Chitosanase mutant with chitotetraose production ability and its application

Abstract

The present invention belongs to the field of enzyme engineering technology and discloses chitosanase mutants capable of producing chitotetraose and their applications. Chitosanase (BsCsn46A), a GH46 family glycoside hydrolase derived from Bacillus subtilis, whose amino acid sequence is shown in SEQ ID NO: 1, was subjected to saturation mutagenesis at amino acid residue 203 to screen for mutants with chitotetraose production potential. The resulting chitosanase mutants are E203M, E203S, E203R, E203H, E203N, E203C, E203Q, E203G, E203T, E203V, and E203P. Hydrolysis analysis confirmed that the resulting mutants possess the ability to produce chitotetraose.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme engineering, and specifically relates to a chitosanase mutant whose hydrolysis product contains chitotetraose. The invention is specifically described as obtaining the mutant by screening mutations at key sites of a substrate channel, and the hydrolysis product contains chitotetraose. Background Art

[0002] Chitosan (CS) is a cationic, naturally alkaline polysaccharide composed of glucosamine (GlcN) linked by β-1,4-glycosidic bonds. Chitosan oligosaccharides (COS), also known as oligosaccharides, are oligomers of glucosamine linked by β-1,4-glycosidic bonds. They are the only positively charged cationic alkaline oligosaccharides found in nature, with a degree of polymerization typically ranging from 2-20 and a molecular weight ≤3200 Da. COS exhibits good water solubility at physiological pH, ease of absorption, and high biological activity. Chitosan oligosaccharides have excellent biosafety and exhibit immunomodulatory, anti-inflammatory, antibacterial, anti-tumor, antioxidant, and lipid-lowering properties. Therefore, they hold broad application prospects in the food, medical, green agriculture, and cosmetics industries.

[0003] Currently, the production of chitosan oligosaccharides primarily involves chemical acid hydrolysis, physical degradation, and enzymatic hydrolysis. Chemical acid hydrolysis hydrolyzes both the O-glycosidic and N-acetyl bonds of chitosan, presenting numerous challenges such as difficult control and loss of activity. Physical degradation produces high-purity products, but yields are low, limiting large-scale production. Enzymatic hydrolysis offers advantages such as mild reaction conditions, high stability, no environmental pollution, and economically viable, large-scale, sustainable production. Consequently, enzymatic hydrolysis has become a current research hotspot in the production of chitosan oligosaccharides.

[0004] Chitosanase (EC3.2.1.132), also known as N-acetylglucosamine hydrolase, specifically catalyzes the conversion of chitosan to chitooligosaccharides or glucosamine. It was first described by Monaghan et al. in 1973. Chitosanases can be divided into endo- and exo-chitosanases based on the substrate cleavage method. Chitosanases are primarily distributed in the GH8, GH46, GH75, and GH80 families, with the GH46 family chitosanase being the most extensively studied.

[0005] It has been reported that most of the chitosanase hydrolysis products are a mixture of chitobiose and chitotriose. However, some studies have shown that chitosan oligosaccharides with a degree of polymerization greater than 4 have better biological activity. Therefore, obtaining chitosanase mutants that can produce chitosan oligosaccharides with a high degree of polymerization has great application value. Summary of the Invention

[0006] The inventors have found in their previous studies that Bacillus subtilis ( Bacillus subtilis) The glutamic acid at position 203 of chitosanase BsCsn46A was mutated, which affected its enzyme activity or hydrolysis products (CN116590260A, CN116949015A). On this basis, the inventors investigated the effect of Bacillus subtilis ( Bacillus subtilis ) The substrate channel of chitosanase BsCsn46A was analyzed, and the 203 position was saturated mutated to screen out mutants with the potential to produce chitotetraose.

