A chitosanase mutant producing chitotetraose and its application

Abstract

The present invention discloses a chitosanase mutant and its use in the preparation of chitotetraose, belonging to the field of enzyme engineering. The invention screens for a mutant whose hydrolysis product has the potential to produce chitotetraose by performing site-directed saturation mutagenesis at position 149 of the amino acid sequence (SEQ ID NO: 1) of the Bacillus subtilis chitosanase BsCsn46A. Aspartic acid is mutated to glycine, labeled D149G. The hydrolysis product of the mutant produces chitotetraose in addition to chitobiose and chitotriose.

Description

Technical Field

[0001] The invention relates to a chitosanase mutant whose hydrolysis product contains chitotetraose, specifically to a mutant obtained by site-directed mutagenesis. The hydrolysis product of the mutant has the ability to produce chitotetraose compared with the original chitosanase, and belongs to the field of enzyme engineering. Background Art

[0002] Chitosan is an alkaline polysaccharide derived from the deacetylation of chitin. It is composed of randomly distributed N-acetylglucosamine (GlcNAc) and D-glucosamine (GlcN) units linked by β-1,4-glycosidic bonds. Chitosan exhibits multiple biological activities and good biosafety, making it widely used in food, medicine, and agriculture. However, its poor water solubility and high viscosity limit its application.

[0003] Chitooligosaccharide (COS), a hydrolysis product of chitosan, has immunomodulatory, anti-inflammatory, antibacterial, anti-tumor, neuroprotective, and lipid-lowering properties. Its advantages include low molecular weight, good solubility, easy absorption, and no toxic side effects. Therefore, chitooligosaccharide plays a significant role in various fields, including health foods, biomedicine, cosmetics, chemicals, and agriculture.

[0004] COS can be prepared by physical, chemical, and enzymatic methods. The physical method uses ultrasound and other methods to act on the β-1,4-glycosidic bonds of chitosan to achieve the purpose of degrading the polysaccharide skeleton. The physical method is easy to control and pollution-free, but the product molecular weight is uneven, the yield is low, and the product polymerization degree is too high, resulting in the inability to use chitosan oligosaccharides. Chemical methods include acid hydrolysis and oxidative degradation. Chemical reactions are not easy to control, the cost is high, and they can cause environmental pollution. Enzymatic preparation of COS can be divided into two schemes: non-specific enzymatic hydrolysis and specific enzymatic hydrolysis. Non-specific enzymes are cheap and easy to obtain, with limited catalytic activity; specific enzymes are chitosanase, which is specific.

[0005] Chitosanase (EC.3.2.1.132) is a glycoside hydrolase that specifically hydrolyzes chitosan to produce chitooligosaccharides. It hydrolyzes chitosan's β-1,4-glycosidic bonds to produce chitooligosaccharides. Its advantages lie in the uniformity of the hydrolysis products and mild reaction conditions, thus facilitating the large-scale production of chitooligosaccharides. As research on chitosanase deepens, its crystal structure has been continuously elucidated, furthering our understanding of the relationship between its protein structure and its hydrolysis mechanism. Currently, some studies have shown that chitooligosaccharides with a degree of polymerization greater than 4 exhibit superior activity. However, the majority of chitosanase hydrolysis products reported to date are chitobiose and chitotriose. Summary of the Invention

[0006] The invention carries out molecular transformation on chitosanase, aiming at obtaining chitosanase mutants with changed polymerization degree.

[0007] By using Bacillus subtilis ( Bacillus subtilis ) The substrate channel of chitosanase BsCsn46A was analyzed to obtain key sites that may affect the enzymatic hydrolysis products. Saturation mutagenesis of the key sites was performed to screen out mutants with the ability to produce chitotetraose.

