CN113151226A - Fusion chitinase for efficiently degrading alpha-chitin and related biological material and application thereof - Google Patents
Fusion chitinase for efficiently degrading alpha-chitin and related biological material and application thereof Download PDFInfo
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- C12N9/2405—Glucanases
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- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
Abstract
The invention discloses a fusion chitinase for efficiently degrading alpha-chitin, a related biological material and application thereof. The fusion chitinase is a protein obtained by fusing beta-N-acetylhexosaminidase and chitinase together, wherein the activity of the chitinase is higher than that of the beta-N-acetylhexosaminidase and the chitinase; the chitinase is D1) or D2): D1) a protein having an amino acid sequence of amino acid residues 53 to 322 of SEQ ID No. 2; D2) a fusion protein obtained by labeling the carboxyl terminal or/and amino terminal fusion protein of the protein shown in D1). The fusion chitinase has degradation effect on various types of chitin, and has high activity on colloidal chitin, alpha-chitin and fungal cell wall chitin; has the ability to completely hydrolyze chitin to produce N-acetylglucosamine. The invention can be widely used in biological medicine, biological agriculture, cosmetics, energy industry and the like.
Description
Technical Field
The invention relates to a fusion chitinase for efficiently degrading alpha-chitin in the biological field, a related biological material and application thereof.
Background
Chitin (Chitin) is a long-chain polymer formed by connecting N-acetyl-D-glucosamine through beta-1, 4-glycosidic bonds, and is the second most abundant renewable natural carbon-based high molecular compound next to cellulose on the earth. Meanwhile, chitin is also the second largest nitrogen-containing compound second to proteins in the world. The chitin is widely present in exoskeletons of marine organisms, squid cartilage, fungi, insects and internal structures of invertebrates, is an important raw material for preparing chitosan and glucosamine, and has wide application in the aspects of medicine, chemical industry, environmental protection, feed additives and health-care food.
The alpha-chitin is in different existing forms, is arranged in parallel in a polymerized single chain trans form and mainly exists in exoskeletons of arthropods such as shrimps and crabs, and has wide intermolecular hydrogen bonds, is hard and is not easy to hydrolyze; the polymerized single chains of the beta-chitin are arranged in a parallel mode and mainly exist in squid cartilage, and the beta-chitin lacks intermolecular hydrogen bonds, has good flexibility and is easy to generate crystal expansion and hydrolysis, so that enzyme is easy to combine with the crystal and act; gamma-chitin, which consists of two parallel single polymeric chains and one antiparallel single polymeric chain, is present in beetle larvae and cephalopods. Fungi are also one of the main sources of chitin in nature, which is present in the propagules (hyphae, spores, etc.) and cell walls of fungi.
The colloidal chitin is artificially strongly acid-treated chitin, is different from a naturally-occurring chitin form, has a loose structure and is easy to hydrolyze; alpha-chitin is the main existing form of natural chitin resources, has a compact structure and is difficult to hydrolyze and utilize.
The production of high value-added chemicals and fuels by biomass conversion is the key to realizing low-carbon sustainable development economy in the future. The degradation product of chitin has wide applicability and economic value. Chitin hydrolysate N-acetylglucosamine (GlcNAc) is widely used for the treatment of inflammatory bowel disease, multiple necrosis and skin atrophy; the glucosamine derivative can be used for regenerating articular cartilage and treating chronic enteritis; the N-acetylglucosamine has sweet taste, and can replace saccharides such as glucose and the like for producing foods and health care products due to the antioxidation and the stimulation of the immune activity of the organism; meanwhile, the chitosan oligosaccharide and the N-acetylglucosamine can also be used for feeds, cosmetics, biofuels and soil fertilizers, and have high economic value. At present, chitin oligosaccharide and N-acetylglucosamine are mainly produced by a strong acid hydrolysis chemical method with high pollution and low efficiency, and a microbial enzyme method for hydrolyzing chitin has the characteristics of environmental friendliness, low cost and high repeatability, so that a bioconversion technology taking chitin as a substrate is the direction of industrial development.
The Chinese patent application with the application number of 201910716758.8 and the publication number of CN110387364A discloses recombinant chitinase (fusion chitinase) His-SaChi18E-HEX-His for efficiently degrading chitin. The fusion enzyme is composed of a chitinase Sachi18E of a GH18 family and a beta-N-acetylhexosaminidase SaHEX.
The His-SaChi18E-HEX-His hydrocolloid chitin system reached a maximum GlcNAc yield of 98.5% at 4 hours of reaction, and the enzyme catalysis efficiency of His-SaChi18E-HEX-His on the hydrocolloid chitin (the efficiency of hydrocolloid chitin) was 2 times that of the combination of His-SaChi18E-His and His-SaHEX-His.
Disclosure of Invention
The invention aims to solve the technical problem of how to obtain the fusion chitinase for efficiently degrading alpha-chitin in one step.
In order to solve the above technical problems, the present invention provides a fusion protein.
The fusion protein provided by the invention is a protein obtained by fusing a chitinase SacHIB of a GH19 family and a beta-N-acetylhexosaminidase SaHEX together, wherein the chitin degrading activity of the protein is higher than that of the single beta-N-acetylhexosaminidase and the single chitinase, and the chitinase can be D1) or D2): D1) a protein having an amino acid sequence of amino acid residues 53 to 322 of SEQ ID No. 2; D2) a fusion protein obtained by labeling the carboxyl terminal or/and amino terminal fusion protein of the protein shown in D1).
In the above fusion protein, both the β -N-acetylhexosaminidase and the chitinase may be derived from Streptomyces, such as Streptomyces alfalfalfalfa.
In the above fusion protein, the β -N-acetylhexosaminidase may be a protein of A1) or A2):
A1) a protein whose amino acid sequence is amino acid residue 323-833 of SEQ ID No. 2;
A2) a fusion protein obtained by labeling the carboxyl terminal or/and amino terminal fusion protein of the protein shown in A1).
In the above fusion protein, the fusion protein may be any one of F1) -F3):
F1) a protein having the amino acid sequence of SEQ ID No. 2;
F2) a protein having an amino acid sequence of positions 53-833 of SEQ ID No. 2;
F3) a fusion protein obtained by labeling the carboxyl-terminal or/and amino-terminal fusion protein of the protein shown in F1) or F2).
Wherein SEQ ID No.2 consists of 841 amino acid residues. F1) The fusion protein of (a) is named as His-SaChiB-HEX-His; F2) the name of the fusion protein is SaChiB-HEX.
In the above fusion protein, the tag protein (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracing and/or purification of the target protein. The tag protein can be Flag tag protein, His tag protein, MBP tag protein, HA tag protein, myc tag protein, GST tag protein and/or SUMO tag protein, etc.
The biological materials related to the fusion protein also belong to the protection scope of the invention.
The biomaterial related to the fusion protein may be at least one of the following B1) -B7):
B1) a nucleic acid molecule encoding the fusion protein;
B2) an expression cassette comprising the nucleic acid molecule of B1);
B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);
B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;
B5) a transgenic plant cell line containing B1) the nucleic acid molecule, or a transgenic plant cell line containing B2) the expression cassette, or a transgenic plant cell line containing B3) the recombinant vector;
B6) a transgenic plant tissue containing B1) the nucleic acid molecule, or a transgenic plant tissue containing B2) the expression cassette, or a transgenic plant tissue containing B3) the recombinant vector;
B7) a transgenic plant organ containing B1) the nucleic acid molecule, or a transgenic plant organ containing B2) the expression cassette, or a transgenic plant organ containing B3) the recombinant vector.
In the above biological material, B1) the nucleic acid molecule may be B11) or B12) as follows:
B11) the coding sequence is a DNA molecule of SEQ ID No. 1;
B12) the nucleotide sequence is the DNA molecule at position 157-2499 of SEQ ID No. 1.
In the above biological material, SEQ ID No.1 consists of 2526 nucleotides, and its coding sequence is nucleotides 1 to 2526. B11) Is His-SaChiB-HEX-His gene; B12) is a SacHIB-HEX gene.
In the above-mentioned biological material, the recombinant vector described in B3) may be specifically pET30a (+) -SachB-HEX, pET30a (+) -SachB-HEX is a recombinant expression vector obtained by replacing a fragment (small fragment including an EcoRI recognition site and an XhoI recognition site) between EcoRI and XhoI recognition sites of pET30a (+) with DNA having a nucleotide sequence of the 151 th 2505 th site of SEQ ID No.1, and leaving the other sequence of pET30a (+) unchanged.
