Chitosanase and its use

Abstract

The application provides a chitosanase and application thereof, and relates to the technical field of enzyme engineering. The amino acid sequence of the chitosanase is shown as SEQ ID NO. 1. The chitosanase prepared by the application can efficiently and controllably catalyze chitosan hydrolysis to generate chitooligosaccharide with a specific polymerization degree. The enzyme is obtained by screening from an extreme environment through metagenomic technology, has a novel sequence, unreported catalytic characteristics 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 for solving the problems of single product and low purity in the traditional method, and lay a technical foundation for the development in the fields of medicine, food and agriculture.

Description

TECHNICAL FIELD

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

[0002] Chitosanase is an enzyme specially used for catalyzing the hydrolysis of beta-1, 4-glycosidic bond of chitosan, and can generate chitooligosaccharide with different degrees of polymerization (DP). Chitooligosaccharide has wide application potential in the fields of medicine, food, agriculture and the like, and its biological activities such as antibacterial activity, antioxidant activity and immunomodulatory activity have been widely studied. However, the traditional chitosan degradation method is difficult to realize the controllable production of chitooligosaccharide, 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 chitooligosaccharide 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 catalytic dimers 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] Chitooligosaccharide has attracted much attention due to its unique biological activity, 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 chitooligosaccharide 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 chitosanase and application thereof. The present application provides chitosanase, which can produce chitooligosaccharide with different degrees of polymerization through modification of chitosanase, and the prepared chitosanase provides a new opportunity for the application in the fields of medicine, food and agriculture. Meanwhile, the present application combines the existing molecular biology and enzyme engineering technology, and will lay a foundation for the industrial production of chitooligosaccharide.

[0006] In order to achieve the purpose of the present application, the following technical scheme is adopted:

[0007] The present application provides a chitosanase, and the amino acid sequence of the chitosanase is shown in SEQ ID NO. 1.

[0008] SEQ ID NO. 1

[0009] MKLLRINFPI ALLFALAAVP ARGQAWEQVK AAAGGDKAPE AA VPAVAAARP AAEGLSPEQ KRRADMLISV FENSTLELQY AYVENLHDGR GYTAGRAGFC SGCGDLLLVA ERYARLKPGN ALVKFLPRLR ELARLASAST KGLAGFPAA WAAAARDPLF RGAQDAVSDE LY YMPALAYA GQLGLTKDFS RIALYEASIQ HGLGEDPDGL PSMIRKASKA AKPPAAGGDE KAWLNEFLKV RRAALAYAAD PATRKEWAES VGRADAMLA IAAAGNLDF AGPVAVNPYG GQFTIP

[0010] The present application also provides a polynucleotide encoding the chitosanase.

[0011] The present application also provides a recombinant vector comprising the polynucleotide.

[0012] Further, the recombinant vector comprises any one of a cloning vector or an expression vector.

[0013] The present application also provides a host cell comprising the polynucleotide or the recombinant vector.

[0014] Further, the host cell is a prokaryotic cell or a eukaryotic cell.

[0015] The present application also provides a recombinant strain comprising the polynucleotide or the recombinant vector.

[0016] The present application also provides a biocatalyst comprising the chitosanase with the amino acid sequence as shown in SEQ ID NO. 1.

[0017] The present application also provides the use of the chitosanase in the preparation of chitooligosaccharides.

[0018] The present application has the following technical effects:

[0019] The chitosanase prepared by the present application can efficiently and controllably catalyze the hydrolysis of chitosan to generate chitooligosaccharides with specific polymerization degrees. The enzyme is obtained by metagenomic technology from an extreme environment, has a novel sequence, unreported catalytic properties, and can precisely regulate the product composition, meeting the preparation requirements of specific high-purity chitooligosaccharides. These characteristics make the enzyme have important application value in the large-scale production of chitooligosaccharides, 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

[0020] 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 description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0021] Figure 1 Sequence alignment of chitosanase EluCsn-46A-2 after removing signal peptide and reference sequence BsCsn46A;

[0022] Figure 2 Structure of chitosanase EluCsn-46A-2 predicted by AlphaFold3;

[0023] Figure 3 Nucleic acid electropherogram of chitosanase EluCsn-46A-2;

[0024] Figure 4 Crude protein map of chitosanase EluCsn-46A-2;

[0025] Figure 5 Pure protein map of chitosanase EluCsn-46A-2;

