Chitosanase based on structure-oriented design and application thereof

Abstract

The application provides a chitosanase based on structure-oriented design and an application thereof, and relates to the technical field of enzyme engineering. The amino acid sequence is shown as SEQ ID NO. 1. The enzyme is derived from the modification of a high-salt extreme environment metagenome, is obtained based on structure-oriented design, has unique substrate recognition and catalytic characteristics, can accurately regulate a hydrolysis site, and can directionally prepare di-, tri- and penta-chitosan oligosaccharides. The product composition is controllable. The problems of insufficient substrate recognition accuracy of the existing chitosanase, uncontrollable product polymerization degree and difficulty in directionally preparing specific high-activity chitosan oligosaccharides are solved.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a chitosanase based on structure-guided design 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, existing chitosanases have insufficient substrate recognition accuracy, uncontrollable product polymerization degree, and difficulty in the targeted preparation of specific highly active chitosan oligosaccharides. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a chitosanase based on structure-guided design and its applications. This solves the problems of insufficient substrate recognition accuracy, uncontrollable product polymerization degree, and difficulty in the targeted preparation of specific highly active chitosan oligosaccharides in existing chitosanases.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a chitosanase based on structure-guided design, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] SEQ ID NO.1 VTQTTNDIYPKALQITTTLENSDTNLQYNYCSFLDDNRGYTFGAVGFTTGTYDGRRLISYYTELNPSNALAKYLPALAKIDAAPHTYNGGDSNPNIDGLDGFKETVEGINDPLWKEAQLYMIKEEYWNSAVSAWNEIGANYELTLAFLYDTNVRLGKDGM QSVISSATLACDGTPANGVDESTYLSELLKARNVKLRNEGLGDVNRNAGFLLLLGSGNVNLNTPFTFVAYGDEFTIMNELNTTTFIPVPELKSEPMPVYFPSTPTIPAVLPTTTPTDVVPIPTEIVTTTKSTAQESLYSLLSRASSQNNPSTLITFLSRNV The present invention also provides a polynucleotide encoding the above-mentioned 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 biocatalyst comprising chitosanase as shown in SEQ ID NO.1, which is an amino acid sequence as described above.

[0013] The present invention also provides an application of a structure-guided design chitosanase in the preparation of chitosan oligosaccharides, wherein the amino acid sequence of the chitosanase is shown in SEQ ID NO.1.

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

[0015] The present invention has the following technical effects: Based on the structural heterogeneity analysis of glycoside hydrolases (such as GH46 catalyzing binary inversion and GH75 configuration reversal), this paper addresses the randomness of hydrolysis caused by the excessive flexibility of the substrate binding pocket in existing chitosanases. It proposes a structure-guided chitosanase and its applications. This enzyme optimizes the steric hindrance and hydrophobicity of the catalytic microenvironment through precise substitution of amino acid residues in key regions, significantly enhancing the enzyme's specificity for chitosan chain length recognition. This transforms traditional broad-spectrum hydrolysis into site-directed cleavage of specific β-1,4-glycosidic bonds, enabling controllable conversion from a broadly distributed mixture to high-purity chitosan oligosaccharides with specific degrees of polymerization. It can produce chitosan oligosaccharides with specific degrees of polymerization. Existing technologies for chitosanases suffer from problems such as a wide degree of polymerization distribution, low yield of target dimer to trimer chitosan oligosaccharides, and difficulty in meeting the requirements for high-purity large-scale preparation. This provides reliable technical support for refined production in the pharmaceutical, food, and agricultural fields. 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-64 predicted by AlphaFold3; Figure 2 Electrophoresis image of chitosanase GEEGC-CNS-64; Figure 3 Crude protein diagram of chitosanase GEEGC-CNS-64; Figure 4 Image of purified protein of chitosanase GEEGC-CNS-64; Figure 5 Standard curve and fitting equation of chitosanase GEEGC-CNS-64; Figure 6 The DNS colorimetric reaction of chitosanase GEEGC-CNS-64, where the left figure is the blank control and the right figure is chitosanase GEEGC-CNS-64; Figure 7 TLC spectrum of chitosanase GEEGC-CNS-64 products; Figure 8 Effects of different pH values ​​on the activity of chitosanase GEEGC-CNS-64; Figure 9 Effects of different ions on the activity of chitosanase GEEGC-CNS-64; Figure 10 Effects of different temperatures on the activity of chitosanase GEEGC-CNS-64; Figure 11 : Graph showing the change in reaction rate of chitosanase GEEGC-CNS-64 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 chitosanase based on structure-guided design, wherein the amino acid sequence of the chitosanase is shown in SEQ ID NO.1.

