVYGDEFSLDQ Secondly, the present invention provides a polynucleotide encoding the above-mentioned novel chitosanase.

[0021] Thirdly, the present invention provides a recombinant vector comprising the above-mentioned polynucleotides.

[0022] In some embodiments, the recombinant vector includes either a cloning vector or an expression vector.

[0023] Fourthly, the present invention also provides a host cell comprising the above-mentioned polynucleotide or recombinant vector.

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

[0025] Fifthly, the present invention also provides a recombinant strain comprising the above-mentioned polynucleotide or recombinant vector.

[0026] In a sixth aspect, the present invention also provides a biocatalyst comprising chitosanase as shown in SEQ ID NO.1, which contains the amino acid sequence described above.

[0027] In a seventh aspect, the present invention provides the application of a novel chitosanase with high catalytic performance in the preparation of chitosan oligosaccharides, the amino acid sequence of which is shown in SEQ ID NO.1.

[0028] In some embodiments, the chitosan oligosaccharide is a disaccharide, trisaccharide, or tetrasaccharide.

[0029] The following is a detailed explanation using specific embodiments: Example 1 Based on the metagenomic sample CRR807416 from a high-salt environment, the target protein was obtained after removing the signal peptide using SignalP software and then modifying it. Its structure was predicted using AlphaFold3. Figure 1 As shown, due to its origin in an extreme environment, this enzyme may possess good stability. The protein is named GEEGC-CNS-263, with "263" in the figure referring to "GEEGC-CNS-263". Its amino acid sequence is: GLIGSDLPQDGDHVSEPDQGNSSAEISEGLRNPTTKDRAARIISSFENSTTEIQYGFAKDIGDGRGITAGRAGFTSRTHDLLLVVERYREKSPGNTLEKFIAPLRAVDGTDSTAGLSGFIETWQTAALTDPALREVQDAVVDELYFNPAVQAAEKLHIRSPLGQLIFWDSITQHGGGTDPDGLPAMTTEVESEYGKAGGQYTEREWLQRFLEVRREHLSYAADPTTREAWKRSVPRVDALESLVLNNNLALAPPIRWRVYGDEFSLDQ.

[0030] The protein has a maximum sequence similarity of 52.63% with existing sequences in the NCBI database.

[0031] Example 2 Based on the amino acid sequence of the target protein and the codon bias of the host cell, the coding gene of the target protein was reverse-translated and artificially synthesized. It was then cloned into an expression vector to construct a recombinant plasmid. The transformation experiments verified the results as follows: Figure 2 As shown, the plasmid construction was successful.

[0032] I. Plasmid Transformation 1. Plasmid pretreatment Centrifuge the recombinant plasmid solution at 12,000×g for 1 min at 4℃ and discard the supernatant.

[0033] Add 20 μL of sterile ultrapure water (DNase / RNase-free) to fully resuspend the plasmid precipitate.

[0034] 2. Thermal shock conversion Add 2 μL of plasmid solution to 100 μL of pre-chilled E. coli BL21(DE3) competent cells and incubate on ice for 30 min. Heat shock at 42℃ for 45 s, then immediately incubate on ice for 2 min.

[0035] Add 900 μL of sterile LB medium and incubate at 37°C and 220 rpm for 1 h with shaking.

[0036] 3. Coating screening After centrifugation (5,000×g, 5 min), discard 900 μL of supernatant and retain approximately 100 μL of resuspended bacterial cells.

[0037] Spread all bacterial culture onto LB agar plates containing 50 μg / mL kanamycin and incubate upside down at 37°C for 12-16 h.

[0038] II. Validation of Positive Clones (1) Template preparation: Pick a single colony and suspend it in 10 μL of sterile water, spot it on an antibiotic plate, and incubate it at 37 °C. The remaining bacterial suspension is used for PCR.