[0026] Figure 6 Standard curve and fitting equation of chitosanase EluCsn-46A-2;

[0027] Figure 7 DNS color reaction of chitosanase EluCsn-46A-2, wherein the left graph is a blank control and the right graph is chitosanase EluCsn-46A-2;

[0028] Figure 8 TLC product spectrum of chitosanase EluCsn-46A-2;

[0029] Figure 9 Effect of different pH values on the enzyme activity of chitosanase EluCsn-46A-2;

[0030] Figure 10 Effect of different ions on the enzyme activity of chitosanase EluCsn-46A-2;

[0031] Figure 11 Effect of different temperatures on the enzyme activity of chitosanase EluCsn-46A-2;

[0032] Figure 12 Reaction rate of chitosanase EluCsn-46A-2 versus time graph. Detailed Implementation

[0033] 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.

[0034] In a first aspect, the present invention provides a chitosanase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0035] SEQ ID NO.1

[0036] MKLLRINFPIALLFALAAVPARGQAWEQVKAAAGGDKAPEAAVPAVAAARPAAEGLSPEQKRRADMLISVFENSTLELQYAYVENLHDGRGYTAGRAGFCSGCGDLLLVAERYARLKPGNALVKFLPRLRELARLASASTKGLAGF PAAWAAAARDPLFRGAQDAVSDELYYMPALAYAGQLGLTKDFSRIALYEASIQHGLGEDPDGLPSMIRKASKAAKPPAAGGDEKAWLNEFLKVRRAALAYAADPATRKEWAESVGRADAMLAIAAAGNLDFAGPVAVNPYGGQFTIP

[0037] Secondly, the present invention provides a polynucleotide encoding the above-mentioned chitosanase.

[0038] Thirdly, the present invention provides a recombinant vector comprising the above-mentioned polynucleotides.

[0039] In some embodiments, the recombinant vector includes either a cloning vector or an expression vector.

[0040] Fourthly, the present invention also provides a host cell comprising the above-mentioned polynucleotide or recombinant vector.

[0041] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0042] Fifthly, the present invention also provides a recombinant strain comprising the above-mentioned polynucleotide or recombinant vector.

[0043] 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.

[0044] In a seventh aspect, the present invention also provides the application of the above-mentioned chitosanase in the preparation of chitosan oligosaccharides.

[0045] The following is a detailed explanation using specific embodiments:

[0046] Example 1

[0047] SRR13189623, originating from the extreme environment of an acidic mine, was downloaded from the NCBI public database. TrimGalore was used for quality control to remove adapter sequences and low-quality reads (quality score <20), yielding clean reads. MEGAHIT was then used to assemble the clean reads. The protein sequence was obtained by predicting open reading frames using the prodigal tool. The protein sequence is: MKLLRINFPIALLFALAAVPARGQAWEQVKAAAGGDKAPEAAVPAVAAARPAAEGLSPEQKRRADMLISVFENSTLELQYAYVENLHDGRGYTAGRAGFCSGCGDLLLVAERYARLKPGNALVKFLPRLRELARLASASTKGLAGFPAAWAAAARDPLFRGAQDAVSDELYYMPALAYAGQLGLTKDFSRIALYEASIQHGLGEDPDGLPSMIRKASKAAKPPAAGGDEKAWLNEFLKVRRAALAYAADPATRKEWAESVGRADAMLAIAAAGNLDFAGPVAVNPYGGQFTIP*

[0048] Diamond alignment yielded a sequence with a similarity of 51.3%, most similar to the NCBI public database: MAG: chitosanase, partial [Phycisphaerales bacterium] GenBank: MCA9253620.1. AlphaFold3 predicted its structure, but its related enzyme activity and catalytic characteristics had not been previously reported. Figure 1 This is a sequence alignment diagram of chitosanase EluCsn-46A-2 with the reference sequence BsCsn46A; the structure of chitosanase EluCsn-46A-2 is shown below. Figure 2 As shown, the low sequence similarity indicates that EluCsn-46A-2 is a highly novel chitosanase, likely possessing unique structure and function.

[0049] Example 2

[0050] Through metagenomic comparison and screening, a chitosanase capable of degrading chitosan was identified and named EluCsn-46A-2. The chitosanase gene sequence was synthesized after removing the signal peptide using SignalP software and used to construct a plasmid. Experiments were conducted on the constructed plasmid to determine its successful construction; the experimental results are as follows. Figure 3 As shown.

