Chitosan enzyme mutant with capacity of producing chitotetraose and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and discloses a chitosanase mutant with chitotetraose production capacity and application of the chitosanase mutant. GH46 family glycoside hydrolase chitosanase (BsCsn46A) derived from bacillus subtilis has an amino acid sequence as shown in SEQ ID NO: 1, the 203th amino acid residue of the BsCsn46A is subjected to saturated mutation, mutants with the chitotetraose production potential are screened out, and the obtained chitosanase mutants are E203M, E203S, E203R, E203H, E203N, E203C, E203Q, E203G, E203T, E203V and E203P. Through hydrolysis result analysis, the obtained mutant has the capacity of producing chitotetraose.

Description

Technical Field

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

[0002] Chitosan (CS) is a cationic natural alkaline polysaccharide formed by connecting glucosamine (GlcN) through β-1,4-glycosidic bonds. Chitosan oligosaccharide (COS), also known as oligosaccharide, is an oligomer of glucosamine connected by β-1,4-glycosidic bonds. It is the only positively charged cationic alkaline oligosaccharide in nature, with a degree of polymerization of 2-20 and a molecular weight of ≤3200Da. COS has the characteristics of good water solubility at physiological pH, easy absorption, and high biological activity. Chitosan oligosaccharide has good biosafety and has the effects of immunomodulation, anti-inflammatory, antibacterial, anti-tumor, antioxidant, and hypolipidemic, so it has broad application prospects in food, medical, green agriculture, and cosmetics industries.

[0003] At present, the production of chitosan oligosaccharides mainly includes chemical acid hydrolysis, physical degradation, and biological enzymatic hydrolysis. In the process of chemical acid hydrolysis, the O-glycosidic bond and N-acetyl bond of chitosan will be hydrolyzed, and there are many problems such as difficult to control the conditions and loss of activity; the physical degradation method has high product purity, but low yield, and large-scale production is limited; the enzymatic hydrolysis process of the biological enzymatic hydrolysis method has the advantages of mild reaction conditions, high stability, no environmental pollution, and can achieve large-scale sustainable production economically. Therefore, the enzymatic hydrolysis method has become one of the current research hotspots in the production of chitosan oligosaccharides.

[0004] Chitosanase (EC3.2.1.132), also known as N-acetylglucosamine hydrolase, can specifically catalyze chitosan to generate chitooligosaccharides or glucosamine. It was first proposed by Monaghan et al. in 1973. According to the different ways of cutting on the substrate, it can be divided into endo-chitosanase and exo-chitosanase. Chitosanase is mainly distributed in GH8, GH46, GH75 and GH80 families, among which the GH46 family chitosanase has been studied most deeply.

[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 previously found 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 conducted a study on 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 mutant is a chitosanase BsCsn46A in which the 203rd glutamic acid is mutated to methionine, which is marked as E203M; a chitosanase BsCsn46A ... The amino acid sequence of Bacillus subtilis chitosanase BsCsn46A is SEQ ID NO: 1, and the nucleotide sequence is SEQ ID NO: 2. The amino acid sequence of the chitosanase mutant E203M is SEQ ID NO: 3, and the nucleotide sequence is SEQ ID NO: 4; the amino acid sequence of the chitosanase mutant E203S is SEQ ID NO: 5, and the nucleotide sequence is SEQ ID NO: 6; the amino acid sequence of the chitosanase mutant E203R is SEQ ID NO: 7, and the nucleotide sequence is SEQ ID NO: 8; the amino acid sequence of the chitosanase mutant E203H is SEQ ID NO: 9, and the nucleotide sequence is SEQ ID NO: 10; the amino acid sequence of the chitosanase mutant E203N is SEQ ID NO: 11, and the nucleotide sequence is SEQ ID NO: 12; the amino acid sequence of the chitosanase mutant E203C is SEQ ID NO: 13, and the nucleotide sequence is SEQ ID NO: 14; the amino acid sequence of the chitosanase mutant E203Q is SEQ ID NO: 15, and the nucleotide sequence is SEQ ID 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 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 by Swiss-Model online software to obtain the spatial structure of chitosanase; the key site (E203) affecting the hydrolysis degree of the product is obtained by substrate channel simulation and molecular docking analysis; primers for site-directed mutation are designed, chitosanase gene is obtained by PCR amplification, and a recombinant vector is formed with the expression vector pET-28a, and then transferred into Escherichia coli E. coli BL21 (DE3); after IPTG induction, the expression was collected, the cells were broken by ultrasound and then the supernatant was collected by centrifugation, and the protein was 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 the advantage of being applied to hydrolysis to produce active oligosaccharide chitotetraose.

