Chitosanase mutant CsnW27-HF, plasmid, recombinant bacteria and application thereof

Abstract

The present application relates to a kind of chitosanase mutant CsnW27-HF, plasmid, recombination bacteria and its application, belong to enzyme engineering technical field, the amino acid sequence of the chitosanase mutant as shown in SEQ ID NO:1, the amino acid sequence of wild-type chitosanase CsnW27 as shown in SEQ ID NO:2.Compared with wild-type chitosanase CsnW27, the 82th and 99th amino acid are subjected to site-directed mutagenesis.The present application also provides plasmid and recombination engineering bacteria comprising the chitosanase mutant CsnW27-HF gene, the enzyme activity of the mutant is increased by 2.36 times compared with wild type.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a chitosanase mutant CsnW27-HF, plasmid, recombinant bacteria and its applications. Background Technology

[0002] Chitosan oligosaccharides are the only naturally occurring cationic basic oligosaccharides, obtained from chitosan through enzymatic or chemical degradation. Their degree of polymerization is mostly concentrated between 2 and 20 glucosamine units. Compared to traditional polysaccharides like chitin and chitosan, chitosan oligosaccharides not only possess excellent water solubility but also exhibit various physiological functions such as antibacterial activity, immunomodulation, and metabolic improvement. Furthermore, their high biocompatibility and good environmental compatibility have made them important raw materials for functional foods, pharmaceuticals, and biomaterials.

[0003] Chitosanase (EC 3.2.1.132), a class of hydrolases capable of efficiently and specifically cleaving the β-1,4-glycosidic bonds of chitosan, is widely found in microorganisms and plant tissues. This enzyme is distributed across multiple families in the glycosidic hydrolase classification system, including GH5, GH7, GH8, GH46, GH75, and GH80. Notably, the GH46, GH75, and GH80 families are unique to chitosanases, exhibiting high specificity for chitosan substrates. Among these, the three-dimensional structure and catalytic mechanism of GH46 family enzymes have been studied most systematically, providing a theoretical basis for subsequent enzyme molecular modification.

[0004] The decomposition of chitosan by chitosanase to prepare chitosan oligosaccharides is considered an important route for green biomanufacturing. However, this process still faces significant bottlenecks in its industrialization: existing chitosanases generally have low catalytic efficiency, poor thermal and operational stability, and produce enzymatic hydrolysates with a wide degree of polymerization, making it difficult to achieve controllable synthesis of the target oligosaccharides. Therefore, continuously exploring novel chitosanase resources and improving their catalytic performance remains a key step in promoting the development of this technology. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a chitosanase mutant CsnW27-HF, plasmid, recombinant bacteria and its application. By modifying the key amino acid residues in chitosanase that affect its catalytic activity, the catalytic activity of chitosanase is improved, laying the foundation for its industrial application.

[0006] This invention is achieved through the following technical solution:

[0007] A chitosanase mutant, CsnW27-HF, has the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence of wild-type chitosanase CsnW27 is shown in SEQ ID NO:2. Compared to wild-type chitosanase CsnW27, site-directed mutations were made at amino acids 82 and 99.

[0008] Furthermore, the wild-type chitosanase CsnW27 of the chitosanase CsnW27-HF is derived from... Paenibacillus hamazuiensis (GenBank ID: WP_275983455.1).

[0009] The present invention also provides a gene encoding the chitosanase mutant CsnW27-HF, the nucleotide sequence of which is shown in SEQ ID NO.3, and the nucleotide sequence of wild-type chitosanase CsnW27 is shown in SEQ ID NO:4.

[0010] The present invention also provides a recombinant plasmid carrying the gene shown in SEQ ID NO.3, and the expression vector is preferably pET-28a(+).

[0011] This invention also provides a recombinant engineered strain obtained by transformation containing the above-mentioned recombinant plasmid, wherein the preferred expression host is... E. coli BL21(DE3).

[0012] The present invention also provides an enzyme preparation containing the above-mentioned chitosanase mutant CsnW27-HF.

[0013] The present invention also provides the application of the chitosanase mutant CsnW27-HF in the preparation of chitosan oligosaccharides.