[0051] I. Plasmid Transformation

[0052] 1. Plasmid pretreatment

[0053] Centrifuge the recombinant plasmid solution at 12,000×g for 1 min at 4℃ and discard the supernatant.

[0054] Add 20 μL of sterile ultrapure water (DNase / RNase-free) to fully resuspend the plasmid precipitate.

[0055] 2. Thermal shock conversion

[0056] 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°C for 45 s (avoid excessive cell damage), then immediately incubate on ice for 2 min.

[0057] Add 900 μL of sterile LB medium (antibiotic-free) and incubate at 37°C and 220 rpm for 1 hour with shaking.

[0058] 3. Coating screening

[0059] After centrifugation (5,000×g, 5 min), discard 900 μL of supernatant and retain approximately 100 μL of resuspended bacterial cells.

[0060] Spread all bacterial culture onto LB agar plates containing 50 μg / mL kanamycin and incubate upside down at 37°C for 12-16 h.

[0061] II. Validation of Positive Clones

[0062] 1. Colony PCR

[0063] (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.

[0064] (2) PCR system (20 μL): 2×TaqMasterMix: 10 μL, forward / reverse primers (10 μM): 1 μL each, template: 1 μL, sterile water: 7 μL

[0065] (3) Reaction program: Pre-denaturation: 95 ℃ for 15 min, Cycling: 95 ℃ for 30 s → 60 ℃ for 30 s → 72 ℃ for 1 min / kb, for a total of 30 cycles, Final extension: 72 ℃ for 5 min

[0066] (4) Electrophoresis verification:

[0067] Take 5 μL of PCR product and perform 1.5% agarose gel electrophoresis (120V, 20min), and confirm the target band by UV imaging.

[0068] (5) After the sequencing verification is correct, select the positive clones in step (1), inoculate them into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, culture at 37℃ and 200 rpm for 12-16 h, and store them in 25% glycerol at -80℃.

[0069] III. Fermentation-induced expression

[0070] (1) Add 50 μL of glycerol bacteria to 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture at 37°C and 200 rpm for 12-16 h with shaking. At the same time, set up a negative control (E. coli BL21 competent cells can be selected), add 50 μL of competent cell solution to 5 mL of LB liquid medium without 50 μg / mL kanamycin, and culture at 37°C and 200 rpm for 12-16 h with shaking.

[0071] (2) Transfer 5 mL of bacterial culture at a 1:50 inoculation rate to 50 mL of LB medium (containing 50 μg / mL kanamycin) and incubate at 37℃ and 200 rpm until OD600 = 0.6-0.8 (approximately 2-3 hours).

[0072] Add IPTG to a final concentration of 0.3 mM (0.1~1 mM), transfer to 16℃-17℃, and induce at 160 rpm for 16-18 h.

[0073] IV. Protein Extraction and Detection

[0074] 1. Bacterial cell collection and lysis

[0075] 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.

[0076] Cell disruption was performed using the hammer enzyme kit (refer to the instructions). The resulting supernatant (crude enzyme solution) was obtained from the hammer enzyme disruption.

[0077] 2. SDS-PAGE analysis

[0078] (1) Collect the supernatant and precipitate of the hammer crushing liquid. Resuspend the precipitate in 1 mL of 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.

[0079] (2) Centrifuge at 12000 rpm for 10 min. Use the supernatant for sample loading. The loading order is: protein marker - fermentation broth supernatant - lysis broth supernatant - lysis broth precipitate.

[0080] (3) Gel preparation method: Refer to the 12.5% ​​SDS-PAGE denaturing acrylamide color gel rapid preparation kit.

[0081] (4) Electrophoresis conditions are low voltage gel pressing (80V constant voltage until bromophenol blue enters the separating gel): 12.5% ​​separating gel, then run the separating gel at 120V. When the blue band reaches the bottom, electrophoresis for about 90 minutes, then stop running the gel.

[0082] Staining and destaining: Stain with Coomassie Brilliant Blue R-250 for 30 min, then destain with a destaining solution (10% acetic acid + 40% ethanol) until the background is transparent to obtain a gel image of the crude chitosanase EluCsn-46A-2 protein. The experimental results are as follows: Figure 4 As shown, the crude protein molecular weight of chitosanase EluCsn-46A-2 is between 33 and 25, close to 29, which is consistent with the expected molecular weight (29.30), proving that chitosanase EluCsn-46A-2 has been correctly expressed. This experiment demonstrates that this gene can be solublely expressed in the *E. coli* system and can be efficiently purified by affinity chromatography, providing a guarantee for subsequent research.