[0008] The chitosanase mutant of the present invention undergoes site-directed mutagenesis at position 149 of the amino acid sequence of Bacillus subtilis chitosanase BsCsn46A, mutating aspartic acid to glycine, and is labeled D149G. The amino acid sequence of Bacillus subtilis chitosanase BsCsn46A is SEQ ID NO: 1, and the nucleotide sequence is SEQ ID NO: 2. The chitosanase mutant D149G has an amino acid sequence of SEQ ID NO: 3, and a nucleotide sequence of SEQ ID NO: 4.

[0009] SEQ ID NO: 1

[0010] 1 AGLNKDQKRRAEQLTSIFEN

[0011] 21 GTTEIQYGYVERLDDGRGYT

[0012] 41 CGRAGFTTATGDALEVVEVY

[0013] 61 TKAVPNSKLKKYLPELRRLA

[0014] 81 KEESDDTSNLKGFASAWKSL

[0015] 101 ANDKEFRAAQDKVNDHLYYQ

[0016] 121 NAMKRSDNAGLKTALARAVM

[0017] 141 YDTVIQHGDGDDPDSFYALI

[0018] 161 KRTNKKAGGSPKDGIDEKKW

[0019] 181 LNKFLDVRYDDLMNPANHDT

[0020] 201 RDEWRESVARVDVLRSIAKE

[0021] 221 NNYNLNGPIHVRSNEYGNFV

[0022] 241 IP。

[0023] SEQ ID NO:2

[0024] GCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTTGAAAACGGCACAACGGAGATCCAATATGGATATGTAGAGCGATTGGATGACGGGCGAGGCTATACATGCGGTCGGGCAGGCTTTACAACGGCTACCGGGGATGCATTGGAAGTAGTGGAAGTATACACAAAGGCAGTTCCGAATAGCAAACTGAAAAAGTATCTGCCTGAATTGCGCCGTCTGGCCAAGGAAGAAAGCGATGATACAAGCAATCTCAAGGGATTCGCTTCTGCCTGGAAGTCGCTTGCAAATGATAAGGAATTTCGCGCCGCTCAAGACAAAGTAAATGACCATTTGTATTATCAGAATGCCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTGATGTACGATACGGTTATTCAGCATGGCGATGGTGATGACCCTGACTCTTTTTATGCCTTGATTAAACGTACGAACAAAAAAGCGGGCGGATCACCTAAAGACGGAATAGACGAGAAGAAGTGGTTGAATAAATTCTTGGACGTACGCTATGACGATCTGATGAATCCGGCCAATCATGACACCCGTGACGAATGGAGAGAATCAGTTGCCCGTGTGGACGTGCTTCGCTCTATCGCCAAGGAGAACAACTATAATCTAAACGGACCGATTCATGTTCGTTCAAACGAGTACGGTAATTTTGTAATCCCTTAA。