[0039] (2) PCR system (20 μL): 2×TaqMasterMix: 10 μL, forward / reverse primers (10 μM): 1 μL each, template: 1 μL, sterile water: 7 μL; (3) PCR reaction program: pre-denaturation: 95 ℃ for 15 min, cycling: 95 ℃, 30s→60 ℃, 30s→72 ℃, 1 min / kb, for a total of 30 cycles, final extension: 72 ℃, 5 min.

[0040] (4) Electrophoresis verification: Take 5 μL of PCR product and perform 1.5% agarose gel electrophoresis (120 V, 20 min), and confirm the target band by UV imaging.

[0041] (5) After the sequencing verification is correct, pick a positive single colony, inoculate it into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture at 37℃ and 200 rpm for 12-16 h with shaking. Then store it in 25% glycerol at -80℃.

[0042] III. Fermentation-induced expression (1) Add 50 μL of glycerol bacteria to 5 mL of LB liquid medium containing 50 μg / mL kanamycin and culture at 37 ℃ with shaking at 200 rpm for 12-16 h. At the same time, set up a negative control (E. coli BL21 competent cells without plasmid) by adding 50 μL of competent cell solution to 5 mL of LB liquid medium without 50 μg / mL kanamycin and culture at 37 ℃ with shaking at 200 rpm for 12-16 h.

[0043] (2) Transfer 5 mL of bacterial culture to 50 mL of LB medium (containing 50 μg / mL kanamycin) at an inoculation rate of 1:50, and incubate at 37℃ and 200 rpm until OD600 = 0.6-0.8 (approximately 2-3 h).

[0044] (3) Add IPTG to a final concentration of 0.3 mM (0.1~1 mM), and transfer to 16-17℃ and 160 rpm for induction for 16-18 h.

[0045] IV. Protein Extraction and Detection 1. Bacterial cell collection and lysis Weigh the empty centrifuge tube, collect the fermentation broth, centrifuge (105,000×g, 10 min) to collect the cells, retain 1 mL of the fermentation broth supernatant, weigh the centrifuge tube containing the cells, and calculate the wet weight of the cells.

[0046] Cell disruption was performed using the Hammer Reduction Kit (ACE BR0005). The resulting supernatant (crude enzyme solution) was obtained from the Hammer Reduction Kit.

[0047] 2. SDS-PAGE analysis (1) Collect the supernatant and precipitate of the hammer crushing liquid. Resuspend the precipitate in 1 mL buffer solution and analyze the protein composition of the fermentation broth supernatant, crushing liquid supernatant and crushing liquid precipitate using SDS-PAGE technology. The specific method is as follows: Take 20 μL of sample and mix with 5 μL of 4× loading buffer, stain with protein staining agent at a ratio of sample:staining agent = 4:1, and boil for 10 min.

[0048] (2) Centrifuge at 12000 rpm for 10 min. Use the supernatant for sample loading. The loading order is: protein marker - fermentation broth supernatant - lysate supernatant - lysate precipitate.

[0049] (3) Gel preparation method: Refer to the 12.5% ​​SDS-PAGE denatured acrylamide color gel rapid preparation kit.

[0050] (4) Low voltage gel pressing (80 V constant voltage until bromophenol blue enters the separating gel), then run the separating gel at 120 V. When the blue band reaches the bottom, electrophoresis for about 90 minutes, then stop running the gel.

[0051] (5) Staining and destaining: Coomassie Brilliant Blue R-250 was used for staining for 30 min. After destaining with destaining solution (10% acetic acid + 40% ethanol) until the background was transparent, a gel image of the crude chitosanase GEEGC-CNS-263 protein was obtained. The experimental results are as follows: Figure 3 As shown, the molecular weight of the crude chitosanase GEEGC-CNS-263 protein is between 33 and 25, which is consistent with the expected molecular weight (30.35), proving that chitosanase GEEGC-CNS-263 has been correctly expressed.

[0052] 4. Protein purification 1. Sample Preparation Collect the supernatant and precipitate from the hammer crushing fluid, and resuspend the precipitate in 1 mL of buffer solution.