[0083] 4. Protein purification

[0084] 1. Sample Preparation

[0085] Collect the supernatant and precipitate from the hammer crushing fluid, and resuspend the precipitate in 1 mL of buffer solution.

[0086] 2. Magnetic bead pretreatment

[0087] Balanced magnetic beads:

[0088] Take an appropriate amount of Ni-NTA magnetic beads (according to the manufacturer's recommended ratio), place them on a magnetic rack for 1 minute to adsorb, and then discard the storage solution.

[0089] Wash three times with binding buffer at a volume of 5 times that of the magnetic beads, discarding the supernatant after each 1-minute adsorption.

[0090] 3. Protein binding

[0091] 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.

[0092] 4. Wash away contaminating proteins

[0093] 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.

[0094] 5. Target protein elution

[0095] Add elution buffer equal to the volume of the magnetic beads and incubate at room temperature with shaking for 10-15 minutes.

[0096] Adsorbed on a magnetic rack for 1 min, the eluent (containing His-tagged protein) was collected.

[0097] Repeat the elution process 1-2 times and combine the eluents. This yields a pure enzyme solution.

[0098] 5. SDS-PAGE analysis

[0099] (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.

[0100] (2) Centrifuge at 12000 rpm for 10 min. Use the supernatant for sample loading. The loading order is: protein marker - lysate supernatant - lysate precipitate.

[0101] (3) Gel preparation method: Refer to the 12.5% ​​SDS-PAGE denaturing acrylamide color gel rapid preparation kit.

[0102] (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.

[0103] Staining and destaining: Coomassie Brilliant Blue R-250 staining for 30 min, followed by destaining with a destaining solution (10% acetic acid + 40% ethanol) until the background is transparent to obtain a gel image of the purified chitosanase EluCsn-46A-2 protein. The experimental results are as follows. Figure 5 As shown, we can see that the molecular weight of the purified chitosanase EluCsn-46A-2 protein is between 33 and 25, close to 29, which is consistent with the expected molecular weight (29.30), proving that chitosanase EluCsn-46A-2 has been correctly expressed.

[0104] Example 3

[0105] Chitosanase activity assay

[0106] Determination of reducing sugar content in hydrolysate

[0107] 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, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 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.

[0108] Fitting equation: With concentration as the x-axis (x, mg / mL) and OD value as the y-axis, a linear regression equation y = ax + b is fitted, R0 2 ≥0.99, enzyme activity was calculated based on the standard curve and the fitted equation, as shown in the figure. Figure 6 As shown.

[0109] Enzyme activity assay (DNS colorimetric method): 1 g of chitosan was hydrolyzed with 200 mM pH 6.5 acetate-sodium acetate buffer to obtain a 1% chitosan solution. The crude or purified enzyme solution was diluted appropriately with distilled water. 100 μL of the diluted enzyme solution was added to 200 μL of the 1% chitosan solution, and the volume was adjusted to 1 mL with acetate-sodium acetate buffer. The mixture was thoroughly mixed and reacted in a 45℃ water bath for 20 min. The reaction was terminated by adding 2 mL of DNS solution. After boiling for 5 min to develop color, the volume was adjusted to 5 mL with distilled water. The mixture was centrifuged at 12000 rpm for 5 min, and the absorbance was measured at 540 nm. Each group was tested in triplicate. The blank control consisted of inactivated enzyme. The experimental procedure was as follows: Figure 7 As shown, the left image is the blank control, and the right image is the chitosanase EluCsn-46A-2. Enzyme activity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per milliliter of enzyme solution per minute at 40°C. The final enzyme activity of chitosanase EluCsn-46A-2 was measured to be 43.42 U / g.

