CN114736889B - Chitosan mutant with improved stability of N-terminal mutant enzyme and application thereof - Google Patents

Chitosan mutant with improved stability of N-terminal mutant enzyme and application thereof Download PDF

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CN114736889B
CN114736889B CN202210377637.7A CN202210377637A CN114736889B CN 114736889 B CN114736889 B CN 114736889B CN 202210377637 A CN202210377637 A CN 202210377637A CN 114736889 B CN114736889 B CN 114736889B
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CN114736889A (en
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郭静
高文君
赵晗
满在伟
蔡志强
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Changzhou University
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01132—Chitosanase (3.2.1.132)

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Abstract

The invention relates to the technologies of gene cloning, site-directed mutagenesis and gene recombination, in particular to a chitosan enzyme mutant with improved N-terminal mutant enzyme stability and application thereof, belonging to the field of enzyme engineering. The invention mutates proline at the 2 nd position of the N end of SaCsn46A into glycine (P2G), and the mutant is expressed in escherichia coli Rosetta. By comparison with the wild-type chitosanase, the temperature stability of the mutant enzyme was found to be improved by a factor of 1.53 at 50℃compared with the temperature stability of the wild-type. The results of thin layer chromatography showed that the enzyme hydrolysis products were mainly glucosamine GlcN and (GlcN) 2 Illustrating that mutant chitosanase industrially produces GlcN and (GlcN) under mild conditions 2 Has good application prospect in the aspect.