[0007] The present invention provides a mutant recombinant chitosanase modified by site-directed mutagenesis and in Escherichia coli E. coliThe chitosanase mutants are expressed in BL21(DE3). The chitosanase BsCsn46A glutamate 203 is mutated to methionine, labeled E203M; to serine, labeled E203S; to arginine, labeled E203R; to histidine, labeled E203H; to asparagine, labeled E203N; to cysteine, labeled E203C; to glutamine, labeled E203Q; to glycine, labeled E203G; to threonine, labeled E203T; to valine, labeled E203V; and to proline, labeled E203P. 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 E203M has an amino acid sequence of SEQ ID NO: 3, and a nucleotide sequence of SEQ ID NO: 4; the chitosanase mutant E203S has an amino acid sequence of SEQ ID NO: 5, and a nucleotide sequence of SEQ ID NO: 6; the chitosanase mutant E203R has an amino acid sequence of SEQ ID NO: 7, and a nucleotide sequence of SEQ ID NO: 8; the chitosanase mutant E203H has an amino acid sequence of SEQ ID NO: 9, and a nucleotide sequence of SEQ ID NO: 10; the chitosanase mutant E203N has an amino acid sequence of SEQ ID NO: 11, and a nucleotide sequence of SEQ ID NO: 12; the chitosanase mutant E203C has an amino acid sequence of SEQ ID NO: 13, and a nucleotide sequence of SEQ ID NO: 14; the chitosanase mutant E203Q has an amino acid sequence of SEQ ID NO: 15, and a nucleotide sequence of SEQ ID NO: NO:16; the amino acid sequence of the chitosanase mutant E203G is SEQ ID NO:17, and the nucleotide sequence is SEQ ID NO:18; the amino acid sequence of the chitosanase mutant E203T is SEQ ID NO:19, and the nucleotide sequence is SEQ ID NO:20; the amino acid sequence of the chitosanase mutant E203V is SEQ ID NO:21, and the nucleotide sequence is SEQ ID NO:22; the amino acid sequence of the chitosanase mutant E203P is SEQ ID NO:23, and the nucleotide sequence is SEQ ID NO:24.

[0008] The present invention provides a recombinant vector carrying the gene encoding the chitosanase mutant and a recombinant bacterium for transforming / transfecting the recombinant vector.

[0009] The specific mutation method of the present invention is as follows: chitosanase (BsCsn46A) is simulated using Swiss-Model online software to obtain the spatial structure of chitosanase; then the key site (E203) affecting the hydrolysis degree of the product is obtained through substrate channel simulation and molecular docking analysis; primers for site-directed mutagenesis are designed, and the chitosanase gene is obtained by PCR amplification. The chitosanase gene is then combined with the expression vector pET-28a to form a recombinant vector and transformed into Escherichia coli. E. coli BL21 (DE3); after IPTG-induced expression, the bacteria were collected, the cells were disrupted by ultrasound, and the supernatant was collected by centrifugation. The protein was then purified by Ni-NTA affinity chromatography to obtain the purified chitosanase mutant.

[0010] The present invention also provides the use of the chitosanase mutant in catalyzing the hydrolysis of chitosan to produce chitooligosaccharides. Specifically, during the catalytic hydrolysis of chitosan by the mutant of the present invention, the hydrolysis product contains chitotetraose.

[0011] Compared with wild chitosanase, the chitosanase mutant provided by the invention has advantages in being applied to hydrolysis to produce active oligosaccharide chitotetraose.

[0012] Thin layer chromatography analysis revealed that the obtained chitosan-producing mutants had different chitosan-producing time periods: mutants E203M, E203S, E203R, and E203H did not produce chitosan after 5 h of hydrolysis, and mutants E203N, E203C, E203Q, E203G, E203T, E203V, and E203P did not produce chitosan after 3 h of hydrolysis. Compared with the original enzyme, all of the mutants took longer to produce chitosan, indicating that they had the potential to produce chitosan. The present invention screened out chitosanase that produces high-polymerization degree chitosan oligosaccharides based on saturated mutations at key sites of the substrate channel, and the chitosanase can be used for the industrial preparation of chitosan, which has obvious advantages in the preparation of active oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Thin layer chromatograms of chitosan degradation products of wild-type chitosanase and mutant E203A.