[0053] 2. Magnetic bead pretreatment Take an appropriate amount of Kangma Bio 20% His Monster Ni-NTA magnetic bead suspension and use it according to the manufacturer's instructions: 50-250 μL of magnetic bead suspension for every 0.2-1 mg of target protein; place the magnetic beads on a magnetic rack for 1 min to adsorb, and then completely discard the storage solution.

[0054] 3. Protein binding Mix the lysate supernatant and the precipitate resuspension separately with the equilibrated magnetic beads, and incubate with gentle shaking at room temperature for 30-60 min (or at 4°C for 1-2 h). Adsorb onto the magnetic rack for 1 min, and discard the unbound liquid.

[0055] 4. Wash away contaminating proteins Wash three times with 5 times the volume of washing buffer for the magnetic beads. Add buffer and gently resuspend the magnetic beads. Adsorb onto a magnetic rack for 1 min, then discard the supernatant.

[0056] 5. Target protein elution Add elution buffer equal to the volume of the magnetic beads and incubate at room temperature with shaking for 10-15 minutes.

[0057] Adsorbed on a magnetic rack for 1 min, the eluent (containing His-tagged protein) was collected.

[0058] Repeat the elution process 1-2 times and combine the eluents. This yields a pure enzyme solution.

[0059] 6. SDS-PAGE analysis (1) Mix 20 μL of sample with 5 μL of 4× loading buffer, stain with protein staining agent at a ratio of sample:staining agent = 4:1, and boil for 5 min. (2) Centrifuge at 12000 rpm for 10 min. Use the supernatant for sample loading. The loading order is: protein marker - lysate supernatant - lysate precipitate. (3) Gel preparation method: Refer to the 12.5% ​​SDS-PAGE denaturing acrylamide color gel rapid preparation kit. (4) Low voltage gel pressing (80 V constant voltage until bromophenol blue enters the separating gel), then run the separating gel at 120 V. When the blue band reaches the bottom, electrophoresis for about 90 minutes, then stop running the gel.

[0060] (5) Staining and destaining: Coomassie Brilliant Blue R-250 staining for 30 min, followed by destaining with destaining solution (10% acetic acid + 40% ethanol) until the background is transparent to obtain a protein gel image of pure chitosanase GEEGC-CNS-263 protein. The experimental results are as follows: Figure 4 As shown, the molecular weight of the purified chitosanase GEEGC-CNS-263 protein is between 33 and 25, close to 30, which is consistent with the expected molecular weight (30.35). This proves that chitosanase GEEGC-CNS-263 has been correctly expressed. The experiment confirms that this gene can be effectively expressed and purified to produce an active chitosanase, providing a material basis for subsequent functional studies.

[0061] Example 3 Chitosanase activity assay Determination of reducing sugar content in hydrolysate Preparation of the standard curve: Prepare a 1 mg / mL glucosamine hydrochloride standard solution (accurately weigh 100 mg of standard and dilute to 100 mL). Pipette 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 1.0 mL of the standard solution into 25 mL stoppered colorimetric tubes, and bring the volume to 3 mL with distilled water. Then, mix each tube with 2 mL of DNS reagent and shake well. Develop the solution in a boiling water bath for 10 min, cool under running water, and dilute to 5 mL with distilled water. Measure the absorbance at 540 nm. Use distilled water with DNS reagent as a blank.

[0062] Fitting equation: Plotting concentration on the x-axis (x, mg / mL) and OD value on the y-axis, a linear regression equation was fitted: y = 1.533x + 0.5824, R². 2 ≥0.99, enzyme activity was calculated based on the standard curve and the fitted equation, as shown in the figure. Figure 5 As shown.