[0014] The beneficial effects of this invention compared to the prior art are as follows:

[0015] This invention addresses the low catalytic activity of existing chitosanases. Starting with CsnW27, multiple sites potentially affecting its catalytic activity were mutated, resulting in a double mutant, CsnW27-HF, where arginine at position 82 and tyrosine at position 99 were mutated to histidine and phenylalanine, respectively. The resulting mutant exhibited 2.36-fold increased enzyme activity compared to the wild type, placing it at a relatively high level among currently available chitosanases. This discovery and the resulting mutant with enhanced catalytic activity lay the foundation for the development, modification, and industrial application of chitosanases. Attached Figure Description

[0016] Figure 1 Figure 1 shows the effect of temperature on the activity of the chitosanase mutant CsnW27-HF.

[0017] Figure 2 The graph shows the effect of pH on the activity of the chitosanase mutant CsnW27-HF.

[0018] Figure 3 Figure 1 shows the effect of temperature on the stability of the chitosanase mutant CsnW27-HF enzyme.

[0019] Figure 4 Figure showing the effect of pH on the stability of the chitosanase mutant CsnW27-HF enzyme;

[0020] Figure 5 This is a graph showing the analysis of the hydrolysis products of chitosan by the chitosanase mutant CsnW27-HF. Detailed Implementation

[0021] The method of the present invention will be further described below with reference to the accompanying drawings and embodiments. However, the experimental conditions used in the embodiments can be selected based on existing technologies. For experimental methods where specific conditions are not specified in the embodiments, they can generally be operated under conventional conditions or according to the conditions recommended by the manufacturer.

[0022] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0023] Example 1: Preparation of recombinant chitosanase CsnW27 strain

[0024] To discover chitosanases with high catalytic activity, this invention uses the chitosanase with the highest reported activity as a template to perform gene mining in NCBI, screening for a chitosanase derived from... Paenibacillus hamazuiensis A chitosanase with high enzyme activity (GenBank ID: WP_275983455.1), whose sequence is shown in SEQ ID NO.2, was named chitosanase CsnW27. The full-length gene was synthesized and ligated into the expression vector pET-28a(+) to obtain a recombinant plasmid. The plasmid was then transformed into the host cell. E. coli Recombinant chitosanase CsnW27 was obtained from BL21(DE3) competent cells.

[0025] Example 2: Construction of chitosanase mutant CsnW27-HF

[0026] Using the chitosanase CsnW27 plasmid as a template, PCR amplification was performed using primer pairs R82HF / R82HR and Y99FF / Y99FR as listed in Table 1, respectively. The products were then subjected to... Dpn After digestion with enzyme I, it is converted to E. coliIn BL21(DE3), chitosanase mutants CsnW27 / 82H and CsnW27 / 99F were constructed. Compared with wild-type chitosanase CsnW27, CsnW27 / 82H mutates arginine at position 82 to histidine, and CsnW27 / 99F mutates tyrosine at position 99 to phenylalanine.

[0027] Table 1 Primer Sequences

[0028] .

[0029] Example 3: Induction, expression, and purification of wild-type chitosanase and its mutants

[0030] Wild-type chitosanase and its mutant recombinant strains were inoculated into LB liquid medium containing kanamycin (100 μg / mL) and cultured at 37°C with shaking until OD reached. 600 The values ​​were 0.6–0.8. IPTG was then added to a final concentration of 0.1 mM, and the temperature was adjusted to 20°C for further induction of expression for 18 h. Bacterial cells were collected by centrifugation at 4°C. After sonication, the cells were purified using a Ni-NTA affinity chromatography column. Non-specifically adsorbed proteins were first washed away with NPI-20 buffer, followed by elution with NPI-200. Finally, ultrafiltration was used to remove salts and concentrate the protein, yielding purified wild-type chitosanase and its mutants.

[0031] The amino acid sequence of the chitosanase mutant CsnW27-HF is SEQ ID NO:1:

[0032] MKSVLVMLLLTMLFLSGCTKTVPEETAKTDNAGQISVLSAGLNANQKRRAEQLISLFENGTLVLQYGYAERLHDGRGITCGHAGFTTGTGDAYEVVKLFTDEVPGNKLAKYLPELKRLLTARNKDDVSRLSGFIRDWSSLG NNAVFRSVQDRVVDDMYYKPSERYSNNLNLATPLARAVIYDTIIQHGDGDDPDGLRSLINRTNQAMGGSPKQGIDEKKWLAKFLDVRRADLQHPADRSSQDVWAQSVGRVDVFKYIAAKGNYRLDGPIRIRTRDYNVTIP;

[0033] The amino acid sequence of wild-type chitosanase CsnW27 is SEQ ID NO:2:

[0034] MKSVLVMLLLTMLFLSGCTKTVPEETAKTDNAGQISVLSAGLNANQKRRAEQLISLFENGTLVLQYGYAERLHDGRGITCGRAGFTTGTGDAYEVVKLYTDEVPGNKLAKYLPELKRLLTARNKDDVSRLSGFIRDWSSLGNNAVFRSVQDRVVDDMYYKPSERYSNNLNLATPLARAVIYDTIIQHGDGDDPDGLRSLINRTNQAMGGSPKQGIDEKKWLAKFLDVRRADLQHPADRSSQDVWAQSVGRVDVFKYIAAKGNYRLDGPIRIRTRDYNVTIP;

[0035] Nucleotide sequence of chitosanase mutant CsnW27-HF SEQ ID NO:3:

[0036] ATGAAATCTGTTCTGGTTATGCTGCTGCTGACCATGCTGTTCCTGTCTGGTTGCACCAAAACCGTTCCGGAAGAAACCGCTAAAACCGACAACGCTGGTCAGATCTCTGTTCTGTCTGCTGGTCTGAACGCTAACCAGAAACGTCGTGCTGAACAGCTGATCTCTCTGTTCGAAAACGGTACCCTGGTTCTGCAGTACGGTTACGCTGAACGTCTGCACGACGGTCGTGGTATCACCTGCGGTCATGCTGGTTTCACCACCGGTACCGGTGACGCTTACGAAGTTGTTAAACTGTTCACCGACGAAGTTCCGGGTAACAAACTGGCTAAATACCTGCCGGAACTGAAACGTCTGCTGACCGCTCGTAACAAAGACGACGTTTCTCGTCTGTCTGGTTTCATCCGTGACTGGTCTTCTCTGGGTAACAACGCTGTTTTCCGTTCTGTTCAGGACCGTGTTGTTGACGACATGTACTACAAACCGTCTGAACGTTACTCTAACAACCTGAACCTGGCTACCCCGCTGGCTCGTGCTGTTATCTACGACACCATCATCCAGCACGGTGACGGTGACGACCCGGACGGTCTGCGTTCTCTGATCAACCGTACCAACCAGGCTATGGGTGGTTCTCCGAAACAGGGTATCGACGAAAAAAAATGGCTGGCTAAATTCCTGGACGTTCGTCGTGCTGACCTGCAGCACCCGGCTGACCGTTCTTCTCAGGACGTTTGGGCTCAGTCTGTTGGTCGTGTTGACGTTTTCAAATACATCGCTGCTAAAGGTAACTACCGTCTGGACGGTCCGATCCGTATCCGTACCCGTGACTACAACGTTACCATCCCG;

[0037] Nucleotide sequence of wild-type chitosanase CsnW27, SEQ ID NO:4:

[0038] ATGAAATCTGTTCTGGTTATGCTGCTGCTGACCATGCTGTTCCTGTCTGGTTGCACCAAAACCGTTCCGGAAGAAACCGCTAAAACCGACAACGCTGGTCAGATCTCTGTTCTGTCTGCTGGTCTGAACGCTAACCAGAAACGTCGTGCTGAACAGCTGATCTCTCTGTTCGAAAACGGTACCCTGGTTCTGCAGTACGGTTACGCTGAACGTCTGCACGACGGTCGTGGTATCACCTGCGGTCGTGCTGGTTTCACCACCGGTACCGGTGACGCTTACGAAGTTGTTAAACTGTACACCGACGAAGTTCCGGGTAACAAACTGGCTAAATACCTGCCGGAACTGAAACGTCTGCTGACCGCTCGTAACAAAGACGACGTTTCTCGTCTGTCTGGTTTCATCCGTGACTGGTCTTCTCTGGGTAACAACGCTGTTTTCCGTTCTGTTCAGGACCGTGTTGTTGACGACATGTACTACAAACCGTCTGAACGTTACTCTAACAACCTGAACCTGGCTACCCCGCTGGCTCGTGCTGTTATCTACGACACCATCATCCAGCACGGTGACGGTGACGACCCGGACGGTCTGCGTTCTCTGATCAACCGTACCAACCAGGCTATGGGTGGTTCTCCGAAACAGGGTATCGACGAAAAAAAATGGCTGGCTAAATTCCTGGACGTTCGTCGTGCTGACCTGCAGCACCCGGCTGACCGTTCTTCTCAGGACGTTTGGGCTCAGTCTGTTGGTCGTGTTGACGTTTTCAAATACATCGCTGCTAAAGGTAACTACCGTCTGGACGGTCCGATCCGTATCCGTACCCGTGACTACAACGTTACCATCCCG。

