Chitosan enzyme mutant for producing chitotetraose and application thereof

Abstract

The invention discloses a chitosanase mutant and application thereof in preparation of chitotetraose, and belongs to the field of enzyme engineering. According to the invention, site-specific saturation mutation is carried out on the 149th site of an amino acid sequence (SEQ ID NO: 1) of bacillus subtilis chitosanase BsCsn46A to screen out a mutant with chitotetraose potential in hydrolysate, aspartic acid is mutated into glycine, the glycine is marked as D149G, and the hydrolysate of the mutant comprises chitobiose, chitotriose and chitotetraose.

Description

Technical Field

[0001] The invention relates to a chitosanase mutant whose hydrolyzate has chitotetraose, specifically to a mutant obtained by site-directed mutagenesis, wherein the hydrolyzate 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 obtained by deacetylation of chitin, which is composed of randomly distributed N-acetylglucosamine (GlcNAc) units and D-glucosamine (GlcN) units linked by β-1,4-glycosidic bonds. Chitosan has multiple biological activities and good biosafety, and is widely used in food, medicine, agriculture and other fields. However, the application of chitosan is limited due to its poor water solubility and high viscosity.

[0003] Chitooligosaccharide (COS) is a hydrolysis product of chitosan, which has the effects of immunomodulation, anti-inflammatory, antibacterial, anti-tumor, neuroprotection, and hypolipidemic. Chitooligosaccharide has the advantages of small molecular weight, good solubility, easy absorption by the body, and no toxic side effects, so it plays a huge role in many fields such as health food, biomedicine, cosmetics, chemical industry, 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 molecular weight of the product is uneven, the yield is low, and the product polymerization degree is too high, which makes chitosan oligosaccharides unusable. Chemical methods include acid hydrolysis and oxidative degradation. Chemical reactions are not easy to control, the cost is high, and they will cause environmental pollution. The enzymatic preparation of COS can be divided into two schemes: non-specific enzyme hydrolysis and specific enzyme 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 obtain chitooligosaccharides. It produces chitooligosaccharides by hydrolyzing the β-1,4-glycosidic bonds of chitosan. Its advantages are that the hydrolysis products are uniform and the reaction conditions are mild, thus making it possible to produce chitooligosaccharides on a large scale. With the in-depth study of chitosanase, its crystal structure has been continuously analyzed, and the understanding of the relationship between its protein structure and hydrolysis mechanism has been deepened. At present, research reports have shown that chitooligosaccharides with a degree of polymerization greater than 4 have better activity, and most of the chitosanase hydrolysis products reported so far are chitobiose and chitotriose. Summary of the invention

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

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

[0008] The chitosanase mutant of the present invention performs site-directed mutation on the 149th position of the amino acid sequence of Bacillus subtilis chitosanase BsCsn46A, mutates aspartic acid to glycine, and marks it as 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 1 AGLNKDQKRRAEQLTSIFEN 21 GTTEIQYGYVERLDDGRGYT 41 CGRAGFTTATGDALEVVEVY 61 TKAVPNSKLKKYLPELRRLA 81 KEESDDTSNLKGFASAWKSL 101 ANDKEFRAAQDKVNDHLYYQ 121 NAMKRSDNAGLKTALARAVM 141 YDTVIQHGDGDDPDSFYALI 161 KRTNKKAGGSPKDGIDEKKW 181 LNKFLDVRYDDLMNPANHDT 201 RDEWRESVARVDVLRSIAKE 221 NNYNLNGPIHVRSNEYGNFV 241 IPs.

[0010] SEQ ID NO:2 GCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTTGAAAACGGCACAACGGAGATCCAATATGGATATGTAGAGCGATTGGATGACGGGCGAGGCTATACATGCGGTCGGGCAGGCTTTACAACGGCTACCGGGGATGCATTGGAAGTAGTGGAAGTATACACAAAGGCAGTTCCGAATAGCAAACTGAAAAAGTATCTGCCTGAATTGCGCCGTCTGGCCAAGGAAGAAAGCGATGATACAAGCAATCTCAAGGGATTCGCTTCTGCCTGGAAGTCGCTTGCAAATGATAAGGAATTTCGCGCCGCTCAAGACAAAGTAAATGACCATTTGTATTATCAGAATGCCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTGATGTACGATACGGTTATTCAGCATGGCGATGGTGATGACCCTGACTCTTTTTATGCCTTGATTAAACGTACGAACAAAAAAGCGGGCGGATCACCTAAAGACGGAATAGACGAGAAGAAGTGGTTGAATAAATTCTTGGACGTACGCTATGACGATCTGATGAATCCGGCCAATCATGACACCCGTGACGAATGGAGAGAATCAGTTGCCCGTGTGGACGTGCTTCGCTCTATCGCCAAGGAGAACAACTATAATCTAAACGGACCGATTCATGTTCGTTCAAACGAGTACGGTAATTTTGTAATCCCTTAA。