[0025] SEQ ID NO:3

[0026] 1 AGLNKDQKRR AEQLTSIFEN

[0027] 21 GTTEIQYGYV ERLDDGRGYT

[0028] 41 CGRAGFTTAT GDALEVVEVY

[0029] 61 TKAVPNSKLK KYLPELRRLA

[0030] 81 KEESDDTSNL KGFASAWKSL

[0031] 101 ANDKEFRAAQ DKVNDHLYYQ

[0032] 121 NAMKRSDNAG LKTALARAVM

[0033] 141 YDTVIQHGGG DDPDSFYALI

[0034] 161 KRTNKKAGGS PKDGIDEKKW

[0035] 181 LNKFLDVRYD DLMNPANHDT

[0036] 201 RDEWRESVAR VDVLRSIAKE

[0037] 221 NNYNLNGPIH VRSNEYGNFV

[0038] 241 IP。

[0039] SEQ ID NO:4

[0040] GCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTTGAAAACGGCACAACGGAGATCCAATATGGATATGTAGAGCGATTGGATGACGGGCGAGGCTATACATGCGGTCGGGCAGGCTTTACAACGGCTACCGGGGATGCATTGGAAGTAGTGGAAGTATACAC AAAGGCATTCCGAATAGCAAACTGAAAAAGTATCTGCCTGAATTGCGCCGTCTGGCCAAGGAAGAAAGCGATGATACAAGCAATCTCAAGGGATTCGCTTCTGCCTGGAAGTCGCTTGCAAATGATAAGGAATTTCGCGCCGCTCAAGACAAAGTAAATGACCATTTGTATTATCAGAATG CCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTGATGTACGATACGGTTATTCAGCATGGCGGAGGTGATGACCCTGACTCTTTTTATGCCTTGATTAAACGTACGAACAAAAAAGCGGGCGGATCACCTAAAGACGGAATAGACGAGAAGAAGTGGTTGAAT AAATTCTTGGACGTACGCTATGACGATCTGATGAATCCGGCCAATCATGACACCCGTGACGAATGGAGAGAATCAGTTGCCCGTGTGGACGTGCTTCGCTCTATCGCCAAGGAGAACAACTATAATCTAAACGGACCGATTCATGTTCGTTCAAACGAGTACGGTAATTTTGTAATCCCTTAA

[0041] The present invention also provides a recombinant vector containing the gene of the chitosanase mutant and a recombinant bacterium containing the recombinant vector, wherein the host bacterium is Escherichia coli.

[0042] The present invention further provides the use of the chitosanase mutant in catalyzing the hydrolysis of chitosan to produce chitooligosaccharides, and more specifically, in catalyzing the hydrolysis of chitosan to produce chitotetraose.

[0043] The specific application method is to collect the bacteria after inducing expression of the recombinant bacteria, ultrasonically disrupt them, use the whole cell liquid of the bacteria or the crude enzyme extract or the purified enzyme liquid to react with chitosan, and control the reaction time within 1 hour.

[0044] The present invention constructs the three-dimensional structure of the enzyme through SWISS-MODEL, analyzes the substrate channel of the enzyme, performs saturation mutation on position 149, a key site affecting the polymerization degree of the product, and screens the saturated mutant library to obtain mutants affecting the polymerization degree of the product, wherein aspartic acid at position 149 is mutated to glycine. Compared with the original enzyme, the hydrolysis time of the enzyme to produce chitotetraose is significantly increased, and the enzyme can be used for the industrial bioenzymatic production of chitosan oligosaccharides with high polymerization degree. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Thin layer chromatograms of chitosan degradation products of wild-type chitosanase and D149E mutant.

[0046] Figure 2 Thin layer chromatograms of chitosan degradation products of chitosanase D149A and D149K mutants.

[0047] Figure 3 Thin layer chromatograms of chitosan degradation products of chitosanase D149R and D149H mutants.

[0048] Figure 4 Thin layer chromatograms of chitosan degradation products of chitosanase D149N and D149C mutants.

[0049] Figure 5 Thin layer chromatograms of chitosan degradation products of chitosanase D149Q and D149G mutants.

[0050] Figure 6 Thin layer chromatograms of chitosan degradation products of chitosanase D149M and D149S mutants.

[0051] Figure 7 Thin layer chromatograms of chitosan degradation products of chitosanase D149T and D149I mutants.

[0052] Figure 8 Thin layer chromatograms of chitosan degradation products of chitosanase D149L and D149F mutants.

[0053] Figure 9 Thin layer chromatograms of chitosan degradation products of chitosanase D149W and D149Y mutants.

[0054] Figure 10 Thin layer chromatograms of chitosan degradation products of chitosanase D149V and D149P mutants. DETAILED DESCRIPTION

[0055] The enzyme's three-dimensional structure was constructed using SWISS-MODEL, and the enzyme's substrate channel was analyzed. Key sites that influence the degree of product polymerization were identified through representative amino acid mutations, and saturation mutations were performed on these key sites. The saturation mutant library was screened to identify superior mutants that influence the degree of product polymerization.