[0110] Example 4: Catalytic characteristics of chitosanase

[0111] Thin-layer chromatography (TLC):

[0112] Add 50 μL of purified enzyme solution to 950 μL of 1% chitosan solution and react for 1 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 6 h, 12 h, 24 h, and 48 h, respectively. After centrifugation at 8000 rpm for 10 min, collect the supernatant and spot it directly onto a silica gel plate using a capillary tube (0.50 cm apart, 1.0 cm from the bottom). Use 5% glucosamine and chitosan oligosaccharide aqueous solutions as standards. Develop the solution in a chromatography tank using isopropanol:ammonia:water = 15:7.5:4 V / V / V. After development, dry the solution 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 EluCsn-46A-2 is shown below. Figure 8 As shown, within 48 hours, the enzymatic hydrolysis products of chitosanase EluCsn-46A-2 were consistently dominated by (GlcN)2-(GlcN)5, with (GlcN)2-(GlcN)4 as the core product. No large amount of monosaccharides (glucosamine) were generated. With the extension of reaction time, the proportion of high DP products (such as (GlcN)5) decreased significantly, while the proportion of low DP products (such as (GlcN)2-(GlcN)3) increased slightly. However, the overall product composition remained stable, proving that the enzyme can precisely regulate the product DP to meet the high-efficiency preparation requirements of specific chitosan oligosaccharides (such as (GlcN)2-(GlcN)4).

[0113] This experiment demonstrates that chitosanase EluCsn-46A-2 exhibits a precise endonuclease pattern and excellent product controllability, enabling stable production of chitosan oligosaccharides within a specific range. This solves the technical problem of product heterogeneity and has extremely high application value.

[0114] Example 5: pH tolerance of chitosanase

[0115] 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 reaction was carried out for 15 min at pH=2, 3, 4, 5, and 6, respectively. The corresponding enzyme activities were measured using the DNS method. The enzyme activity values ​​measured under the optimal temperature conditions were taken as the quotient standard, and the relative enzyme activities under other conditions were calculated. The experimental results are as follows: Figure 9 As shown, from Figure 9As can be seen, the activity of chitosanase EluCsn-46A-2 is low at pH=2 and 3, and high at pH=4, 5, and 6. Furthermore, the activity gradually increases with increasing pH, reaching its peak at pH=6. The optimal pH for chitosanase EluCsn-46A-2 is 6, indicating that this enzyme has the highest catalytic efficiency in a slightly acidic environment and good adaptability to neutral to slightly acidic environments (pH 4-6). This experiment demonstrates that EluCsn-46A-2 is an enzyme adapted to weakly acidic environments, which facilitates its application in various industrial settings.

[0116] Example 6: Ion tolerance of chitosanase

[0117] To determine the effect of different ions on chitosanase activity, the ion concentration in the reaction solution was adjusted to 0.5 mmol / L. 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). Mn was detected at 45℃ for 15 min (the optimal temperature and pH were determined). 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+ (CaCl2•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 EluCsn-46A-2 is Mn. 2+ Co 2+ Fe 3+Enhancing the relative enzyme activity to over 100% (up to approximately 130%) can improve the enzyme's catalytic efficiency.

[0118] Ions that inhibit the activity of chitosanase EluCsn-46A-2 include: Ca... 2+ NH4 + Ni 2+ 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 EluCsn-46A-2 include: Al 3+ Cu 2+ When the relative enzyme activity is close to 100%, the enzyme exhibits strong tolerance. This experiment clarified the influence of different metal ions on enzyme activity, providing an important basis for selecting appropriate reaction conditions in practical applications.

[0119] Example 7: Temperature tolerance of chitosanase

[0120] 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℃, 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 EluCsn-46A-2 increases with increasing temperature from 20℃ to 50℃, reaching its peak at 50℃. From 50℃ to 80℃, the activity gradually decreases with increasing temperature. The optimal temperature for chitosanase EluCsn-46A-2 is 50℃; it is easily inactivated above 50℃. This confirms that EluCsn-46A-2 is a thermophilic enzyme. The optimal temperature of 50℃ is higher than that of many room-temperature enzymes, which is beneficial for accelerating the reaction rate at higher temperatures and may reduce microbial contamination.

[0121] Example 8: Changes in the reaction rate of chitosanase over time

[0122] 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, 10 min, 15 min, and 25 min. The experimental results are as follows: Figure 12 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 EluCsn-46A-2 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.

[0123] The above experiments demonstrate that this invention successfully screened a chitosanase, EluCsn-46A-2, 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.

[0124] 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. Chitosanase, 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 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, Chitosanase containing the amino acid sequence shown in SEQ ID NO.

1.

9. The use of the chitosanase as described in claim 1 in the preparation of chitosan oligosaccharides.

Images