[0014] Figure 2 Thin layer chromatograms of chitosan degradation products of chitosanase mutants E203N and E203C.

[0015] Figure 3 Thin layer chromatograms of chitosan degradation products of chitosanase mutants E203Q and E203G.

[0016] Figure 4 Thin layer chromatograms of chitosan degradation products of chitosanase mutants E203M and E203S.

[0017] Figure 5Thin layer chromatograms of chitosan degradation products of chitosanase mutants E203T and E203I.

[0018] Figure 6 Thin layer chromatograms of chitosan degradation products of chitosanase mutants E203L and E203F.

[0019] Figure 7 Thin layer chromatograms of chitosan degradation products of chitosanase mutants E203W and E203Y.

[0020] Figure 8 Thin layer chromatograms of chitosan degradation products of chitosanase mutants E203V and E203P.

[0021] Figure 9 Thin layer chromatograms of chitosan degradation products of chitosanase mutants E203D and E203R.

[0022] Figure 10 This is a thin layer chromatogram of the chitosan degradation products of the chitosanase mutant E203H. DETAILED DESCRIPTION

[0023] Previous studies (CN116590260A) identified position 203 in the substrate channel as a potential site affecting catalytic activity. Saturation mutagenesis at position 203 was performed to screen for mutants that produced the hydrolysis product, chitotetraose. Among these, the chitosanase mutant E203K, in which glutamic acid at position 203 was mutated to lysine, has been shown to produce chitotetraose and was not further investigated in this study. The construction methods of chitosanase mutants E203A and E203W are described in CN116949015A.

[0024] 1. The primer sequences were designed using the online software PrimerX, and the mutant chitosanase gene was obtained by genetic engineering.

[0025] Table 1: Primer sequences for the saturation mutant library

[0026] Primer name Primer usage Primer (5–3') E203CF E203C <![CDATA[CAATCATGACACCCGTGAC TGC TGGAGAGAATCAGTTGCC]]> E203CR E203C <![CDATA[GGCAACTGATTCTCTCCA GCA GTCACGGGTGTCATGATTG]]> E203DF E203D <![CDATA[CACCCGTGAC GAC TGGAGAGAATCAG]]> E203DR E203D <![CDATA[CTGATTCTCTCCA GTC GTCACGGGTG]]> E203FF E203F <![CDATA[CAATCATGACACCCGTGAC TTT TGGAGAGAATCAGTTGCC]]> E203FR E203F <![CDATA[GGCAACTGATTCTCTCCA AAA GTCACGGGTGTCATGATTG]]> E203GF E203G <![CDATA[GACACCCGTGAC GGA TGGAGAGAATC]]> E203GR E203G <![CDATA[GATTCTCTCCA TCC GTCACGGGTGTC]]> E203HF E203H <![CDATA[CAATCATGACACCCGTGAC CAT TGGAGAGAATCAGTTGCC]]> E203HR E203H <![CDATA[GGCAACTGATTCTCTCCA ATG GTCACGGGTGTCATGATTG]]> E203IF E203I <![CDATA[CAATCATGACACCCGTGAC ATC TGGAGAGAATCAGTTGCC]]> E203IR E203I <![CDATA[GGCAACTGATTCTCTCCA GAT GTCACGGGTGTCATGATTG]]> E203LF E203L <![CDATA[CAATCATGACACCCGTGAC TTG TGGAGAGAATCAGTTGCC]]> E203LR E203L <![CDATA[GGCAACTGATTCTCTCCA CAA GTCACGGGTGTCATGATTG]]> E203MF E203M <![CDATA[CAATCATGACACCCGTGAC ATG TGGAGAGAATCAGTTGCC]]> E203MR E203M <![CDATA[GGCAACTGATTCTCTCCA CAT GTCACGGGTGTCATGATTG]]> E203NF E203N <![CDATA[CAATCATGACACCCGTGAC AAT TGGAGAGAATCAGTTGCC]]> E203NR E203N <![CDATA[GGCAACTGATTCTCTCCA ATT GTCACGGGTGTCATGATTG]]> E203PF E203P <![CDATA[CAATCATGACACCCGTGAC CCT TGGAGAGAATCAGTTGCC]]> E203PR E203P <![CDATA[GGCAACTGATTCTCTCCA AGG GTCACGGGTGTCATGATTG]]> E203QF E203Q <![CDATA[CAATCATGACACCCGTGAC CAG TGGAGAGAATCAGTTGCC]]> E203QR E203Q <![CDATA[GGCAACTGATTCTCTCCA CTG GTCACGGGTGTCATGATTG]]> E203RF E203R <![CDATA[CATGACACCCGTGAC CGC TGGAGAGAATCAGTTG]]> E203RR E203R <![CDATA[CAACTGATTCTCTCCA GCG GTCACGGGTGTCATG]]> E203SF E203S <![CDATA[CAATCATGACACCCGTGAC AGC TGGAGAGAATCAGTTGCC]]> E203SR E203S <![CDATA[GGCAACTGATTCTCTCCA GCT GTCACGGGTGTCATGATTG]]> E203TF E203T <![CDATA[CATGACACCCGTG ACA CATGGAGAGAATCAG]]> E203TR E203T <![CDATA[CTGATTCTCTCCATG TGT CACGGGTGTCATG]]> E203VF E203V <![CDATA[GACACCCGTGAC GTA TGGAGAGAATCAG]]> E203VR E203V <![CDATA[CTGATTCTCTCCA TAC GTCACGGGTGTC <!-- 3 -->]]> E203YF E203Y <![CDATA[CAATCATGACACCCGTGAC TAT TGGAGAGAATCAGTTGCC]]> E203YR E203Y <![CDATA[GGCAACTGATTCTCTCCA ATA GTCACGGGTGTCATGATTG]]>