In the above biological material, the recombinant microorganism of B4) may be a recombinant microorganism in which a gene encoding the protein is introduced into a recipient microorganism, and the recipient microorganism may be any one of C1) to C4):
C1) a prokaryotic microorganism;
C2) bacteria of the enterobacteriaceae family;
C3) an Escherichia bacterium;
C4) coli, e.g. e.coli BL21(DE 3).
Among the above-mentioned biomaterials, the recombinant microorganism of B4) may be specifically a recombinant escherichia coli expressing a recombinant protein (named as His-SaChiB-HEX-His) having an amino acid sequence of SEQ ID No.2, which is obtained by introducing the pET30a (+) -SaChiB-HEX into escherichia coli e.coli BL21(DE 3).
Of the above-mentioned biological materials, B5) to B7) may or may not include propagation material.
The invention also provides a method for preparing the fusion protein.
The method for preparing the fusion protein provided by the invention comprises the following steps: expressing the encoding gene of the fusion protein in an organism to obtain the fusion protein; the organism is a microorganism, a plant or a non-human animal.
In the above method, the microorganism may be any one of C1) -C4):
C1) a prokaryotic microorganism;
C2) bacteria of the enterobacteriaceae family;
C3) an Escherichia bacterium;
C4) coli, e.g. e.coli BL21(DE 3).
The application of the fusion protein in chitinase or chitin hydrolysis also belongs to the protection scope of the invention. The use may be for non-disease diagnostic purposes, non-disease prophylactic purposes and/or non-disease therapeutic purposes.
The chitin may be alpha-chitin, colloidal chitin, and/or fungal cell wall chitin.
The fusion protein, the biological material and the application of the method in preparing the chitinase preparation also belong to the protection scope of the invention.
The chitinase preparation may hydrolyze alpha-chitin, colloidal chitin, and/or fungal cell wall chitin.
The invention fuses chitinase SacHIB derived from Streptomyces alfalfa and beta-N-acetylhexosaminidase SaHEX derived from Streptomyces alfa to obtain recombinant protein His-SacHIB-HEX-His with chitinase activity higher than that of independent chitinase His-SacHIB-His and independent beta-N-acetylhexosaminidase His-SaHEX-His. Experiments prove that the optimum pH value of the fusion chitinase is 7.0, and the fusion chitinase has higher enzyme activity (more than 60 percent of enzyme activity) at the temperature of 30 ℃ and the pH value of 4.0-7.5; the pH stability is good, and the pH is stable between 4.5 and 8.5; the optimum temperature is 30 ℃, and the enzyme activity is more than 60% at 20-50 ℃. The chitinase specific activities (U/mol enzyme protein) of His-SaChiB-HEX-His, His-SaChiB-His and His-SaHEX-His to colloidal chitin are 2135.94 +/-265.98U/mu mol enzyme protein, 823.78 +/-14.52U/mu mol enzyme protein and 250.49 +/-0.56U/mu mol enzyme protein respectively. The yield of GlcNAc reached the maximum of 99.6% in the His-SaChiB-HEX-His hydrolyzed alpha-chitinase system at 8 hours of the reaction, and the yield of GlcNAc reached the maximum of 94.5% in the His-SaChiB-His + His-SaHEX-His hydrolyzed alpha-chitinase system at 24 hours of the reaction, and the enzyme catalytic efficiency (efficiency of hydrolyzing alpha-chitin) of the fusion chitinase His-SaChiB-HEX-His on alpha-chitin was 3.2 times that of the combination of His-SaChiB-His and His-SaHEX-His. The His-SaChiB-His hydrolyzed alpha-chitin system reached a maximum GlcNAc yield of 84.8% at 12 hours of reaction, and the catalytic efficiency of the fusion chitinase His-SaChiB-HEX-His on alpha-chitin (efficiency of hydrolyzing alpha-chitin) was 1.8 times that of His-SaChiB-His. His-SaHEX-His alone cannot hydrolyze alpha-chitin (FIG. 2). The His-SaChi18E-HEX-His hydrolyzed alpha-chitin system reached a maximum GlcNAc yield of 89.1% at 24 hours of reaction, and the catalytic efficiency of the fusion chitinase His-SaChiB-HEX-His on alpha-chitin (efficiency of hydrolyzing alpha-chitin) was 3.4 times that of His-SaChi18E-HEX-His (fig. 3). The results show that the specific activity of His-SaChiB-HEX-His hydrolyzed alpha-chitin is 162.05 +/-38.64U/mu mol, and the specific activity of His-SaChi18E-HEX-His hydrolyzed alpha-chitin is 115.75 +/-27.61U/mu mol. Indicating that the chitinase activity of His-SaChiB-HEX-His for hydrolyzing alpha-chitin is 1.4 times that of His-SaChi 18E-HEX-His.
The fusion chitinase His-SaChiB-HEX-His and SaChiB-HEX have degradation effect on various types of chitin, and have higher activity on colloidal chitin, alpha-chitin and fungal cell wall chitin; has the ability to completely hydrolyze chitin to produce N-acetylglucosamine; compared with other reported chitinase systems, the method has the highest reaction efficiency, and can be applied to the industrial enzymatic decomposition of chitin to prepare the beta-N-acetyl-D-glucosamine. The fused chitinase can be widely used in biomedicine (such as producing N-acetylglucosamine), biological agriculture (such as food), biological agriculture (such as feed additive), cosmetics, energy industry and the like.
Drawings
FIG. 1 is an SDS-PAGE analysis of a protein of interest. M is a protein molecular weight standard; 1 is a nickel column purified target protein sample obtained from BL21(DE3)/pET30a (+) -SaChiB thalli; 2, a nickel column purified target protein sample obtained from BL21(DE3)/pET30a (+) -SaHEX thalli; 3 is a nickel column purified target protein sample obtained from BL21(DE3)/pET30a (+) -SaChiB-HEX bacteria.
FIG. 2 shows the yields of N-acetylglucosamine (GlcNAc) at various times of reaction of His-SaChiB-HEX-His hydrolyzed alpha-chitin system (indicated as "His-SaChiB-HEX-His" in the figure), His-SaChiB-His hydrolyzed alpha-chitin system (indicated as "His-SaChiB-His" in the figure), and His-SaChiB-His + His-SaHEX-His hydrolyzed alpha-chitin system (indicated as "His-SaChiB-His + His-SaHEX-His" in the figure).
FIG. 3 shows the yields of N-acetylglucosamine (GlcNAc) at different times of reaction of His-SaChiB-HEX-His hydrolyzed alpha-chitin system (indicated as "His-SaChiB-HEX-His") and His-SaChi18E-HEX-His hydrolyzed alpha-chitin system (indicated as "His-SaChi 18E-HEX-His").
FIG. 4 is His-SaChiB-HEX-His waterHPLC chromatogram of alpha-chitin system reaction for 8 hours. In the figure, the upper diagram is an HPLC chromatogram of a standard obtained by mixing DP1, DP2, DP3, DP4, DP5 and DP6 at a mass ratio of 1:1, and DP1, DP2, DP3, DP4, DP5 and DP6 are respectively a standard GlcNAc, (GlcNAc)2、(GlcNAc)3、(GlcNAc)4、(GlcNAc)5And (GlcNAc)6(ii) a The lower panel is an HPLC chromatogram of a His-SaChiB-HEX-His hydrolyzed alpha-chitin system for 8 hours.
FIG. 5 is an HPLC chromatogram of a His-SaChiB-His + His-SaHEX-His hydrolyzed alpha-chitin system reaction for 24 hours. In the figure, the upper diagram is an HPLC chromatogram of a standard obtained by mixing DP1, DP2, DP3, DP4, DP5 and DP6 at a mass ratio of 1:1, and DP1, DP2, DP3, DP4, DP5 and DP6 are respectively a standard GlcNAc, (GlcNAc)2、(GlcNAc)3、(GlcNAc)4、(GlcNAc)5And (GlcNAc)6(ii) a The lower panel is an HPLC chromatogram of a His-SaChiB-His + His-SaHEX-His hydrolysis alpha-chitin system reaction for 24 hours.
FIG. 6 is an HPLC chromatogram of a His-SaChiB-His hydrolyzed alpha-chitin system reaction for 12 hours. In the figure, the upper diagram is an HPLC chromatogram of a standard obtained by mixing DP1, DP2, DP3, DP4, DP5 and DP6 at a mass ratio of 1:1, and DP1, DP2, DP3, DP4, DP5 and DP6 are respectively a standard GlcNAc, (GlcNAc)2、(GlcNAc)3、(GlcNAc)4、(GlcNAc)5And (GlcNAc)6(ii) a The lower panel is an HPLC chromatogram of a His-SaChiB-His hydrolyzed alpha-chitin system for 12 hours.