[0056] The original enzyme was the highly active chitosanase BsCsn46A obtained in the early stages of the experiment. The key site identified by analysis was position 149 in the amino acid sequence of BsCsn46A. The primers for the mutant were designed using the online software PrimerX. The sequence is shown in the table below. The chitosanase gene was then synthesized using molecular biology methods.

[0057] PCR amplification was performed using chitosanase BsCsn46A gene SEQ ID NO: 2 as a template and primer sequences as shown in Table 1.

[0058] Table 1. Primer sequences for the saturation mutant library

[0059] Primer name Primer usage Primer (5–3') D149CF D149C <![CDATA[GGTTATTCAGCATGGC TGC GGTGATGACCCTGAC]]> D149CR D149C <![CDATA[GTCAGGGTCATCACC GCA GCCATGCTGAATAACC]]> D149EF D149E <![CDATA[GTTATTCAGCATGGC GAA GGTGATGACCCTGACTC]]> D149ER D149E <![CDATA[GAGTCAGGGTCATCACC TTC GCCATGCTGAATAAC]]> D149FF D149F <![CDATA[GGTTATTCAGCATGGC TTT GGTGATGACCCTGAC]]> D149FR D149F <![CDATA[GTCAGGGTCATCACC AAA GCCATGCTGAATAACC]]> D149GF D149G <![CDATA[GTTATTCAGCATGGC GGA GGTGATGACCCTGAC]]> D149GR D149G <![CDATA[GTCAGGGTCATCACC TCC GCCATGCTGAATAAC]]> D149HF D149H <![CDATA[GTTATTCAGCATGGC CAT GGTGATGACCCTG]]> D149HR D149H <![CDATA[CAGGGTCATCACC ATG GCCATGCTGAATAAC]]> D149IF D149I <![CDATA[GTTATTCAGCATGGC ATC GGTGATGACCCTGAC]]> D149IR D149I <![CDATA[GTCAGGGTCATCACC GAT GCCATGCTGAATAAC]]> D149LF D149L <![CDATA[GTTATTCAGCATGGC TTG GGTGATGACCCTGAC]]> D149LR D149L <![CDATA[GTCAGGGTCATCACC CAA GCCATGCTGAATAAC]]> D149MF D149M <![CDATA[GTTATTCAGCATGGC ATG GGTGATGACCCTGAC]]> D149MR D149M <![CDATA[GTCAGGGTCATCACC CAT GCCATGCTGAATAAC]]> D149NF D149N <![CDATA[GTTATTCAGCATGGC AAT GGTGATGACCCTG]]> D149NR D149N <![CDATA[CAGGGTCATCACC ATT GCCATGCTGAATAAC]]> D149PF D149P <![CDATA[GTTATTCAGCATGGC CCT GGTGATGACCCTG]]> D149PR D149P <![CDATA[CAGGGTCATCACC AGG GCCATGCTGAATAAC]]> D149QF D149Q <![CDATA[GTTATTCAGCATGGC CAG GGTGATGACCCTGAC]]> D149QR D149Q <![CDATA[GTCAGGGTCATCACC CTG GCCATGCTGAATAAC]]> D149RF D149R <![CDATA[GTTATTCAGCATGGC CGC GGTGATGACCCTGAC]]> D149RR D149R <![CDATA[GTCAGGGTCATCACC GCG GCCATGCTGAATAAC]]> D149SF D149S <![CDATA[GTTATTCAGCATGGC AGC GGTGATGACCCTGAC]]> D149SR D149S <![CDATA[GTCAGGGTCATCACC GCT GCCATGCTGAATAAC]]> D149TF D149T <![CDATA[GTTATTCAGCATGGC ACA GGTGATGACCCTGAC]]> D149TR D149T <![CDATA[GTCAGGGTCATCACC TGT GCCATGCTGAATAAC]]> D149VF D149V <![CDATA[GTTATTCAGCATGGC GTA GGTGATGACCCTGAC]]> D149VR D149V <![CDATA[GTCAGGGTCATCACC TAC GCCATGCTGAATAAC]]> D149YF D149Y <![CDATA[GTTATTCAGCATGGC TAT GGTGATGACCCTG]]> D149YR D149Y <![CDATA[CAGGGTCATCACC ATA GCCATGCTGAATAAC]]> D149AF D149A <![CDATA[GTTATTCAGCATGGC GCC GGTGATGACCCTGAC]]> D149AR D149A <![CDATA[GTCAGGGTCATCACC GGC GCCATGCTGAATAAC <!-- 4 -->]]> D149WF D149W <![CDATA[GGTTATTCAGCATGGC TGG GGTGATGACCCTGAC]]> D149WR D149W <![CDATA[GTCAGGGTCATCACC CCA GCCATGCTGAATAACC]]> D149KF D149K <![CDATA[GGTTATTCAGCATGGC AAA GGTGATGACCCTGACTC]]> D149KR D149K <![CDATA[GAGTCAGGGTCATCACC TTT GCCATGCTGAATAACC]]>