[0039] Example 4. Activity detection of chitosanase CsnW27 and mutant CsnW27-HF

[0040] The chitosanase CsnW27 and the mutant CsnW27-HF obtained in Example 3 were tested for enzyme activity using the DNS method. The specific procedure was as follows: 190 μL of colloidal chitosan (1%) was taken, and then 10 μL of chitosanase CsnW27 or the mutant CsnW27-HF were added respectively. The mixture was incubated at 37°C for 20 min, then 300 μL of DNS reagent was added, followed by a boiling water bath for 5 min. After cooling to room temperature, the absorbance at 540 nm was measured. Enzyme activity (U) is defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute under standard conditions. The enzyme activity of wild-type chitosanase CsnW27 was measured to be 7830.04 U / mg, and the enzyme activity of the mutant CsnW27-HF was 18519.84 U / mg, which is 2.36 times that of wild-type chitosanase.

[0041] Example 5: Enzymatic Properties Detection of Chitosanase Mutant CsnW27-HF

[0042] (1) Effects of temperature and pH on the activity of chitosanase mutant CsnW27-HF

[0043] 10 μL of the obtained chitosanase mutant CsnW27-HF was added to 190 μL of colloidal chitosan (1%), and enzymatic reactions were carried out at 20-80℃ to study the effect of temperature on enzyme activity. The obtained chitosanase mutant CsnW27-HF enzyme solution was diluted with different buffers: citrate buffer (20 mM, pH 3.0-6.0), phosphate buffer (20 mM, pH 6.0-8.0), Tris-HCl buffer (100 mM, pH 8.0-9.0), and Gly-NaOH buffer (20 mM, pH 9.0-10.0), and then the reaction was carried out at 37℃ to study the effect of pH on enzyme activity. The highest enzyme activity was taken as 100%, and the results are as follows: Figure 1 and Figure 2 As shown in the figure. The results show that the optimal temperature for the chitosanase mutant CsnW27-HF is 50℃ and the optimal pH is 6.0.

[0044] (2) Effects of temperature and pH on the stability of chitosanase mutant CsnW27-HF enzyme

[0045] The obtained chitosanase mutant CsnW27-HF was incubated at 20℃, 30℃, and 40℃ for different times, and its residual enzyme activity was tested to study the effect of temperature on its stability. The obtained chitosanase mutant CsnW27-HF was also incubated in phosphate buffers (pH 6.0-8.0) at different pH values ​​for different times, and then the residual enzyme activity was measured to study the effect of pH on its stability. The results are as follows: Figure 3 and Figure 4 As shown, CsnW27-HF retained 56.9% of its initial enzyme activity when stored at 40℃ for 2 h. It exhibited maximum stability at pH 7.0, retaining 81.6% of its activity after 24 h of incubation.

[0046] Example 6: Analysis of hydrolysis products of chitosanase mutant CsnW27-HF

[0047] The hydrolysis products of the chitosanase mutant CsnW27-HF were analyzed using colloidal chitosan (1%) as the substrate. Samples were taken at different time points, and an equal volume of ethanol was added followed by centrifugation. A suitable amount of sample was then spotted onto a silica gel plate. A mobile phase of n-propanol-25% ammonia-water (8:3:1, v / v / v) was used, and a 0.1% ethanol-ninhydrin solution was used as the colorimetric reagent. Results are as follows: Figure 5 As shown, the initial hydrolysis products of the chitosanase mutant CsnW27-HF contain chitobiose, chitotriose, and chitotetraose. As the reaction proceeds, the chitotetraose is gradually degraded, and the final product consists of chitobiose and chitotriose.

[0048] The above embodiments are only for illustrating the technical concept and features of the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make various modifications or alterations to the present invention, and these equivalent forms are also covered within the scope defined by the appended claims.

Claims

1. A chitosanase mutant CsnW27-HF, characterized in that, The amino acid sequence of the chitosanase mutant is shown in SEQ ID NO:

1.

2. The gene encoding the chitosanase mutant CsnW27-HF of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. A recombinant plasmid, characterized in that, The plasmid carries the gene described in claim 2, and the expression vector is pET-28a(+).

4. The recombinant engineered strain obtained by transformation with the recombinant plasmid according to claim 3, characterized in that, The host is E. coli BL21(DE3).

5. An enzyme preparation, characterized in that, The enzyme preparation contains the chitosanase mutant CsnW27-HF as described in claim 1.

6. The application of the chitosanase mutant CsnW27-HF according to claim 1 in the preparation of chitosan oligosaccharides.

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