FIG. 7 is an HPLC chromatogram of a His-SaChi18E-HEX-His hydrolyzed alpha-chitin system for 24 hours. In the figure, the upper diagram is an HPLC chromatogram of a standard obtained by mixing DP1, DP2, DP3, DP4, DP5 and DP6 at a mass ratio of 1:1, and DP1, DP2, DP3, DP4, DP5 and DP6 are respectively a standard GlcNAc, (GlcNAc)2、(GlcNAc)3、(GlcNAc)4、(GlcNAc)5And (GlcNAc)6(ii) a The lower panel is an HPLC chromatogram of a His-SaChi18E-His hydrolyzed alpha-chitin system for 24 hours.
FIG. 8 shows the results of pH optimum assay of the fusion chitinase His-SaChiB-HEX-His.
FIG. 9 shows the results of pH stability assay of the fusion chitinase His-SaChiB-HEX-His.
FIG. 10 shows the results of temperature optima measurement of the fusion chitinase His-SaChiB-HEX-His.
FIG. 11 shows the results of measuring the temperature stability of the fusion chitinase His-SaChiB-HEX-His.
FIG. 12 shows the results of Thin Layer Chromatography (TLC). In the figure, M is standard GlcNAc, (GlcNAc)2、(GlcNAc)3、(GlcNAc)4、(GlcNAc)5And (GlcNAc)6(ii) a 1.2, 3 and 4 are each (GlcNAc)2His-SaChiB-His hydrolysis (GlcNAc)2Product of (3), His-SaHEX-His hydrolysis (GlcNAc)2And His-SaChiB-HEX-His hydrolysis (GlcNAc)2The product of (1).
FIG. 13 shows the morphological changes of His-SaChiB-HEX-His hydrolyzed alpha-chitin analyzed by electron microscopy. A is untreated, B is enzymolysis for 1h, and C is enzymolysis for 8 h.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples, the expression vector pET30a (+) was a product of invitrogen (usa), and the competent cell e.coli BL21(DE3) was a product of transcgen Biotech Co.
The standard N-acetylglucosamine (GlcNAc) used in the examples described below was BBI Life Sciences, having a product number of A602245-0025.
The colloidal chitins in the following examples were all processed as follows: weighing 10g of chitin, adding 200mL of concentrated hydrochloric acid, stirring by a magnetic stirrer until the chitin is dissolved, adding 500mL of 50% ethanol aqueous solution, stirring for 5min, standing at 4 ℃ for 24 h, centrifuging, collecting precipitate, adding distilled water to adjust the pH value to 7, and finally diluting to 500mL by using distilled water to obtain colloidal chitin liquid with the chitin content of 0.02 g/mL. Wherein the chitin is a product of the company Limited in the Industrial bioengineering (Shanghai) and has a product number of A500659-0100.
The alpha-Chitin from shell shells in the following examples is a product from Sigma, cat # C9213-500G.
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
Example 1 preparation of fusion chitinases SachB-HEX and His-SachB-HEX-His and determination of enzymatic efficiency of alpha-chitin
Deleting a signal peptide (amino acid residues 1-26) of Streptomyces alfalfa chitinase of GenBank accession Number WP-076682988 (12-APR-2018) to obtain mature chitinase (the amino acid sequence is amino acid residues 27-296 of GenBank accession Number WP-076682988 (12-APR-2018), and naming the mature chitinase as SacHIB; the signal peptide (amino acid sequence is the amino acid residues from 1 to 25) of Streptomyces beta-N-acetylhexosaminidase of GenBank accession Number AYR18867.1(12-NOV-2018) was deleted to obtain mature beta-N-acetylhexosaminidase (amino acid sequence is the amino acid residues from 26 to 536 of GenBank accession Number AYR18867.1(12-NOV-2018), which was named as SaHEX).
The mature chitinase SacHIB and the mature beta-N-acetylhexosaminidase SaHEX are fused together to obtain a fusion protein which is named as SacHIB-HEX. The amino acid sequence of SaChiB-HEX is the protein at the 53 th to 833 th positions of SEQ ID No. 2.
Adopting pET30a (+) as an expression vector to carry out prokaryotic expression of the SaChiB-HEX in E.coli BL21(DE3) to obtain a fusion protein His-SaChiB-HEX-His (the amino acid sequence is SEQ ID No.2) containing the SaChiB-HEX.
pET30a (+) is used as an expression vector to carry out nuclear expression on SacHIB (the amino acid sequence is amino acid residues from 27 th to 296 th of GenBank accession Number WP-076682988 (12-APR-2018), namely amino acid residues from 53 th to 322 th of SEQ ID No.2) in E.coli BL21(DE3) to obtain fusion protein His-SacHIB-His (protein obtained by deleting amino acid residues from 323 rd and 833 th of SEQ ID No.2 and keeping other amino acid residues of SEQ ID No.2 unchanged) containing the SacHIB. Wherein the His-SaChiB-His is a control protein of His-SaChiB-HEX-His, and the only difference is that the His-SaChiB-His is a protein obtained by deleting SaHEX in the His-SaChiB-HEX-His; the sabib is a control protein of the sabib-HEX, and the only difference is that the sabib is a protein obtained by deleting the SaHEX in the sabib-HEX.
pET30a (+) is used as an expression vector to carry out nuclear expression on SaHEX (the amino acid sequence is amino acid residues 26-536 of GenBank accession Number AYR18867.1(12-NOV-2018), namely amino acid residue 323-833 of SEQ ID No.2) in E.coli BL21(DE3) to obtain a fusion protein His-SaHEX-His (a protein obtained by deleting amino acid residues 53-322 of SEQ ID No.2 and keeping other amino acid residues of SEQ ID No.2) containing the SaHEX as a control. Wherein the His-SaHEX-His is a control protein of His-SaChiB-HEX-His, and the only difference is that the His-sachix-His is a protein obtained by deleting SaChiB in the His-SaChiB-HEX-His; SaHEX is a control protein for SaChiB-HEX, the only difference being that SaHEX is a protein obtained by deleting SaChiB in SaChiB-HEX.
It is specifically clarified below that the enzyme catalytic efficiency of SacHIB-HEX and His-SacHIB-HEX-His on alpha-chitin is superior to that of SacHIB, His-SacHIB-His, SaHEX and His-SaHEX-His. The specific experimental methods and experimental results are as follows:
1. preparation of recombinant bacteria
1.1 three fusion genes, namely His-SachB-hex-His gene, His-SachB-His gene and His-Sahex-His gene, are prepared in the step.
The nucleotide sequence of the His-SachiB-hex-His gene is shown as SEQ ID No.1, and the SEQ ID No.1 consists of 2526 nucleotides. The His-SachB-hex-His gene contains a SachB-hex gene, and the nucleotide sequence of the SachB-hex gene is the 157 th and 2499 th nucleotides of SEQ ID No. 1. The His-SacHIB-HEX-His gene shown in SEQ ID No.1 encodes the protein His-SacHIB-HEX-His shown in SEQ ID No. 2.
The His-SacHIB-His gene is a control gene of the His-SacHIB-hex-His gene, and the His-SacHIB-His gene is a DNA molecule obtained by deleting the 967-th and 2499-th nucleotides (Sahex gene) of SEQ ID No. 1. The His-SachiB-His gene contains a SachiB gene, and the nucleotide sequence of the SachiB gene is 157 th and 966 th nucleotides of SEQ ID No. 1. The His-SacHIB-His gene encodes protein His-SacHIB-His, and the His-SacHIB-His is obtained by deleting amino acid residues at position 323-833 of SEQ ID No.2 and keeping other amino acid residues of SEQ ID No.2 unchanged. The SachB gene encodes protein SachB, and the amino acid sequence of the SachB is the 27 th to 296 th amino acid residues of GenBank accession Number WP-076682988 (12-APR-2018), namely the 53 th to 322 th amino acid residues of SEQ ID No. 2.