[0011] SEQ ID NO:3 1 AGLNKDQKRR AEQLTSIFEN 21 GTTEIQYGYV ERLDDGRGYT 41 CGRAGFTTAT GDALEVVEVY 61 TKAVPNSKLK KYLPELRRLA 81 KEESDDTSNL KGFASAWKSL 101 ANDKEFRAAQ DKVNDHLYYQ <h2 style=";text-align:left;direction:ltr">121 Names of the People<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 141 YDTVIQHGGG DDPDSFYALI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 161 KRTNKKAGGS PKDGIDEKKW<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 181 LNKFLDVRYD DLMNPANHDT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 201 RDEWRESVAR VDVLRSIAKE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 221 NNYNLNGPIH VRSNEYGNFV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 241 IP.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0012] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:4<h2 style=";text-align:left;direction:ltr"> GCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTTGAAAACGGCACAACGGAGATCCAATATGGATATGTAGAGCGATTGGATGACGGGCGAGGCTATACATGCGGTCGGGCAGGCTTTACAACGGCTACCGGGGATGCATTGGAAGTAGTGGAAGTATACAC AAAGGCATTCCGAATAGCAAACTGAAAAAGTATCTGCCTGAATTGCGCCGTCTGGCCAAGGAAGAAAGCGATGATACAAGCAATCTCAAGGGATTCGCTTCTGCCTGGAAGTCGCTTGCAAATGATAAGGAATTTCGCGCCGCTCAAGACAAAGTAAATGACCATTTGTATTATCAGAATG CCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTGATGTACGATACGGTTATTCAGCATGGCGGAGGTGATGACCCTGACTCTTTTTATGCCTTGATTAAACGTACGAACAAAAAAGCGGGCGGATCACCTAAAGACGGAATAGACGAGAAGAAGTGGTTGAAT AAATTCTTGGACGTACGCTATGACGATCTGATGAATCCGGCCAATCATGACACCCGTGACGAATGGAGAGAATCAGTTGCCCGTGTGGACGTGCTTCGCTCTATCGCCAAGGAGAACAACTATAATCTAAACGGACCGATTCATGTTCGTTCAAACGAGTACGGTAATTTTGTAATCCCTTAA 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.

[0013] 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.

[0014] The specific application method is to collect the bacteria after inducing the 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.

[0015] The invention constructs the three-dimensional structure of the enzyme through SWISS-MODEL, analyzes the substrate channel of the enzyme, performs saturation mutation on the key site 149 that affects the polymerization degree of the product, screens the saturated mutant library to obtain mutants that affect the polymerization degree of the product, wherein the aspartic acid at the 149th position is mutated to glycine, and compared with the original enzyme, the action time of the enzyme for hydrolyzing and producing chitotetraose is significantly increased, and the enzyme can be used for the industrialized bioenzymatic method to produce chitosan oligosaccharides with high polymerization degree. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Thin layer chromatograms of chitosan degradation products of wild-type chitosanase and D149E mutant.

[0017] Figure 2 Thin layer chromatograms of chitosan degradation products of chitosanase D149A and D149K mutants.

[0018] Figure 3 Thin layer chromatograms of chitosan degradation products of chitosanase D149R and D149H mutants.

[0019] Figure 4 Thin layer chromatograms of chitosan degradation products of chitosanase D149N and D149C mutants.

[0020] Figure 5 Thin layer chromatograms of chitosan degradation products of chitosanase D149Q and D149G mutants.

[0021] Figure 6 Thin layer chromatograms of chitosan degradation products of chitosanase D149M and D149S mutants.

[0022] Figure 7 Thin layer chromatograms of chitosan degradation products of chitosanase D149T and D149I mutants.

[0023] Figure 8 Thin layer chromatograms of chitosan degradation products of chitosanase D149L and D149F mutants.

[0024] Fig. 9 3. It is the thin layer chromatogram of chitosan degradation products of chitosanase D149W and D149Y mutants.