[0012] Through thin layer chromatography analysis, it was found that the obtained mutants producing chitosan had different chitosan production times during hydrolysis. Mutants E203M, E203S, E203R, and E203H did not produce chitosan after 5 h of hydrolysis, and E203N, E203C, E203Q, E203G, E203T, E203V, and E203P did not produce chitosan after 3 h of hydrolysis. Compared with the original enzyme, all of them had longer chitosan production times during hydrolysis, and were mutants with the potential to produce chitosan. The chitosanase producing high-polymerization degree chitosan oligosaccharides was screened out based on saturated mutations at key sites of the substrate channel, and the chitosanase can be used for industrial preparation of chitosan, which has obvious advantages in preparing 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] Fig. 9 Thin layer chromatograms of chitosan degradation products of chitosanase mutants E203D and E203R.

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

[0023] Previous studies (CN116590260A) have identified position 203 located in the substrate channel as a potential site affecting catalytic activity; saturation mutation of position 203 was performed to screen out mutants with the hydrolysis product chitosan tetraose. Among them, the chitosanase mutant E203K in which the glutamic acid at position 203 was mutated to lysine has been confirmed to have the ability to produce chitosan tetraose, and is no longer studied in the present invention. The construction methods of chitosanase mutants E203A and E203W refer to CN116949015A.

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

[0025] Table 1: Primer sequences for the saturation mutant library 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]]> 2. Construction of recombinant plasmids and recombinant bacteria Table 2. Inverse PCR system 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 Reverse PCR amplification conditions: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing at 65 °C for 1 min, extension at 68 °C for 10 min, 15 cycles; insulation at 4 °C.

[0026] The amplified PCR product was digested with 0.7 μL DpnI for 1.5 h to remove the plasmid template. The competent E. coli DH5α was placed in an ice bath and 10 μL of the above reaction mixture was added to form a transformation system to 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. Glycerol culture was stored at -80℃.

[0027] 3. Protein induced expression and purification The mutant strain obtained above was inoculated into 10 mL of liquid LB medium at a one thousandth inoculation rate 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.

[0028] The above bacterial solution was collected and centrifuged with a refrigerated centrifuge to collect the bacteria, the cells were broken with an ultrasonic cell disruptor, and the supernatant was collected and placed on ice. The nickel column was taken out of the refrigerator, turned upside down to make the nickel column fluffy, and the column was washed with pure water. The collected supernatant was passed through the nickel column twice, and the unbound protein was fully eluted with the loading buffer, and then the protein was eluted with the elution buffer (20 mM Tris-HCl, 0.5 M NaCl, 0.08 M imidazole, 10% glycerol, pH8.0), and the eluted protein sample was collected. The protein content in the enzyme solution can be determined by the Broadford method, and the protein content in the enzyme solution can be determined by the protein quantification reagent. The kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. (Bradford Reagent: E211-01).

[0029] 4. DNS method for enzyme activity determination The reaction system is: 1475 μL pH buffer, 18 μL 100 mM Mn 2+ , 500 μL 1% colloidal chitosan solution, add 25 μL purified enzyme solution, under the optimal pH and temperature conditions, the reaction system was water bathed for 5 min, 1.5 mL DNS solution was added to terminate the reaction, and then boiled in boiling water for 5 min, and finally distilled water was used to make the volume 25 mL, cooled and allowed to stand for 1 h, and then the absorbance of the sample was measured at a wavelength of 520 nm using a UV spectrophotometer. Prepare 1 mg / mL N-acetylglucosamine standard solution to draw a standard curve. Calculate chitosanase activity according to the standard curve equation. Chitosanase activity is defined as the amount of enzyme required to produce 1 µmol reducing sugar in 1 min for 1 mL of crude enzyme solution under the above reaction conditions, which is 1 enzyme activity unit (U / mL). The enzyme activity of the mutant enzyme is shown in the following table.