Description

Chitosan mutant with improved stability of N-terminal mutant enzyme and application thereof
Technical Field
The invention relates to a gene cloning, site-directed mutagenesis and gene recombination technology, in particular to a chitosan enzyme mutant with improved N-terminal mutant enzyme stability and application thereof, belonging to the field of enzyme engineering.
Background
Chitosan is a cationic polysaccharide linked by glucosamine (GlcN) through a beta-1, 4-glycosidic linkage. Chitosan is a deacetylated product of chitin, widely existing in shells and cartilage of lower plant fungi, algal cells, shellfish, mollusks (such as salmon, squid), is the second largest natural high molecular compound next to cellulose on earth, and is a renewable biological resource.
The chitosan has excellent functions and excellent biological activities of resisting bacteria, resisting tumors, improving immunity and the like, so that the chitosan has wide application prospect in the fields of foods, medicines, agriculture, cosmetics and the like. However, many functional properties of chitosan are reported to be exhibited only when degraded to a certain extent, and most of its degradation products are chitosan oligosaccharides, which not only have all properties of chitosan, but also are readily soluble in water, have hygroscopicity and moisture retention, and can be well applied to food, medicine, agriculture and cosmetics.
The methods for producing chitosan oligosaccharide mainly comprise three methods: physical, chemical, enzymatic hydrolysis. In view of the safety, degradation efficiency and environmental protection of the product, the enzymolysis method is generally adopted for degrading chitosan in industrialization. In addition, the enzyme hydrolysis method has strong specificity, mild reaction conditions and easy preparation, and has become a research hot spot in recent years.
Chitosanase is a hydrolase specially used for degrading chitosan into chitosan oligosaccharide, and is prepared according to the carbohydrate activity enzyme database @www.cazy.org) It is known that chitanases are classified as Glycoside Hydrolases (GH) families 46, 75, 80 and 8.GH46, GH75 and GH80 currently contain only chitosanase, while the GH8 family contains some other glycoside hydrolases. It is known that fungal-derived chitosanase is mainly distributed in the GH75 family, whereas bacterial-derived chitosanase is mainly of the GH46 family, and a minority is of the GH80 family. Avermectin is an aerobic bacterium and mainly produces avermectin and ivermectin. Heggset has reported that a chitosanase of the Streptomyces avermitilis GH75 family mainly produces long oligomers (DP.gtoreq.2). However, no report about gene cloning and characteristics of the streptomyces avermitilis GH46 family chitosan enzyme exists at present, and a new chitosan enzyme SaCsn46A is cloned by utilizing a PCR technology in the early stage of the research, and has a wide substrate spectrum, but the temperature stability is required to be improved.
Disclosure of Invention
The invention aims to find a chitosan enzyme mutant with improved N-terminal mutant enzyme stability by carrying out molecular transformation on chitosan enzyme, so that the enzymolysis method is better applied to industrial production of chitosan oligosaccharide.
The invention particularly provides a chitosan enzyme mutant with improved N-terminal mutant enzyme stability, and the amino acid sequence of the chitosan enzyme mutant is SEQ NO.3. The invention also provides a gene for encoding the chitosan enzyme mutant, and the nucleotide sequence of the gene is SEQ NO.4.
The invention also provides a recombinant plasmid which comprises the gene.
The invention also provides a host cell comprising the gene or the recombinant plasmid.
The invention provides a chitosan enzyme mutant with improved N-terminal mutant enzyme stability, which is expressed in escherichia coli Rosetta.
The chitosan enzyme mutant with improved N-terminal mutant enzyme stability is characterized in that proline at 2 nd position of chitosan enzyme SaCsn46A is mutated into glycine, and the mutation is marked as P2G.
The mutant method comprises the following steps:
1. two primers are designed, the chitosan enzyme SaCsn46A gene is amplified, the signal peptide is removed, the protein sequence of the SaCsn46A is SEQ NO.1, and the gene sequence is SEQ NO.2. Cloning the amplified target gene into an expression vector pET-28a to construct a recombinant plasmid pET-SaCsn46a.
The two primers are respectively: upstream primer CGGGATCCGCACCCGTCGGCCTGGACGAC, downstream primer CCCAAGCTTTCAGCCGATGTGGTAGCTGTC
2. Simulating chitosanase (SaCsn 46A) by using Swiss-Model online software to obtain a space structure of the chitosanase;
3. the unfolding free energy change of each mutant amino acid of SaCsn46A was calculated using the popmulic prediction software to determine the key amino acid positions associated with the stability of SaCsn46A.
4. Designing a site-directed mutagenesis primer, obtaining a mutant chitosan enzyme gene with a sequence of SEQ NO.4 through a PCR technology, cloning a target gene into an expression vector pET-28a, and obtaining a recombinant vector pET-P2G containing the mutant chitosan enzyme gene sequence;
the two primers are respectively: upstream primer CGGGATCCGCAGGCGTCGGCCTGGACGAC and downstream primer CCCAAGCTTTCAGCCGATGTGGTAGCTGTC。
The cleavage sites are BamHI and HindIII.
5. Transferring the recombinant vector in the step 4 into escherichia coli for induction culture, collecting thalli after centrifugation, performing ultrasonic cell disruption, and performing protein purification by using a Ni-NTA affinity chromatographic column to obtain mutant chitosanase, wherein the amino acid sequence of the mutant chitosanase is SEQ NO.3.