[0020] SEQ DI NO.1 VTQTTNDIYPKALQITTTLENSDTNLQYNYCSFLDDNRGYTFGAVGFTTGTYDGRRLISYYTELNPSNALAKYLPALAKIDAAPHTYNGGDSNPNIDGLDGFKETVEGINDPLWKEAQLYMIKEEYWNSAVSAWNEIGANYELTLAFLYDTNVRLGKDGM QSVISSATLACDGTPANGVDESTYLSELLKARNVKLRNEGLGDVNRNAGFLLLLGSGNVNLNTPFTFVAYGDEFTIMNELNTTTFIPVPELKSEPMPVYFPSTPTIPAVLPTTTPTDVVPIPTEIVTTTKSTAQESLYSLLSRASSQNNPSTLITFLSRNV Secondly, the present invention provides a polynucleotide encoding the above-mentioned 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 an application of a structure-guided design-based chitosanase in the preparation of chitosan oligosaccharides, wherein the amino acid sequence of the chitosanase is shown in SEQ ID NO.1.

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

[0029] The following is a detailed explanation using specific embodiments: Example 1 Based on the metagenomic sample CRR807418 from a high-salt environment, the target protein was synthesized and optimized after removing the signal peptide using SignalP software, and 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 target protein is named GEEGC-CNS-64, and "64" in the figure refers to "GEEGC-CNS-64". The protein sequence is VTQTTNDIYPKALQITTTLENSDTNLQYNYCSFLDDNRGYTFGAVGFTTGTYDGRRLISYYTELNPSNALAKYLPALAKIDAAPHTYNGGDSNPNIDGLDGFKETVEGINDPLWKEAQLYMIKEEYWNSAVSAWNEIGANYELTLAFLYDTNVRLGKDGMQSVISSATLACDGTPANGVDESTYLSELLKARNVKLRNEGLGDVNRNAGFLLLLGSGNVNLNTPFTFVAYGDEFTIMNELNTTTFIPVPELKSEPMPVYFPSTPTIPAVLPTTTPTDVVPIPTEIVTTTKSTAQESLYSLLSRASSQNNPSTLITFLSRNV.

[0030] The protein has a maximum sequence similarity of 51.76% 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 ℃ 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℃. 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.

[0040] (3) PCR reaction program: pre-denaturation: 95 ℃ for 15 min, cycling: 95 ℃, 30s→60 ℃, 30s→72 ℃, 1 min / kb, a total of 30 cycles, final extension: 72 ℃ for 5 min.

[0041] (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.

[0042] (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℃.

[0043] 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℃ and 200 rpm for 12-16 h with shaking. 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℃ and 200 rpm for 12-16 h with shaking.

[0044] (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).

[0045] (3) Add IPTG to a final concentration of 0.3 mM (0.1~1 mM), transfer to 16-17℃ and 160 rpm for induction for 16-18 h.

[0046] 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.

[0047] Cell disruption was performed using the Hammer Reduction Kit (ACE BR0005). The resulting supernatant (crude enzyme solution) was obtained from the Hammer Reduction Kit.

[0048] 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.

[0049] (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.

[0050] (3) Gel preparation method: Refer to the 12.5% ​​SDS-PAGE denatured acrylamide color gel rapid preparation kit.

[0051] (4) Low voltage gel pressing (80 V constant voltage until bromophenol blue enters the separating gel), then run the separating gel at 120 V, and then run the separating gel at 120 V until the blue band reaches the bottom. After about 90 minutes of electrophoresis, stop running the gel.

[0052] (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 gel image of crude chitosanase GEEGC-CNS-64 protein. The experimental results are as follows: Figure 3 As shown, the molecular weight of the crude chitosanase GEEGC-CNS-64 protein is between 40 and 33, which is consistent with the expected molecular weight (35.99), proving that chitosanase GEEGC-CNS-64 has been correctly expressed.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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 beads. Attach to a magnetic rack for 1 min, then discard the supernatant.

[0057] 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.

[0058] Adsorbed on a magnetic rack for 1 min, the eluent (containing His-tagged protein) was collected.

[0059] Repeat the elution process 1-2 times and combine the eluents. This yields a pure enzyme solution.

[0060] 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) The electrophoresis conditions are low voltage gel pressing (80V constant voltage until bromophenol blue enters the separating gel), followed by 120V running of the separating gel. When the blue band reaches the bottom, electrophoresis is performed for about 90 minutes, and then the gel running is stopped.