[0063] Sample enzyme activity assay (DNS colorimetric method): Dissolve 1 g of chitosan in 200 mM pH 6.5 acetate-sodium acetate buffer to obtain a 1% chitosan solution. Take the crude enzyme solution or purified enzyme solution, dilute it appropriately with distilled water, add 100 μL of the diluted enzyme solution to 200 μL of 1% chitosan solution, and make up to 1 mL with acetate-sodium acetate buffer. Mix well and react in a 45℃ water bath for 20 min. Add 2 mL of DNS solution to terminate the reaction, boil for 5 min to develop color, and then make up to 5 mL with distilled water. Centrifuge at 12000 rpm for 5 min and measure the absorbance at 540 nm. Each group has three replicates. The blank control is an inactivated enzyme. The experimental procedure is as follows: Figure 6 As shown, the left image represents the blank control, and the right image represents chitosanase GEEGC-CNS-263. Enzyme activity is defined as the amount of enzyme required per milliliter of enzyme solution per minute to release 1 μmol of reducing sugar at 40°C; one enzyme activity unit (U) is defined as this amount of enzyme required to react and release 1 μmol of reducing sugar. The final enzyme activity of chitosanase GEEGC-CNS-263 was measured to be 2.6 U / g. This result provides a benchmark for quantifying the enzyme's catalytic efficiency, demonstrating that the enzyme does indeed have the ability to catalyze the hydrolysis of chitosan.

[0064] Example 4: Catalytic characteristics of chitosanase Thin-layer chromatography (TLC): Add 50 μL of purified enzyme solution to 950 μL of 1% chitosan solution, react for about 30 min, then centrifuge at 8000 rpm for 10 min. Collect the supernatant and spot it directly onto a silica gel plate using a capillary tube (5 mm spacing between spots, 10 mm from the bottom). Use 5% glucosamine and chitosan oligosaccharide aqueous solutions as standards. Develop the plate in a chromatography tank using isopropanol:ammonia:water = 15:7.5:4 V / V / V, ammonia concentration 25%. After development, dry the plate, then spray with 1% (w / v) ninhydrin ethanol solution as a colorimetric reagent. Finally, incubate at 105 °C for 20 min. The TLC product spectrum of chitosanase GEEGC-CNS-263 is shown below. Figure 7 As shown, within 2 hours, the enzymatic hydrolysis products of chitosanase GEEGC-CNS-263 are mainly (GlcN)2, (GlcN)3, and (GlcN)4, exhibiting a broad product spectrum and a high number of high-value products. This enzyme uses a classic endo-glucanase approach to hydrolyze the substrate, and its active site can effectively bind to and cleave the internal glycosidic bonds of long-chain chitosan oligosaccharides, thereby releasing products with various degrees of polymerization, ranging from disaccharides to tetrasaccharides, in a balanced manner.

[0065] Example 5: pH tolerance of chitosanase To determine the optimal temperature for chitosanase, 50 μL of engineered E. coli BL21 bacteria (109 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 for 15 min at pH=4.5, 5.0, 5.5, 6.0, 6.5, and 7.0, respectively. The corresponding enzyme activities were measured using the DNS method. The enzyme activity measured under the optimal temperature condition was set as 100%, and the relative enzyme activities under other conditions were calculated. The experimental results are as follows: Figure 8 As shown, from Figure 8 As can be seen, the activity of chitosanase GEEGC-CNS-263 was low at pH = 4.5, 5.0, 5.5, and 7.0, while it was higher at pH = 6.0 and 6.5, reaching its peak at pH = 6.0. The optimal pH for chitosanase GEEGC-CNS-263 is 6.0, indicating that the enzyme exhibits the highest catalytic efficiency in a slightly acidic environment and shows good adaptability to neutral to slightly acidic environments (pH = 4-6). This experiment demonstrates that GEEGC-CNS-263 is an enzyme adapted to weakly acidic environments, which facilitates its application in various industrial settings.