The His-Sahex-His gene is a control gene of the His-SachiB-hex-His gene, and the His-Sahex-His gene is a DNA molecule obtained by deleting the 157 th and 966 th nucleotides (the SachiB gene) of SEQ ID No. 1. The His-Sahex-His gene contains a Sahex gene, and the nucleotide sequence of the Sahex gene is the 967-2499 th nucleotide of SEQ ID No. 1. The His-Sahex-His gene encodes protein His-SaHEX-His, and the His-SaHEX-His is obtained by deleting amino acid residues from 53 th to 322 th positions of SEQ ID No.2 and keeping other amino acid residues of SEQ ID No.2 unchanged. The Sahex gene encodes protein SaHEX, the amino acid sequence of SaHEX is the 26 th to 536 th amino acid residues of GenBank accession Number AYR18867.1(12-NOV-2018), namely the 323 rd and 833 th amino acid residues of SEQ ID No. 2.
1.2 the DNA with the nucleotide sequence of position 151-2505 of SEQ ID No.1 is used for replacing a fragment between EcoRI recognition sites and XhoI recognition sites of pET30a (+) (a small fragment including the EcoRI recognition sites and the XhoI recognition sites), and other sequences of pET30a (+) are kept unchanged to obtain a His-Sachib-hex-His gene recombinant expression vector pET30a (+) -Sachib-hex. pET30a (+) -SachB-hex contains the His-SachB-hex-His gene shown in SEQ ID No. 1.
pET30a (+) -SachB-HEX can express the protein His-SachB-HEX-His with the amino acid sequence of SEQ ID No.2 in E.coli BL21(DE 3).
The His-SachB-hex-His gene in the recombinant expression vector pET30a (+) -SachB-hex is replaced by the His-SachB-His gene to obtain the His-SachB-His gene recombinant expression vector pET30a (+) -SachB. pET30a (+) -Sachib expresses the above-mentioned protein His-SacHIB-His in E.coli BL21(DE 3).
The His-SachiB-hex-His gene in the recombinant expression vector pET30a (+) -SachiB-hex is replaced by the His-Sahex-His gene to obtain the His-Sahex-His gene recombinant expression vector pET30a (+) -Sahex. pET30a (+) -Sahex expresses the above protein His-SaHEX-His in E.coli BL21(DE 3).
1.3 separately transforming the 4 expression vectors pET30a (+) -SachB-hex, pET30a (+) -SachB, pET30a (+) -Sachx and pET30a (+) in step 1.2 into competent cells of Escherichia coli BL21(DE3), respectively. This was spread evenly on LB plates containing kanamycin and cultured at 37 ℃ for 16 hours. The single colony was shake-cultured overnight, plasmid was extracted and sequenced, the recombinant E.coli containing pET30a (+) -SachB-hex was named BL21(DE3)/pET30a (+) -SachB-hex as shown by the sequencing result, the recombinant E.coli containing pET30a (+) -SachB was named BL21(DE3)/pET30a (+) -SachB as shown by the sequencing result, the recombinant E.coli containing pET30a (+) -Sachx was named BL21(DE3)/pET30a (+) -Sachx as shown by the sequencing result, and the recombinant E.coli containing pET30a (+) as shown by the sequencing result was named BL21(DE3)/pET30a (+) (empty vector control).
2. Preparation of fusion proteins SaChiB-HEX and His-SaChiB-HEX-His
The four strains BL21(DE3)/pET30a (+) -Sachib-hex, BL21(DE3)/pET30a (+) -Sachib, BL21(DE3)/pET30a (+) -Sachex and BL21(DE3)/pET30a (+) were inoculated separately with an inoculum size of 0.5% to 3mL LB vial liquid medium (containing 50. mu.g/mL kanamycin sulfate), and were cultured and activated for 12 to 16 hours at 37 ℃ and 220rpm with a shaking shaker. Then, an appropriate amount of activated bacterial liquid is taken according to the inoculation amount of 1 percent and inoculated into a 300mL large bottle LB culture solution (containing 50 mu g/mL kanamycin sulfate), in a shaking table at 37 ℃ and 220rpm, the continuous culture is carried out for 2.5 to 3 hours (the OD600 value of the bacterial liquid is determined to be 0.8 by an ultraviolet spectrophotometer, an LB liquid culture medium containing 50 mu g/mL kanamycin sulfate is used as a blank control), IPTG (filtration sterilization through a 0.22 mu m filter membrane) is added until the content of the IPTG is 0.6mM, and the induction culture is carried out for 6 hours in the shaking table at 30 ℃ and 220 rpm. And transferring the induced culture bacteria liquid to a centrifugal cup, centrifuging for 10min at the rotating speed of 4000rpm, discarding supernatant, re-suspending the bacteria by using 5mL of buffer solution, recovering the bacteria, and collecting the re-suspended bacteria into a 10mL centrifugal tube. In ice waterUnder the bath condition, the heavy suspension is subjected to ultrasonic cell disruption by an ultrasonic cell disruptor. The power of a crushing instrument is set to be 200W, the working time of ultrasonic waves is set to be 4s, the interval time is set to be 3s, and the crushing time is set to be 30 min. Immediately after the disruption, the bacterial solution was centrifuged at 12,000rpm at 4 ℃ for 10min, the supernatant was collected, filtered through a 0.22 μm filter and applied to a solution 1 for preliminary use (solute concentration: 300mmol/L NaCl, 50mmol/L NaH)2PO410mmol/L imidazole, solvent water, pH8.0 solution) in a well-balanced nickel column. The nickel column was loaded onto an AKTA machine using 10 column volumes of solution 1 and 10 column volumes of solution 2 (solute and concentration: 300mmol/L NaCl, 50mmol/L NaH)2PO450mmol/L imidazole, solvent water, pH8.0 solution) washed the contaminating protein in the nickel column and the protein peak was monitored on the AKTA machine. Using solution 3 (solute and its concentration are as follows: 300mmol/L NaCl, 50mmol/L NaH)2PO4350mmol/L imidazole, the solvent is water, and the solution has pH of 8.0) to wash the target protein hung on the nickel column, and an eluted sample in which a peak of the target protein appears is collected using AKTA, and the sample is referred to as a nickel column purified target protein sample. The sample of the target protein purified by the nickel column was analyzed by SDS-PAGE, and it was found that the sample of the target protein purified by the nickel column obtained from BL21(DE3)/pET30a (+) -SacHIB-HEX cells contained the target protein His-SacHIB-HEX-His of 90kDa in size and was obtained from BL21(DE3)
The target protein sample purified by the nickel column obtained from the/pET 30a (+) -SacHIB cell contains the target protein His-SaChiB-His with the size of 33kDa, the target protein sample purified by the nickel column obtained from the BL21(DE3)/pET30a (+) -Sahex cell contains the target protein His-SaHEX-His with the size of 60kDa, and the target protein sample purified by the nickel column obtained from the BL21(DE3)/pET30a (+) cell does not contain the foreign target protein (FIG. 1).
The target protein sample purified by the nickel column was further purified by passing through a molecular sieve using Superdex200 gel column manufactured by GE. The mobile phase used solution 1. After the purification by the molecular sieve, a large amount of imidazole contained in the sample can be removed, and elution peaks are collected to obtain a target protein sample purified by the molecular sieve (His-SaChiB-HEX-His purified by the molecular sieve, His-SaChiB-His purified by the molecular sieve and His-SaHEX-His purified by the molecular sieve). And (3) carrying out mass spectrometry on the His-SaChiB-HEX-His protein purified by the molecular sieve to analyze the amino acid sequence, wherein the result shows that the amino acid sequence of the His-SaChiB-HEX-His is shown as SEQ ID No. 2.
3. The enzyme catalysis efficiency (efficiency of hydrolyzing alpha-chitin) of the alpha-chitin by the SaChiB-HEX and the His-SaChiB-HEX-His is better than that of the His-SaChiB-His, the His-SaHEX-His, the His-SaChiB-His + the His-SaHEX-His and the His-SaChi18E-HEX-His
His-SaChi18E-HEX-His purified by a molecular sieve is prepared by a method of paragraph 0075-0087 of the Chinese invention patent application with the application number of 201910716758.8 and the publication number of CN110387364A as a reference.
Preparing the following 5 hydrolyzed alpha-chitin systems by using the molecular sieve purified His-SaChiB-HEX-His, the molecular sieve purified His-SaChiB-His, the molecular sieve purified His-SaHEX-His and the molecular sieve purified His-SaChi18E-HEX-His obtained in the step 2: His-SaChiB-HEX-His hydrolyzed alpha-chitin system, His-SaChiB-His hydrolyzed alpha-chitin system, His-SaHEX-His hydrolyzed alpha-chitin system, His-SaChiB-His + His-SaHEX-His hydrolyzed alpha-chitin system and His-SaChi18E-HEX-His hydrolyzed alpha-chitin system.