[0030] Table 3. Enzyme activities of mutant enzymes 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 5. Hydrolysis reaction of chitosanase 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 the reaction system. 2+ , place at 37 ℃, 160 rpm shaker for full reaction, centrifuge the hydrolysis system at 5min, 30min, 1h, 3h, 5h, 7h, 9h, and 11h, take 20 μL of the supernatant, boil it for 10 min and retain it.

[0031] Thin layer chromatography. The operation method is: use a pipette or capillary to absorb 2 μL of the hydrolyzed samples at different times and spot them on a high-efficiency silica gel plate. At the same time, use a mixed solution of GlcN2-5 standard and D-glucosamine hydrochloride as a reference. After the spotted silica gel plate is fully dried, place it in a chromatography tank and develop it until the chromatography liquid climbs to 1 cm from the top, which takes about an hour. Take out the plate and dry it without ammonia water, then spray the color developer (0.5% ninhydrin ethanol solution) evenly, blow dry it, and place it in a 110 ℃ oven for 10 to 20 minutes until clear purple-red spots appear.

[0032] Through thin layer chromatography analysis, it was found that the obtained mutants producing chitosan had different chitosan production times during hydrolysis. Mutants E203M, E203S, E203R, and E203H did not produce chitosan after 5 h of hydrolysis, and E203N, E203C, E203Q, E203G, E203T, E203V, and E203P did not produce chitosan after 3 h of hydrolysis. Compared with the original enzyme, all of them had longer chitosan production times during hydrolysis, and were mutants with the potential to produce chitosan. The chitosanase producing high-polymerization degree chitosan oligosaccharides was screened out based on saturated mutations at key sites of the substrate channel, and the chitosanase can be used for industrial preparation of chitosan, which has obvious advantages in preparing active oligosaccharides.

[0033] 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 principle 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, wherein the glutamic acid at position 203 is mutated to methionine, which is denoted as E203M, and its amino acid sequence is shown in SEQ ID NO:3; or the 203rd position is mutated to serine, which is denoted as E203S, and its amino acid sequence is shown in SEQ ID NO:5; or the 203rd position is mutated to arginine, which is denoted as E203R, and its amino acid sequence is shown in SEQ ID NO:7; or the 203rd position is mutated to histidine, which is denoted as E203H, and its amino acid sequence is shown in SEQ ID NO:9; or the 203rd position is mutated to asparagine, which is denoted as E203N, and its amino acid sequence is shown in SEQ ID NO:11; or the 203rd position is mutated to cysteine, which is denoted as E203C, and its amino acid sequence is shown in SEQ ID NO:13; or the 203rd position is mutated to glutamine, which is denoted as E203Q, and its amino acid sequence is shown in SEQ ID NO:15; or the 203rd position mutates to glycine, denoted as E203G, and its amino acid sequence is shown in SEQ ID NO:17; or the 203rd position mutates to threonine, denoted as E203T, and its amino acid sequence is shown in SEQ ID NO:19; or the 203rd position mutates to valine, denoted as E203V, and its amino acid sequence is shown in SEQ ID NO:21; or the 203rd position mutates to proline, denoted as E203P, and its amino acid sequence is shown in SEQ ID NO:

23.

2. A gene encoding the chitosanase mutant for hydrolyzing chitosan to produce chitotetraose as claimed in claim 1.

3. A gene recombination vector carrying the chitosanase mutant according to claim 1 or 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 chitooligosaccharides from chitosan.

6. The use according to claim 5, characterized in that: The chitosanase mutant catalyzes chitosan to produce chitotetraose.

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