[0066] Example 6: Ion tolerance of chitosanase To determine the effect of different ions on chitosanase activity, the ion concentration in the reaction solution was set to 0.5 mmol / L. 50 μL of engineered L. 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 subjected to EDTA and Cu at 45℃ for 15 min (the optimal temperature and pH were determined). 2+ (anhydrous CuSO4), NH4 + ((NH4)2SO4), Co 2+ (CoSO4•7H2O), Fe 2+ (FeSO4•7H2O), Zn 2+ (ZnSO4•7H2O), Cr 3+ (CrCl3•6H2O), Mn 2+ (MnSO4•H2O), Al 3+ (Al2(SO4)3•16H2O), K + (K2SO4), Mg 2+ (MgSO4•7H2O), Fe 3+ (FeCl3•6H2O), Ca 2+The effect of (CaCl₂•2H₂O) plasma on chitosanase activity was investigated using the DNS method to measure the corresponding enzyme activity. The enzyme activity measured under conditions without added ions was taken as the highest percentage baseline, and the relative enzyme activity under other conditions was calculated. Experimental results are as follows: Figure 9 As shown, from Figure 9 As can be seen, the ion that promotes the activity of chitosanase GEEGC-CNS-263 is Mn. 2+ Al 3+ Mg 2+ Equal to making the relative enzyme activity higher than 100% (Mn 2+ The highest concentration (approximately 130%) of the above ions can enhance the catalytic efficiency of enzymes. Ions that inhibit the activity of chitosanase GEEGC-CNS-263 include: Cu. 2+ Fe 3+ When the relative enzyme activity is reduced to below 100% (Cu 2+ The lowest (approximately 24%) of the above ions may inhibit enzyme activity by disrupting enzyme structure or competing for active sites; ions with no significant effect on the enzyme activity of chitosanase GEEGC-CNS-263 include: K + Ca 2+ 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.

[0067] Example 7: Temperature tolerance of chitosanase To determine the optimal temperature for chitosanase, 50 μL of engineered E. 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℃, and 70℃ for 10 min each. The corresponding enzyme activities were measured using the DNS method. The enzyme activity at the optimum temperature was taken as 100%, and the enzyme activities at other temperatures were compared to this value to calculate the relative enzyme activity (%). The experimental results are as follows: Figure 10 As shown, from Figure 10As can be seen, the enzyme activity of chitosanase GEEGC-CNS-263 increases with increasing temperature from 20℃ to 40℃, reaching its peak at 40℃. From 40℃ to 70℃, the enzyme activity gradually decreases with increasing temperature. The optimal temperature for chitosanase GEEGC-CNS-263 is 40℃. The enzyme activity exhibits a single-peak curve with temperature change, slowly decreasing after reaching the optimal temperature. This confirms that GEEGC-CNS-263 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.

[0068] Example 8: Changes in the reaction rate of chitosanase over time To determine the change in chitosanase reaction rate over time, 50 μL of engineered E. coli BL21 bacteria (10 9 The 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 enzyme activity was measured using the DNS method at 1 min, 5 min, 15 min, and 25 min. The experimental results are as follows: Figure 11 As shown, the slope of enzyme activity and time is finally converted into the enzyme conversion rate. It can be seen that the enzyme reaction rate reaches its peak in the early stage (1 min) of the chitosanase GEEGC-CNS-263 reaction; in the later stage of the enzyme reaction (5 min-25 min), the enzyme rate gradually slows down and the slope of the curve decreases.

[0069] The above experiments demonstrate that the present invention successfully screened and designed a novel chitosanase, GEEGC-CNS-263, from extreme environments. Through recombinant expression, purification, and functional verification, it can be seen that this chitosanase has advantages such as novel sequence, high catalytic efficiency, and controllable product. It can solve the problems of single product and low purity in traditional chitosan degradation methods, and provides a reliable tool for the large-scale production of chitosan oligosaccharides with specific degrees of polymerization. It has broad application prospects in the fields of medicine, food, and agriculture.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

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

1.

2. A polynucleotide, characterized in that, The novel chitosanase as described in claim 1 is encoded.

3. A recombinant vector, characterized in that, It contains the polynucleotide as described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector includes either a cloning vector or an expression vector.

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

6. The host cell according to claim 5, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

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

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

1.

9. The application of a novel chitosanase with high catalytic performance in the preparation of chitosan oligosaccharides, characterized in that, The amino acid sequence of the novel chitosanase is shown in SEQ ID NO.1.

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