[0025] Fig.10 Thin layer chromatograms of chitosan degradation products of chitosanase D149V and D149P mutants. DETAILED DESCRIPTION

[0026] The three-dimensional structure of the enzyme was constructed by SWISS-MODEL, the substrate channel of the enzyme was analyzed, and the key sites that affect the polymerization degree of the product were obtained through representative amino acid mutations, and saturation mutations were performed on the key sites. The saturation mutant library was screened to obtain excellent mutants that affect the polymerization degree of the product.

[0027] The original enzyme was the high-activity chitosanase BsCsn46A obtained in the early stage of the experiment. The key site found in the analysis was the 149th site of the amino acid sequence of BsCsn46A. The primer design of the mutant was designed by the online software PrimerX. The sequence is shown in the table below. The chitosanase gene was then synthesized by molecular biology methods.

[0028] PCR amplification was performed using chitosanase BsCsn46A gene SEQ ID NO: 2 as a template and primer sequences as shown in Table 1.

[0029] Table 1. Primer sequences for saturation mutant library 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]]> 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.

[0030] Digest with DpnI for 2 hours to remove the plasmid template. Precool the remaining reaction and take 10 μL, then transform it into E. coli DH5α. Sample the bacteria containing the recombinant gene for sequencing, and compare the sequences to ensure that only the amino acid at the mutation site is mutated. Extract the plasmid from the bacteria with the correct sequencing and then transfer it into the heterologous expression host E. coli BL21. Store the glycerol bacteria in a frozen state.

[0031] The preserved strain was inoculated into 10 mL of liquid LB medium at a one thousandth inoculation volume for activation, and then transferred to 50 mL of liquid LB medium for expansion culture, and then the inducer IPTG (isopropyl-β-D-thiogalactopyranoside) was added to induce protein production.

[0032] Protein purification operation: The bacteria were collected and ultrasonically disrupted by an ultrasonic disruptor; the supernatant was purified by Ni-IDA affinity chromatography. The supernatant was loaded onto the Ni-IDA column, 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, pH 8.0). The eluted protein samples were collected and stored at -20 °C. The separated components were analyzed by SDS-PAGE protein electrophoresis, and the protein content in the enzyme solution was determined by a protein quantitative reagent. The kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. (Bradford Reagent: E211-01).

[0033] 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 1% colloidal chitosan solution, add 25 μL purified enzyme solution, water bath for 5 min under the measured optimal pH and optimal temperature conditions, add 1.5 mL DNS solution to terminate the reaction, boil with boiling water for 5 min, and finally dilute to 25 mL with distilled water, cool and stand for 1 h. No enzyme was added to the blank group. The absorbance value 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 according to the standard curve, and the chitosanase activity was defined as: the amount of enzyme used to generate 1 μmol of reducing sugar per minute is defined as one enzyme activity unit (U). The enzyme activity data of the relevant mutant enzymes are as follows.

[0034] Table 3. Enzyme activities of mutant enzymes 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 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 the 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 it for 10 min and retain it.

[0035] TLC analysis. The operation method of thin layer chromatography is as follows: use a capillary to absorb the hydrolyzate samples at different times, spot them on a high-efficiency silica gel plate, and use a mixed solution of GlcN1-5 standard products as a control. After the spotted silica gel plate is fully dried, place it in a chromatography bar and fully unfold it. Take out the silica gel plate and blow it dry with a hair dryer until there is no ammonia smell, evenly spray the color developer (0.5% ninhydrin-ethanol solution), blow dry, and place it in a 110 ℃ oven until clear purple-red spots appear.

[0036] Combined with the analysis of thin layer chromatography results, mutant D149G did not produce tetraose in the hydrolysis of 3h, and the time of producing tetraose was longer than that of the original enzyme, and it had a better ability to produce tetraose. High DP chitosan oligosaccharides have better biological activity than low DP chitosan oligosaccharides, and the obtained mutants have obvious advantages in the preparation of active oligosaccharides. The mutants that produce tetraose based on the saturated mutation of the enzyme can be used for industrial production, and the enzyme can also be further modified based on this to obtain chitosanase with high enzyme activity and high product polymerization degree.

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 gene of a chitosanase mutant, characterized in that it encodes the chitosanase mutant producing chitotetraose according to claim 1.

3. A gene of the chitosanase mutant as claimed in 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 chitooligosaccharides.

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

9. The use of the chitosanase mutant according to claim 7, characterized in that: The application method is to collect the bacteria after inducing the 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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