[0027] 2. Construction of recombinant plasmids and recombinant bacteria

[0028] Table 2. Inverse PCR system

[0029] 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

[0030] 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.

[0031] The amplified PCR product was digested with 0.7 μL DpnI for 1.5 h to remove the plasmid template. 10 μL of the above reaction mixture was added to the competent E. coli DH5α under ice bath conditions to form a transformation system and complete the transformation. The obtained recombinant bacteria were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The correctly sequenced extracted plasmid was then transformed into E. coli BL21 medium. Glycerol culture was stored at -80℃.

[0032] 3. Protein induced expression and purification

[0033] The mutant strain obtained above was inoculated into 10 mL of liquid LB medium at a thousandth inoculum size for activation and cultured on a shaker at 37°C and 160 rpm for 15 h; then transferred to 50 mL of liquid LB medium for expansion culture for 3 h, and then the inducer was added and cultured on a shaker at 16°C and 160 rpm for 15 h to induce the mutant strain to produce protein.

[0034] The bacterial suspension was collected using a refrigerated centrifuge to collect the cells, disrupted using an ultrasonic cell disruptor, and the supernatant collected by centrifugation and placed on ice. The nickel column was removed from the refrigerator and inverted to fluff the column. The column was washed with pure water and the collected supernatant was passed through the nickel column twice. Unbound proteins were first thoroughly 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. The protein content in the enzyme solution can be determined using the Broadford assay using a protein quantification reagent (Bradford Reagent: E211-01) purchased from Nanjing Novezan Biotechnology Co., Ltd.

[0035] 4. Determination of enzyme activity by DNS method

[0036] The reaction system is: 1475 μL pH buffer, 18 μL 100 mM Mn 2+To 500 μL of 1% colloidal chitosan solution, 25 μL of the purified enzyme solution was added. Under optimal pH and temperature conditions, the reaction system was incubated in a water bath for 5 minutes. The reaction was terminated by adding 1.5 mL of DNS solution, followed by boiling for 5 minutes. Finally, the volume was adjusted to 25 mL with distilled water, cooled, and allowed to stand for 1 hour. The absorbance of the sample was measured at 520 nm using a UV spectrophotometer. A 1 mg / mL N-acetylglucosamine standard solution was prepared for plotting a standard curve. Chitosanase activity was calculated using the standard curve equation. Chitosanase activity is defined as the amount of enzyme required to produce 1 μmol of reducing sugar in 1 minute from 1 mL of crude enzyme solution under the above reaction conditions. One unit (U / mL) of chitosanase activity is calculated. The enzymatic activity of the mutant enzymes is shown in the table below.