The His-SaChiB-HEX-His hydrolyzed alpha-chitin system consists of His-SaChiB-HEX-His, alpha-chitin and 10mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (solvent) with the pH value of 7.0. In the His-SaChiB-HEX-His hydrolysis alpha-chitin system, the content of His-SaChiB-HEX-His is 5nmol/mL, and the content of alpha-chitin is 0.01 g/mL.
The His-SaChiB-His hydrolyzed alpha-chitin system consists of His-SaChiB-His, alpha-chitin and 10mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (solvent) with the pH value of 7.0. In the His-SaChiB-His hydrolysis alpha-chitin system, the content of His-SaChiB-His is 5nmol/mL, and the content of alpha-chitin is 0.01 g/mL.
The His-SaHEX-His hydrolyzed alpha-chitin system consists of His-SaHEX-His, alpha-chitin and 10mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (solvent) with pH of 7.0. In the His-SaHEX-His hydrolysis alpha-chitin system, the content of His-SaHEX-His is 5nmol/mL, and the content of alpha-chitin is 0.01 g/mL.
The His-SaChiB-His + His-SaHEX-His hydrolysis alpha-chitin system consists of His-SaChiB-His, His-SaHEX-His, alpha-chitin and 10mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (solvent) with the pH value of 7.0. In the His-SaChiB-HEX-His hydrolysis alpha-chitin system, the content of His-SaChiB-His is 5nmol/mL, the content of His-SaHEX-His is 5nmol/mL, and the content of alpha-chitin is 0.01 g/mL.
The His-SaChi18E-HEX-His hydrolyzed alpha-chitin system consists of His-SaChi18E-HEX-His, alpha-chitin and 10mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (solvent) with pH of 7.0. In the His-SaChi18E-HEX-His hydrolysis alpha-chitin system, the content of His-SaChi18E-HEX-His is 5nmol/mL, and the content of alpha-chitin is 0.01 g/mL.
The above 5 hydrolyzed alpha-chitin systems were each subjected to the following operations: the hydrolyzed alpha-chitin system was reacted at 30 ℃ and at different reaction times, samples were removed, quenched by the addition of an equal amount of 70% acetonitrile in water, stored at-20 ℃ in a freezer and analyzed by SHODEX Amino-P504E column (Showa Denko), HPLC-UV detector. The detection wavelength is 210nm, the mobile phase is 70% acetonitrile solution, the flow rate is 1mL/min, the column temperature is 25 ℃, and the sample injection amount is 5 mu L. In equal amounts (GlcNAc)nThe mixture (BBI Life Sciences company) was used as a standard and subjected to quantitative analysis by a standard curve method (external standard method). The experiment was repeated three times, with 3 replicates per reaction time set for each hydrolyzed alpha-chitin system per experiment.
The enzyme catalytic efficiency was plotted with the yield of N-acetylglucosamine (GlcNAc) as the ordinate and the reaction time as the abscissa. Here, the yield (%) of N-acetylglucosamine (GlcNAc) is GlcNAc release amount (mg)/[ initial α -chitin concentration (mg) × 1.08] × 100.
The results showed that the maximum GlcNAc yield reached 99.4% for the His-SaChiB-HEX-His hydrolyzed alpha-chitinase system at 8 hours of reaction, the maximum GlcNAc yield reached 94.5% for the His-SaChiB-His + His-SaHEX-His hydrolyzed alpha-chitinase system at 24 hours of reaction, and the enzyme catalysis efficiency (efficiency of hydrolyzing alpha-chitin) of the fusion chitinase His-SaChiB-HEX-His on alpha-chitin was 3.2 times that of the combination of His-SaChiB-His and His-SaHEX-His((99.4% ÷ 8) ÷ (94.5% ÷ 24) ═ 3.2)). His-SaChiB-His hydrolyzed alpha-chitin system at 12 hours of reaction GlcNAc and (GlcNAc)2The yields of these two reducing sugars reached a maximum yield of 84.8%, and the enzymatic efficiency of the fused chitinase His-SaChiB-HEX-His on α -chitin (efficiency of hydrolyzing α -chitin) was 1.8 times that of His-SaChiB-His ((99.4% ÷ 8) ÷ (84.8% ÷ 12) ═ 1.8). His-SaHEX-His alone cannot hydrolyze alpha-chitin (FIG. 2). His-SaChi18E-HEX-His hydrolyzed alpha-chitin system to a maximum GlcNAc yield of 89.1% at 24 hours of reaction, with the catalytic efficiency of the fusion chitinase His-SaChiB-HEX-His on alpha-chitin (efficiency of hydrolyzing alpha-chitin) being 3.4 times that of His-SaChi18E-HEX-His ((99.4% ÷ 8) ÷ (89.1% ÷ 24) ═ 3.4)) (fig. 3).
The His-SaChiB-HEX-His hydrolyzed alpha-chitin system showed GlcNAc (product purity > 98%) as a result of HPLC detection of the reaction for 8 hours, and alpha-chitin was completely hydrolyzed to GlcNAc (fig. 4).
The results of HPLC examination of His-SaChiB-His + His-SaHEX-His hydrolyzed alpha-chitin line at 24 hours of reaction indicated that the hydrolysate was GlcNAc, (product purity > 98%) (FIG. 5).
HPLC detection of His-SaChiB-His hydrolyzed alpha-chitin system at 12 hours of reaction indicated that the hydrolysates included GlcNAc and (GlcNAc)2(FIG. 6).
His-SaChi18E-HEX-His hydrolyzed alpha-chitin system showed by HPLC detection at 24 hours of reaction that the hydrolysis product was GlcNAc (> 98% product purity), and alpha-chitin was completely hydrolyzed to GlcNAc (FIG. 7).
Example 2 assay of chitinase Activity of the fusion chitinase His-SaChiB-HEX-His
The chitinase activity is measured by a 3, 5-dinitrosalicylic acid (DNS) method: to 0.3mL of 50mM disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution having a pH of 7.0, 0.1mL of an enzyme solution (the solvent is 50mM disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution having a pH of 7.0) and 0.2mL of colloidal chitin were added to obtain an enzymatic reaction system, and the enzymatic reaction system was reacted at 30 ℃ for 5min, after completion of the reaction, 0.4mL of DNS was added, boiled for 10min, centrifuged, and then OD540 was measured. 1 enzyme activity unit (U) is defined as the amount of enzyme required to break down colloidal chitin to release 1. mu. mol N-acetamido-D-glucose (GlcNAc) per minute under the above conditions. The experiment was repeated three times.
The results showed that the chitinase specific activities (U/mol of enzyme protein) of the molecular sieve-purified His-SacHIB-HEX-His, the molecular sieve-purified His-SacHIB-His and the molecular sieve-purified His-SaHEX-His obtained in example 1 were 2135.94 + -265.98U/μmol of enzyme protein, 823.78 + -14.52U/μmol of enzyme protein and 250.49 + -0.56U/μmol of enzyme protein, respectively.
Example 3 determination of the Properties of the fusion chitinase His-SaChiB-HEX-His
The properties of the purified His-SaChiB-HEX-His of the molecular sieve obtained in example 1 were determined as follows.
1. Determination of optimum pH and pH stability of fusion chitinase His-SaChiB-HEX-His
1.1 optimal pH determination of the fusion chitinase His-SaChiB-HEX-His
The molecular sieve purified His-SaChiB-HEX-His obtained in example 1 was subjected to an enzymatic reaction under buffers of various pH to determine the optimum pH thereof. The buffers used were respectively: 50mmol/L disodium hydrogenphosphate-citric acid buffer (pH 4.0-7.0), 50mmol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH 6.0-8.0), 50mmol/L LTris-HCl buffer (pH8.0-9.0), and 50mmol/L glycine-sodium hydroxide buffer (pH 9.0-10.0). The chitinase activity of His-SaChiB-HEX-His in the above buffer was measured by the 3, 5-dinitrosalicylic acid (DNS) method in example 2, except that 50mM disodium hydrogenphosphate-sodium dihydrogenphosphate buffer at pH7.0 in example 2 was replaced with the corresponding buffer described above, and the other procedures were the same. The chitinase activity of His-SaChiB-HEX-His at pH7.0 was taken as 100%. The experiment was repeated three times.
The results showed that the optimum pH of His-SaChiB-HEX-His was 7.0, and the enzyme activity was maintained at 60% or more of the maximum enzyme activity in the range of pH4.0 to 7.5 (FIG. 8).