[0060] Table 2. Inverse PCR system

[0061] Reagent name Volume (μL) template 2 PCR Buffer 5 dNTPs (10 mM) 1 Upstream / downstream primers (100 mM) 0.3 each PfuDNA polymerase 1.5 <![CDATA[ddH2O]]> 40 Total volume 50

[0062] Reverse PCR amplification conditions were as follows: pre-denaturation at 95°C for 3 min; 15 cycles of denaturation at 95°C for 30 s, annealing at 65°C for 1 min, and extension at 68°C for 10 min; and insulation at 4°C.

[0063] Digest with DpnI for 2 hours to remove the plasmid template. Pre-cool 10 μL of the remaining reaction mixture and transform it into E. coli DH5α. Sequence a sample of cells containing the recombinant gene and compare the sequences to ensure that only the amino acid at the mutation site has been mutated. Extract the plasmid from cells with correct sequencing results and transform it into the heterologous expression host E. coli BL21. Store the glycerol stock in a frozen container.

[0064] The preserved strain was inoculated into 10 mL of liquid LB medium at a rate of one thousandth for activation, and then transferred to 50 mL of liquid LB medium for expansion culture. The inducer IPTG (isopropyl-β-D-thiogalactopyranoside) was then added to induce protein production.

[0065] Protein purification procedures: Bacteria were harvested and disrupted by sonication. The supernatant was purified by Ni-IDA affinity chromatography. The supernatant was loaded onto a Ni-IDA column. Unbound proteins were first eluted with loading buffer, followed by elution with elution buffer (20 mM Tris-HCl, 0.5 M NaCl, 0.08 M imidazole, 10% glycerol, pH 8.0). The eluted protein sample was collected and stored at -20°C. The separated fractions were analyzed by SDS-PAGE electrophoresis, and the protein content in the enzyme solution was determined using a protein quantification reagent purchased from Nanjing Novezan Biotechnology Co., Ltd. (Bradford Reagent: E211-01).

[0066] The purified enzyme was tested for enzyme activity by DNS method. The reaction system consisted of 1475 μL pH buffer, 18 μL 100 mM Mn 2+ , 500 μL of 1% colloidal chitosan solution, add 25 μL of the purified enzyme solution, incubate in a water bath for 5 minutes under the measured optimal pH and optimal temperature conditions, add 1.5 mL of DNS solution to terminate the reaction, then boil in boiling water for 5 minutes, and finally dilute to 25 mL with distilled water, cool and let stand for 1 hour. No enzyme was added to the blank group. The absorbance of the sample was measured at a wavelength of 520 nm using a UV-visible spectrophotometer. The reducing end content of the product released by the catalytic reaction was calculated based on the standard curve, and the chitosanase activity was defined as the amount of enzyme used to generate 1 μmol of reducing sugar per minute, which was defined as one enzyme activity unit (U). The enzymatic activity data of the relevant mutant enzymes are as follows.