[0037] Table 3. Enzyme activities of mutant enzymes

[0038] mutant enzyme Optimum pH Optimum temperature / ℃ Specific enzyme activity (U / mg) WT 6.0 50 15620.46 E203F 6.0 50 1179.81 E203W 6.0 55 302.60 E203Y 6.0 45 2573.59 E203P 6.0 55 5306.96 E203N 6.0 60 5495.22 E203Q 6.0 60 8649.99 E203M 6.0 60 3636.80 E203T 6.0 60 4979.64 E203A 6.2 60 16309.98 E203D 6.4 50 2365.39 E203R 6.4 55 3072.79 E203H 6.4 55 1269.50 E203I 6.4 55 3643.35 E203L 6.4 55 2412.94 E203G 6.4 40 932.72 E203S 6.4 60 1861.64 E203V 6.6 60 5302.42 E203C 6.8 55 8457.77

[0039] 5. Chitosanase hydrolysis reaction

[0040] 450 μL of 1% colloidal chitosan solution, 20 U of purified enzyme solution, 500 μL of pH 6.6 phosphate buffer and 9 μL of 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.

[0041] Thin-layer chromatography (TLC) is performed by pipetting 2 μL of the hydrolyzed samples obtained at different times using a pipette or capillary tube and spotting them on a high-performance silica gel plate. A mixed solution of a GlcN2-5 standard and D-glucosamine hydrochloride is used as a control. After the spotted silica gel plate is thoroughly dried, place it in a chromatography tank and allow the chromatography solution to reach 1 cm from the top. This should take approximately one hour. After drying the plate free of ammonia, spray it evenly with a color developer (0.5% ninhydrin in ethanol), air dry it, and bake it in a 110°C oven for 10 to 20 minutes until clear purple-red spots appear.

[0042] Thin layer chromatography analysis revealed that the obtained chitosan-producing mutants had different chitosan-producing time periods: mutants E203M, E203S, E203R, and E203H did not produce chitosan after 5 h of hydrolysis, and mutants E203N, E203C, E203Q, E203G, E203T, E203V, and E203P did not produce chitosan after 3 h of hydrolysis. Compared with the original enzyme, all of the mutants took longer to produce chitosan, indicating that they had the potential to produce chitosan. The present invention screened out chitosanase that produces high-polymerization degree chitosan oligosaccharides based on saturated mutations at key sites of the substrate channel, and the chitosanase can be used for the industrial preparation of chitosan, which has obvious advantages in the preparation of active oligosaccharides.

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A chitosanase mutant capable of producing chitotetraose, characterized in that: The chitosanase mutant is a chitosanase BsCsn46A derived from Bacillus subtilis, in which the glutamic acid at position 203 is mutated to methionine, denoted as E203M, and its amino acid sequence is shown in SEQ ID NO: 3; or the glutamic acid at position 203 is mutated to serine, denoted as E203S, and its amino acid sequence is shown in SEQ ID NO: 5; or the arginine at position 203 is mutated to E203R, and its amino acid sequence is shown in SEQ ID NO: 7; or the histidine at position 203 is mutated to E203H.

2. A gene encoding the chitosanase mutant that hydrolyzes chitosan to produce chitotetraose according to claim 1.

3. A gene recombination vector according to claim 2.

4. A host bacteria comprising the recombinant vector as claimed in claim 3.

5. Use of the chitosanase mutant according to claim 1 in catalyzing the production of chitotetraose from chitosan.

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