1.2 determination of pH stability of the fusion chitinase His-SaChiB-HEX-His
The molecular sieve purified His-SaChiB-HEX-His obtained in example 1 was treated in buffers of the following different pH at 37 ℃ for 60 min: 50mmol/L disodium hydrogenphosphate-citric acid buffer (pH 4.0-7.0), 50mmol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH 6.0-8.0), 50mmol/L Tris-HCl buffer (pH8.0-9.0), and 50mmol/L glycine-sodium hydroxide buffer (pH 9.0-10.0).
The chitinase activity of the treated His-SaChiB-HEX-His was then determined using the 3, 5-dinitrosalicylic acid (DNS) method of example 2. The chitinase activity of His-SaChiB-HEX-His at pH7.0 was 100%. The experiment was repeated three times.
The chitinase His-SaChiB-HEX-His was fused and treated in the above buffers with various pH values at 37 ℃ for 60min, and then the enzyme activity was measured in a buffer system with pH7.0 at 30 ℃ to investigate the pH tolerance of the enzyme. The results show that the fused chitinase is stable at pH 4.0-9.0, and the residual enzyme activity is more than 60% after the fused chitinase is treated in the pH range for 60min, which indicates that the enzyme has better pH stability (figure 9).
2. Determination of optimal temperature and thermal stability of fusion chitinase His-SaChiB-HEX-His
2.1 optimal temperature determination of the fusion chitinase His-SaChiB-HEX-His
The chitinase activity of the His-SaChiB-HEX-His purified by the molecular sieve obtained in example 1 was measured by the 3, 5-dinitrosalicylic acid (DNS) method in example 2, except that the reaction temperature of 30 ℃ was changed to 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 65 ℃ respectively, and the other operations were the same. The chitinase activity of His-SaChiB-HEX-His at 30 ℃ is 100%. The experiment was repeated three times.
The results indicated that the optimal temperature for His-SaChiB-HEX-His was 30 deg.C (FIG. 10).
2.2 measurement of the thermal stability of the fusion chitinase His-SaChiB-HEX-His
The purified His-SaChiB-HEX-His obtained in example 1 was allowed to stand at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃ for 1 hour, and then the chitinase activity of the treated His-SaChiB-HEX-His was measured by the 3, 5-dinitrosalicylic acid (DNS) method in example 2. The chitinase activity of His-SaChiB-HEX-His treated at 30 deg.C for 0min is 100%. The experiment was repeated three times.
The results showed that His-SaChiB-HEX-His had better stability at 30-40 deg.C (FIG. 11).
3. Enzyme kinetic parameters of fusion chitinase His-SaChiB-HEX-His
Under the optimal reaction condition, 1% colloidal chitin is used as a substrate to react for 5min, 10min, 15 min, 20min, 30min, 60min, 90 min and 120min respectively, and the first-stage reaction time of His-SaChiB-HEX-His is determined. Then, the enzyme activity was measured at 30 ℃ in 50mM disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution of pH7.0 by the 3, 5-dinitrosalicylic acid (DNS) method in example 2 using colloidal chitin of various concentrations (2mg/mL-5mg/mL) as a substrate for the first-order reaction time, the reaction was carried out under optimum conditions and the activity was measured, and K was calculatedm、Vmax、kcatAnd kcat/KmThe value is obtained. Through determination, the K of His-SaChiB-HEX-His takes colloidal chitin as a substrate at the temperature of 30 DEG CmThe value was 11.1mg/mL, the maximum reaction velocity VmaxIs 2289.5U/. mu.mol, kcatValues of 38.12/s and kcat/KmThe value was 3.43 mL/mg. multidot.s.
4. Effect of different Metal ion Chemicals on enzymatic Activity of the fusion chitinase His-SaChiB-HEX-His
The reagents shown in Table 1 were added to the enzymatic reaction system according to the 3, 5-dinitrosalicylic acid (DNS) method in example 2 at a final concentration of 1mmol/L to investigate the effect on the enzyme activity, and the other operations were the same as in example 2. The chitinase activity measured by the 3, 5-dinitrosalicylic acid (DNS) method in example 2 was 100%. The experiment was repeated three times.
The results (Table 1) show that Co2+And Mn2+Has a small amount of promotion effect on enzyme activity, Ag+And Hg+Has obvious inhibition effect on enzyme activity, Zn2+And Cu2+Has little inhibition effect on enzyme activity, and other metal ions and chemical reagents have no influence on the enzyme activity basically, so that the His-SaChiB-HEX-His is an enzyme independent of metal ions.
TABLE 1 Effect of various Metal ions and chemical reagents on the chitinase Activity of His-SaChiB-HEX-His
| Reagent | Relative chitinase Activity (%) |
| KCl | 102％±5.6％ |
| CaCl2 | 89％±4.0％ |
| NaCl | 104％±2.6％ |
| MgCl2 | 96％±12.9 |
| ZnCl | |
| 2 | 60％±10.4％ |
| FeCl2 | 109％±6.3％ |
| FeCl3 | 95％±6.4％ |
| CuSO4 | 26％±5.5％ |
| NiCl2 | 98％±8.6％ |
| CoCl2 | 119％±11.7％ |
| MnCl2 | 136％±13.1 |
| HgCl | |
| 2 | 2％±1.7％ |
| AgNO3 | 13％±9.3％ |
| EDTA | 88％±3.6％ |
| Beta-mercaptoethanol | 108％±7.2％ |
5. TLC analysis of His-SaChiB-HEX-His hydrolysis (GlcNAc)2To the resultant reaction system was added 10. mu.L of 10mg/mL (GlcNAc)2Solution (Qingdao Bozhihui Biotech Co., Ltd.) (solvent 50mM pH7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer), and 50pmol of His-SaChiB-HEX-His enzyme solution (solvent), His-SaHEX-His enzyme solution and His-SaChiB-His enzyme solution were added, respectively, and the system was made up to 30. mu.L with 50mM pH7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and reacted at 30 ℃ for 1 hour after mixing. The product was analyzed by Thin Layer Chromatography (TLC). The analysis results show (fig. 12): His-SaChiB-His No-break down (GlcNAc)2His-SaHEX-His and His-SaChiB-HEX-His can be decomposed (GlcNAc)2The fusion chitinase His-SaChiB-HEX-His has beta-N-acetylhexosaminidase activity.
6. Substrate specificity of His-SaChiB-HEX-His
Under the optimum reaction conditions, the chitinase activity of His-SaChiB-HEX-His was measured at 30 ℃ in 50mM disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution having a pH of 7.0 according to the 3, 5-dinitrosalicylic acid (DNS) method in example 2 using colloidal chitin, alpha-chitin, fungal cell wall chitin, chitosan, cellulose and beta-1, 3-glucan as substrates, respectively. The amount of enzyme required to decompose a substrate under optimum conditions to release 1. mu. mol of reducing sugar per minute is defined as one enzyme activity unit (U).
His-SaChiB-HEX-His has a broad substrate spectrum (Table 2). It is active not only on colloidal chitin, but also on alpha-chitin and fungal chitin, and can hydrolyze chitin in different forms. Meanwhile, the compound also has weak activity on beta-1, 3-glucan, chitosan, cellulose and the like.
TABLE 2 substrate spectra of His-SaChiB-HEX-His
7. Analysis of morphological change of His-SaChiB-HEX-His hydrolyzed alpha-chitin by electron microscope
And (3) carrying out vacuum freeze drying on unhydrolyzed alpha-chitin, His-SaChiB-HEX-His hydrolyzed alpha-chitin for 1h and His-SaChiB-HEX-His hydrolyzed alpha-chitin for 8 h. And taking a proper amount of dried sample, placing the sample on conductive gel for gold spraying treatment, and observing the shape of the sample by using a scanning electron microscope.
The results show that the untreated alpha-chitin surface is smooth and the original pore shape of the surface is irregular (13, a). After 1h of His-SaChiB-HEX-His treatment, the alpha-chitin crystal surface formed visible pores with regular shape (B in FIG. 13). After the His-SaChiB-HEX-His treatment is carried out for 8 hours, the number of holes on the surface of the alpha-chitin particles is obviously increased, and the shapes are regular and smooth (C in figure 13).