[0067] Table 3. Enzyme activities of mutant enzymes

[0068] mutant enzyme Optimum pH Optimum temperature / ℃ Specific enzyme activity (U / mg) WT 6.0 50 15620.46 D149A 6.0 55 7177.20 D149E 6.0 55 5521.51 D149R 6.0 55 1019.54 D149H 6.0 55 1325.61 D149K 6.0 55 1518.98 D149N 6.0 55 4025.39 D149Q 6.0 55 1620.46 D149G 6.0 55 4246.50 D149M 6.0 50 2191.54 D149S 6.0 45 4298.43 D149T 6.0 55 2690.86 D149I 6.0 55 2288.78 D149Y 6.0 55 3961.19 D149V 6.0 55 2986.11 D149C 6.2 55 4099.21 D149W 6.2 55 3198.18 D149P 6.2 55 2110.42 D149F 6.4 55 3304.20 D149L 6.6 55 1375.74

[0069] Hydrolysis reaction: 450 μL 1% colloidal chitosan solution, 20 U purified enzyme solution, 500 μL pH 6.6 phosphate buffer and 9 μL 100 mM Mn were added to 1 mL of reaction system. 2+ , place at 37 ° C, shake at 160 rpm for full reaction, centrifuge the hydrolysis system at 5 min, 30 min, 1 h, 3 h, 5 h, 7 h, 9 h, and 11 h, take 20 μL of the supernatant, boil for 10 min and retain it.

[0070] TLC analysis. The thin-layer chromatography procedure was as follows: Samples of the hydrolyzate obtained at different times were aspirated using a capillary tube and spotted onto a high-performance silica gel plate. A mixed solution of GlcN1-5 standards was used as a control. The spotted silica gel plate was thoroughly dried and then placed on a chromatography bar for full development. The plate was removed and blown dry with a hair dryer until no ammonia odor remained. The plate was evenly sprayed with a color developer (0.5% ninhydrin-ethanol solution), dried, and placed in a 110°C oven until clear purple-red spots appeared.

[0071] Combined with thin-layer chromatography analysis, mutant D149G produced no chitosan after 3 hours of hydrolysis, a longer time than the original enzyme, indicating a greater capacity for chitosan production. High-DP chitosan oligosaccharides exhibited greater bioactivity than low-DP chitosan oligosaccharides, demonstrating a significant advantage in the preparation of active oligosaccharides. Screening for chitosan-producing mutants based on saturated enzyme mutagenesis could be used in industrial production, and this could also be used to further engineer the enzyme to produce chitosanases with high enzymatic activity and a high degree of polymerization.

Claims

1. A chitosanase mutant producing chitotetraose, characterized in that the amino acid sequence of the chitosanase mutant is SEQ ID NO:

3.

2. A chitosanase mutant gene, characterized in that it encodes the chitotetraose-producing chitosanase mutant according to claim 1.

3. A gene of the chitosanase mutant according to claim 2, characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO:

4.

4. A recombinant vector comprising the gene of the chitosanase mutant according to claim 2 or 3.

5. A recombinant bacterium, characterized in that A gene comprising the chitosanase mutant according to claim 2 or 3, or a recombinant vector according to claim 4.

6. The recombinant bacterium according to claim 5, characterized in that The host bacteria of the recombinant bacteria is Escherichia coli.

7. A use of the chitosanase mutant according to claim 1, characterized in that: The chitosanase mutant is used in catalyzing the hydrolysis of chitosan to produce chitotetraose.

8. The use of the chitosanase mutant according to claim 7, characterized in that: The application method is to collect the bacteria after inducing expression of the recombinant bacteria, ultrasonically disrupt them, use the whole cell liquid of the bacteria or the crude enzyme extract or the purified enzyme liquid to react with chitosan, and control the reaction time within 1 hour.

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