[0061] (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 pure chitosanase GEEGC-CNS-64 protein was obtained. The experimental results are as follows: Figure 4 As shown, the molecular weight of the purified chitosanase GEEGC-CNS-64 protein is between 40 and 33, which is consistent with the expected molecular weight (35.99), proving that chitosanase GEEGC-CNS-64 has been correctly expressed. The experiment confirms that the gene can be effectively expressed and purified to produce an active chitosanase, providing a material basis for subsequent functional studies.

[0062] 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, 1.0, and 1.5 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.

[0063] Fitting equation: Plotting concentration (X, mg / mL) on the x-axis and OD value on the y-axis, the fitted linear regression equation is Y = 1.915X - 0.04749, R0. 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.

[0064] 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-64. 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-64 was measured to be 1.0 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.

[0065] Example 4: Catalytic characteristics of chitosanase Thin-layer chromatography (TLC): Add 50 μL of purified enzyme solution to 950 μL of 1% chitosan solution. After reacting for about 1 h, centrifuge at 8000 rpm for 10 min and collect the supernatant. Spot the supernatant directly onto a silica gel plate using a capillary tube (5 mm apart, 10 mm from the bottom). Use 5% glucosamine and chitosan oligosaccharide aqueous solutions as standards. Develop in a chromatography tank using isopropanol:ammonia:water = 15:7.5:4 V / V / V, ammonia concentration 25%. After development, dry the plate and 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-64 is shown below. Figure 7 As shown, the enzymatic hydrolysis products of chitosanase GEEGC-CNS-64 are mainly (GlcN)2, (GlcN)3, and (GlcN)5, exhibiting a broad product spectrum and numerous high-value products. This enzyme employs a classic endo-glucanase hydrolysis method, 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.

[0066] Example 5: pH tolerance of chitosanase To determine the optimal temperature for chitosanase, 50 μL of engineered E. coli BL21 bacteria (10 9A 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-64 continuously increases with increasing pH, reaching its maximum at pH 7.0. Therefore, the optimal pH for chitosanase GEEGC-CNS-64 is 7.0, indicating that this enzyme exhibits the highest catalytic efficiency under neutral conditions and good adaptability under acidic conditions (pH 4.5-7.0). This experiment demonstrates that GEEGC-CNS-64 is an enzyme adapted to neutral environments, which facilitates its application in various industrial settings.

[0067] 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-64 is Mn. 2+ Equivalents, such as Fe, can increase the relative enzyme activity to over 100% (up to approximately 120%), thereby improving the enzyme's catalytic efficiency. Ions that inhibit the activity of chitosanase GEEGC-CNS-64 include Fe. 2+ Fe 3+ etc., so that the relative enzyme activity is lower than 100% (Fe 3+ The lowest (approximately 40%) effect may be achieved by disrupting the enzyme structure or competing for active sites to inhibit enzyme activity; ions with no significant effect on the activity of chitosanase GEEGC-CNS-64 include: EDTA and NH4+. + The relative enzyme activity is close to 100%, indicating strong enzyme tolerance. This experiment shows that GEEGC-CNS-64 has strong tolerance to most metal ions and high overall enzyme activity, providing an important reference for selecting appropriate reaction conditions (such as buffer composition) in practical applications.

[0068] 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 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ 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 10 As can be seen, the enzyme activity of chitosanase GEEGC-CNS-64 is relatively low at 30℃, and reaches its peak at 40℃. Between 50℃ and 80℃, the enzyme activity of chitosanase GEEGC-CNS-64 decreases with increasing temperature. The optimal temperature for chitosanase GEEGC-CNS-64 is 40℃. This confirms that GEEGC-CNS-64 is a room-temperature enzyme, and its optimal temperature is close to that of most enzymes. This is beneficial for accelerating the reaction rate at around 40℃ and may reduce contamination by other microorganisms.

[0069] 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 9The 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-64 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.

[0070] The above experiments demonstrate that this invention successfully screened a chitosanase, GEEGC-CNS-64, 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, providing 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.

[0071] 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 chitosanase based on structure-guided design, characterized in that, The amino acid sequence of the chitosanase is shown in SEQ ID NO.

1.

2. A polynucleotide, characterized in that, The 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 contains 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, Chitosanase containing the amino acid sequence shown in SEQ ID NO.

1.

9. The application of a structure-guided design-based chitosanase in the preparation of chitosan oligosaccharides, characterized in that, The amino acid sequence of the chitosanase is shown in SEQ ID NO.1; The chitosan oligosaccharide is chitobiose, chitotriose, or chitopentose.

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