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences
<120> fusion chitinase for efficiently degrading alpha-chitin, related biological material and application thereof
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 2526
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgcaccatc atcatcatca ttcttctggt ctggtgccac gcggttctgg tatgaaagaa 60
accgctgctg ctaaattcga acgccagcac atggacagcc cagatctggg taccgacgac 120
gacgacaagg ccatggctga tatcggatcc gaattcacgg cgtccgcggc cgcgtgcgcc 180
gcgccatgga gctcgtcctc cgtctacacg ggcggcaaga ccgcctcgca caacgggcac 240
aactggaccg ccaagtggtg gacccagaac gagacgccgg gccgctccga cgtctgggcg 300
gacgcgggcg cctgcggcgg cggcacggat ccgggcaacc ccgacccgtc cggattcgtg 360
gtcagcgagg cccagttcaa ccagatgttc ccgagccgga actcgttcta cacgtacaag 420
ggcctgacgg acgcgctgaa ggcgtacccg gccttcgcca acaccggcag cgacaccgtc 480
aagcgccagg aggcggcggc gttcctcgcc aacgtccacc acgagaccgg cgggctgaag 540
tacatcgtcg agcagaacca ggccaactac ccgcactact gcgacgcgaa ccagccctac 600
ggctgccccg ccgggcaggc cgcgtactac ggccgcggcc cgatccagct cagctggaac 660
ttcaactaca aggccgcggg cgacgcgctc ggcatcgacc tgctgcgcaa cccctacctg 720
gtggagcggg acccggccgt cgcctggaag accggcctct ggtactggaa cacccagtcg 780
ggccccggca ccatgacgcc gcacaacgcc atggtcaacg gcaagggctt cggtgagacc 840
atccgcgcca tcaacggcac cctggagtgc aacggcggca accccgccca ggtgcagagc 900
cgcatcgacc gctacaagca gttcacccag ctcctcggca ccacgccggg ctccaacctg 960
agctgcgccg cccccgcccc cgaggcgacc cggccctccg tcaccccgct cggtgaggtg 1020
gtgcccgctc ccctgaaggc ggaggcgggc ggcgccgggt accagatcac cgccaagacg 1080
cgcatccgcg tcggcgacgg caaccccgac gagcgccgcg tcggcgagta cctcgcccgg 1140
gtgctgcggc cctccaccgg ctacaagctg cccgtcacca gcggagaggg gagcgacggc 1200
atccggctgc gcatcagcgc cgaaccggcg aacaaggtcc tcggcaacga ggggtaccgc 1260
gtcatctccg agcgcggctc cctcaccatc acctcctggt ccggcgccgg cctcttccac 1320
ggcgtccaga cggtccgcca gcaactgccc gccgctgtgg agaagaagtc gaagcagcgc 1380
ggaccctggc ggatcgcggg cggcaccatc aaggacatgc cgcgctatgg ctaccgctcc 1440
acgatgctcg acgtctcacg gcacttcttc accgtcgacc aggtcaagcg ctacatcgac 1500
caggcgtccc tgtacaagat gaacaagctg cacctgcacc tcagcgacga ccagggctgg 1560
cgcatcgcca tcgactcctg gccgcgcctc gcgacccacg gcggctccac ccaggtcggc 1620
ggcggcgagg gcggctacta cacgaaggcc cagtacaagg agatcgtggc ctacgccgcc 1680
tcgcggtaca tggaggtcgt gcccgagatc gacatgccgg ggcacaccaa cgccgcgctc 1740
gcctcgtacg ccgagctgaa ctgcgacggc gtggccccgc cgctctacac cggcaccgcg 1800
gtcggcttca gctcgctgtg cgtgaagaag gacgtcacgt acgacttcgt ggacgacgtg 1860
atccgtgagc tggccgccat gacgccgggc gagtacctgc acatcggcgg cgacgaggcg 1920
cactccacca gccacgagga cttcgtcgcg ttcatggaca aggtgcagcc ggtggtcgcc 1980
aagtacggca agaaggtgat gggctggcac cagctggccg gcgcccggcc cgcgaagggc 2040
gccgtcgccc agtactgggg ttacgacagg acgggtgccg ccgagcgcga gcaggtcgtg 2100
aacgccgcga agaacggcac caagctggtc ctctcgccgg ccgaccgctc ctacctcgac 2160
cacaagtaca ccaaggacac cccgctcggc ctgtcctggg ccggtctcgt cgaggtgcgg 2220
cggtcctacg actgggaccc gggcgcctac ctccagggcg cgcccgcgga cgcggtcatg 2280
ggtgtcgagg cgccgctgtg gacggagacc ctgtcgacct ccgcgcacct ggaccacatg 2340
gcgttcccgc ggcttcccgg gatcgccgag ctcggctggt cgcccgcggc cacgcacgac 2400
tgggacgcgt acaagacgcg gctcgccgcg caggcgcccc gctgggacgc cctgggcatc 2460
ggctactacg agtcgccgca ggtgccctgg cccgccaagc tcgagcacca ccaccaccac 2520
cactga 2526
<210> 2
<211> 841
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile
35 40 45
Gly Ser Glu Phe Thr Ala Ser Ala Ala Ala Cys Ala Ala Pro Trp Ser
50 55 60
Ser Ser Ser Val Tyr Thr Gly Gly Lys Thr Ala Ser His Asn Gly His
65 70 75 80
Asn Trp Thr Ala Lys Trp Trp Thr Gln Asn Glu Thr Pro Gly Arg Ser
85 90 95
Asp Val Trp Ala Asp Ala Gly Ala Cys Gly Gly Gly Thr Asp Pro Gly
100 105 110
Asn Pro Asp Pro Ser Gly Phe Val Val Ser Glu Ala Gln Phe Asn Gln
115 120 125
Met Phe Pro Ser Arg Asn Ser Phe Tyr Thr Tyr Lys Gly Leu Thr Asp
130 135 140
Ala Leu Lys Ala Tyr Pro Ala Phe Ala Asn Thr Gly Ser Asp Thr Val
145 150 155 160
Lys Arg Gln Glu Ala Ala Ala Phe Leu Ala Asn Val His His Glu Thr
165 170 175
Gly Gly Leu Lys Tyr Ile Val Glu Gln Asn Gln Ala Asn Tyr Pro His
180 185 190
Tyr Cys Asp Ala Asn Gln Pro Tyr Gly Cys Pro Ala Gly Gln Ala Ala
195 200 205
Tyr Tyr Gly Arg Gly Pro Ile Gln Leu Ser Trp Asn Phe Asn Tyr Lys
210 215 220
Ala Ala Gly Asp Ala Leu Gly Ile Asp Leu Leu Arg Asn Pro Tyr Leu
225 230 235 240
Val Glu Arg Asp Pro Ala Val Ala Trp Lys Thr Gly Leu Trp Tyr Trp
245 250 255
Asn Thr Gln Ser Gly Pro Gly Thr Met Thr Pro His Asn Ala Met Val
260 265 270
Asn Gly Lys Gly Phe Gly Glu Thr Ile Arg Ala Ile Asn Gly Thr Leu
275 280 285
Glu Cys Asn Gly Gly Asn Pro Ala Gln Val Gln Ser Arg Ile Asp Arg
290 295 300
Tyr Lys Gln Phe Thr Gln Leu Leu Gly Thr Thr Pro Gly Ser Asn Leu
305 310 315 320
Ser Cys Ala Ala Pro Ala Pro Glu Ala Thr Arg Pro Ser Val Thr Pro
325 330 335
Leu Gly Glu Val Val Pro Ala Pro Leu Lys Ala Glu Ala Gly Gly Ala
340 345 350
Gly Tyr Gln Ile Thr Ala Lys Thr Arg Ile Arg Val Gly Asp Gly Asn
355 360 365
Pro Asp Glu Arg Arg Val Gly Glu Tyr Leu Ala Arg Val Leu Arg Pro
370 375 380
Ser Thr Gly Tyr Lys Leu Pro Val Thr Ser Gly Glu Gly Ser Asp Gly
385 390 395 400
Ile Arg Leu Arg Ile Ser Ala Glu Pro Ala Asn Lys Val Leu Gly Asn
405 410 415
Glu Gly Tyr Arg Val Ile Ser Glu Arg Gly Ser Leu Thr Ile Thr Ser
420 425 430
Trp Ser Gly Ala Gly Leu Phe His Gly Val Gln Thr Val Arg Gln Gln
435 440 445
Leu Pro Ala Ala Val Glu Lys Lys Ser Lys Gln Arg Gly Pro Trp Arg
450 455 460
Ile Ala Gly Gly Thr Ile Lys Asp Met Pro Arg Tyr Gly Tyr Arg Ser
465 470 475 480
Thr Met Leu Asp Val Ser Arg His Phe Phe Thr Val Asp Gln Val Lys
485 490 495
Arg Tyr Ile Asp Gln Ala Ser Leu Tyr Lys Met Asn Lys Leu His Leu
500 505 510
His Leu Ser Asp Asp Gln Gly Trp Arg Ile Ala Ile Asp Ser Trp Pro
515 520 525
Arg Leu Ala Thr His Gly Gly Ser Thr Gln Val Gly Gly Gly Glu Gly
530 535 540
Gly Tyr Tyr Thr Lys Ala Gln Tyr Lys Glu Ile Val Ala Tyr Ala Ala
545 550 555 560
Ser Arg Tyr Met Glu Val Val Pro Glu Ile Asp Met Pro Gly His Thr
565 570 575
Asn Ala Ala Leu Ala Ser Tyr Ala Glu Leu Asn Cys Asp Gly Val Ala
580 585 590
Pro Pro Leu Tyr Thr Gly Thr Ala Val Gly Phe Ser Ser Leu Cys Val
595 600 605
Lys Lys Asp Val Thr Tyr Asp Phe Val Asp Asp Val Ile Arg Glu Leu
610 615 620
Ala Ala Met Thr Pro Gly Glu Tyr Leu His Ile Gly Gly Asp Glu Ala
625 630 635 640
His Ser Thr Ser His Glu Asp Phe Val Ala Phe Met Asp Lys Val Gln
645 650 655
Pro Val Val Ala Lys Tyr Gly Lys Lys Val Met Gly Trp His Gln Leu
660 665 670
Ala Gly Ala Arg Pro Ala Lys Gly Ala Val Ala Gln Tyr Trp Gly Tyr
675 680 685
Asp Arg Thr Gly Ala Ala Glu Arg Glu Gln Val Val Asn Ala Ala Lys
690 695 700
Asn Gly Thr Lys Leu Val Leu Ser Pro Ala Asp Arg Ser Tyr Leu Asp
705 710 715 720
His Lys Tyr Thr Lys Asp Thr Pro Leu Gly Leu Ser Trp Ala Gly Leu
725 730 735
Val Glu Val Arg Arg Ser Tyr Asp Trp Asp Pro Gly Ala Tyr Leu Gln
740 745 750
Gly Ala Pro Ala Asp Ala Val Met Gly Val Glu Ala Pro Leu Trp Thr
755 760 765
Glu Thr Leu Ser Thr Ser Ala His Leu Asp His Met Ala Phe Pro Arg
770 775 780
Leu Pro Gly Ile Ala Glu Leu Gly Trp Ser Pro Ala Ala Thr His Asp
785 790 795 800
Trp Asp Ala Tyr Lys Thr Arg Leu Ala Ala Gln Ala Pro Arg Trp Asp
805 810 815
Ala Leu Gly Ile Gly Tyr Tyr Glu Ser Pro Gln Val Pro Trp Pro Ala
820 825 830
Lys Leu Glu His His His His His His
835 840
Claims (10)
1. A fusion protein characterized by: the fusion protein is a protein obtained by fusing beta-N-acetylhexosaminidase and chitinase together, and the activity of chitinase is higher than that of the beta-N-acetylhexosaminidase and the chitinase; the chitinase is D1) or D2):
D1) a protein having an amino acid sequence of amino acid residues 53 to 322 of SEQ ID No. 2;
D2) a fusion protein obtained by labeling the carboxyl terminal or/and amino terminal fusion protein of the protein shown in D1).
2. The fusion protein of claim 1, wherein: the beta-N-acetylhexosaminidase is derived from streptomyces.
3. The fusion protein of claim 1 or 2, wherein: the beta-N-acetylhexosaminidase is A1) or A2):
A1) a protein whose amino acid sequence is amino acid residue 323-833 of SEQ ID No. 2;
A2) a fusion protein obtained by labeling the carboxyl terminal or/and amino terminal fusion protein of the protein shown in A1).
4. The fusion protein according to any one of claims 1 to 3, characterized in that: the fusion protein is any one of F1) -F3):
F1) a protein having the amino acid sequence of SEQ ID No. 2;
F2) a protein having an amino acid sequence of positions 53-833 of SEQ ID No. 2;
F3) a fusion protein obtained by labeling the carboxyl-terminal or/and amino-terminal fusion protein of the protein shown in F1) or F2).
5. The biomaterial related to the fusion protein of any one of claims 1 to 4, which is at least one of the following B1) -B7):
B1) a nucleic acid molecule encoding the fusion protein of any one of claims 1-4;
B2) an expression cassette comprising the nucleic acid molecule of B1);
B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);
B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;
B5) a transgenic plant cell line containing B1) the nucleic acid molecule, or a transgenic plant cell line containing B2) the expression cassette, or a transgenic plant cell line containing B3) the recombinant vector;
B6) a transgenic plant tissue containing B1) the nucleic acid molecule, or a transgenic plant tissue containing B2) the expression cassette, or a transgenic plant tissue containing B3) the recombinant vector;
B7) a transgenic plant organ containing B1) the nucleic acid molecule, or a transgenic plant organ containing B2) the expression cassette, or a transgenic plant organ containing B3) the recombinant vector.
6. The biomaterial of claim 5, wherein: B1) the nucleic acid molecule is B11) or B12) as follows:
B11) the coding sequence is a DNA molecule of SEQ ID No. 1;
B12) the nucleotide sequence is the DNA molecule at position 157-2499 of SEQ ID No. 1.
7. A method of making a fusion protein, comprising: expressing a gene encoding the fusion protein of any one of claims 1 to 4 in an organism to obtain the fusion protein; the organism is a microorganism, a plant or a non-human animal.
8. The method of claim 7, wherein: the microorganism is any one of C1) -C4):
C1) a prokaryotic microorganism;
C2) bacteria of the enterobacteriaceae family;
C3) an Escherichia bacterium;
C4) escherichia coli.
9. Use of the fusion protein of any one of claims 1-4 as a chitinase or for hydrolyzing chitin.
10. Use of the fusion protein of any one of claims 1-4, the biological material of claim 5 or 6, the method of claim 7 or 8 for the preparation of a chitinase preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944787A (en) * | 2020-07-30 | 2020-11-17 | 华南理工大学 | Chitinase fused with carbohydrate binding module as well as preparation method and application thereof |
| CN114645036A (en) * | 2018-07-11 | 2022-06-21 | 河北农业大学 | Antifungal fusion protein formed by fusing chitosanase and chitinase and related biological material and application thereof |
| CN117737037A (en) * | 2024-02-07 | 2024-03-22 | 中国林业科学研究院高原林业研究所 | N-acetylglucosaminidase mutant De266L delta 6 and preparation and application thereof |
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| CN108864303A (en) * | 2018-07-11 | 2018-11-23 | 河北农业大学 | Antimycotic fusion protein and its relevant biological material and application |
| CN110387364A (en) * | 2019-08-05 | 2019-10-29 | 河北农业大学 | A kind of recombinant chitinase and its relevant biological material and application |
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| CN108864303A (en) * | 2018-07-11 | 2018-11-23 | 河北农业大学 | Antimycotic fusion protein and its relevant biological material and application |
| CN110387364A (en) * | 2019-08-05 | 2019-10-29 | 河北农业大学 | A kind of recombinant chitinase and its relevant biological material and application |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114645036A (en) * | 2018-07-11 | 2022-06-21 | 河北农业大学 | Antifungal fusion protein formed by fusing chitosanase and chitinase and related biological material and application thereof |
| CN114645036B (en) * | 2018-07-11 | 2023-08-01 | 河北农业大学 | Antifungal fusion protein formed by fusing chitinase and related biological material and application thereof |
| CN111944787A (en) * | 2020-07-30 | 2020-11-17 | 华南理工大学 | Chitinase fused with carbohydrate binding module as well as preparation method and application thereof |
| CN111944787B (en) * | 2020-07-30 | 2022-03-29 | 华南理工大学 | Chitinase fused with carbohydrate binding module as well as preparation method and application thereof |
| CN117737037A (en) * | 2024-02-07 | 2024-03-22 | 中国林业科学研究院高原林业研究所 | N-acetylglucosaminidase mutant De266L delta 6 and preparation and application thereof |
| CN117737037B (en) * | 2024-02-07 | 2024-04-19 | 中国林业科学研究院高原林业研究所 | N-acetylglucosaminidase mutant De266L delta 6 and preparation and application thereof |
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