The chitosan enzyme SaCsnA used in the invention has a wider substrate spectrum, the temperature stability of the provided mutant is obviously improved, and the temperature stability is improved without influencing other characteristics of the enzyme. Under suitable conditions the mutant enzyme is still capable of hydrolyzing chitosan to GlcN and (GlcN) 2 The mutant chitosanase disclosed by the invention has a good application prospect in industrial production.
Drawings
FIG. 1 shows the temperature stability of wild-type chitosanase and chitosanase mutants in the examples of the present invention.
Detailed Description
The present invention is not limited to the following embodiments, and those skilled in the art can implement the present invention in various other embodiments according to the present invention, or simply change or modify the design structure and thought of the present invention, which fall within the protection scope of the present invention. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The invention is further described in detail below in connection with the examples:
1. confirmation of the chitosanase mutation site
The spatial structure of SaCsn46A is obtained by using Swiss-Model to simulate the three-dimensional structure of SaCsn46A by taking Streptomyces sp.N174 (1CHK_A) as a template, the unfolding free energy change (delta G) of each mutant amino acid of SaCsn46A is calculated by using PoPMuSiC prediction software to assist in designing and improving the stability, and key amino acid sites related to the stability of SaCsn46A are determined, and the corresponding mutant plasmid is obtained by designing primers for PCR because the unfolding free energy change of amino acid at position 2 of the chitosan is relatively large.
The protein sequence of SaCsn46A is shown in SEQ NO. 1.
The two primers are respectively: upstream primer CGGGATCCGCAGGCGTCGGCCTGGACGAC and downstream primer CCCAAGCTTTCAGCCGATGTGGTAGCTGTC。
PCR system:
the template was pET-SaCsn46a.
PCR amplification conditions: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 64℃for 1min, elongation at 68℃for 10min,15 cycles, incubation at 4 ℃.
DpnI digestion template plasmid
20 mu LPCR product was taken and 1 mu LDpnI restriction enzyme was added directly to the PCR product.
3. Transfer into E.coli
10 mu L of the enzyme digestion product is directly transformed into E.coli DH5 alpha competent cells. Recombinant cells carrying the mutant plasmid were sent to Shanghai Bioengineering Co.Ltd for sequencing. The mutant plasmid sequenced correctly was transformed to e.coli BL21.
4. Protein purification
The crude enzyme solution obtained by sonicating cells was applied to a Ni-NTA affinity column, eluted with 0.02M imidazole eluent (0.02M Tris-HCl, pH 8.0,0.5M NaCl,0.02M imidazole and 10% glycerol), then eluted with 0.08M imidazole eluent (0.02M Tris-HCl, pH 8.0,0.5M NaCl,0.08M imidazole and 10% glycerol), the eluent containing the chitinase activity was collected, imidazole was removed by dialysis and the enzyme was stored at-20 ℃.
5. Enzymatic property detection
The enzyme activity is measured by using a DNS method, 1475 mu L of pH buffer solution and 500 mu L of colloidal chitosan solution are added into a 2mL reaction system, 25 mu L of fully mixed enzyme solution is added, the mixture is immediately reacted in a water bath kettle at 40 ℃ for 10min, the mixture is taken out, 1.5mL of DNS solution is added to terminate the reaction, the mixture is boiled in boiling water for 5min, finally distilled water is used for fixing the volume to 25mL, the mixture is cooled to room temperature and then mixed uniformly, the mixture is transferred into a 50mL centrifuge tube, 8000rpm is set for centrifugation for 5min, the OD value of the supernatant at 520nm is measured, and the solution added with 25 mu L of ddH2O is set as a blank group for zeroing.
Definition of enzyme activity: the amount of enzyme required to catalyze the production of reducing sugars corresponding to 1. Mu. Mol of glucosamine in 1 minute at 40℃was 1 enzyme activity unit (U).
(1) Optimum pH
The enzyme activity of the chitosanase was determined in different pH buffers (pH 3.0-8.0) at a temperature of 40 ℃. The highest point of the enzyme activity is taken as 100 percent.
(2) pH stability
Under the condition of 0 ℃, the chitosan enzyme is placed in phosphate buffer solution with pH of 6.8 to be preserved for 2 hours, and the enzyme activity of the chitosan enzyme measured at the beginning is taken as 100%.
(3) Optimum temperature
Under the condition of the optimal pH, the reaction system is respectively placed at 25-80 ℃ for reaction, and the enzyme activity of the chitosanase is measured at a temperature gradient of every 5 ℃. The highest point of the enzyme activity is taken as 100 percent.
(4) Temperature stability
The chitosanase was stored at 55℃and pH 6.8 for 2 hours, and the enzyme activity of the chitosanase measured at the beginning was 100%.
6. Enzymatic properties of wild-type chitosanase and mutants thereof
The enzymatic properties of the wild-type chitosanase and its mutant (mutant P2G, amino acid sequence SEQ NO. 3) are shown in FIGS. 1 and Table 1, and the temperature stability of the mutant P2G is significantly higher than that of the wild-type (improved by about 1.53 times), but the specific enzyme activity is reduced.
The invention utilizes PoPMuSiC software to screen out the sites which possibly have influence on the temperature stability of SaCsn46A, 1 site is selected for mutation, and the functional expression is carried out in escherichia coli Rosetta (DE 3). The study on the biochemical property shows that the temperature stability of the mutant enzyme is slightly improved compared with that of the original enzyme.
TABLE 1 enzymatic Properties of wild-type chitosanase and mutants thereof
SEQ NO.1
S.avermitilis chitosanase protein sequence
APVGLDDPAKKEIAMKLVSSAENSSLDWKAQYKYIEDIGDGRGYTAGIIGFCSGTGDMLDLVELYTQRKPGNVLATYLPALRNVNGGDSHQGLDPGFPGDWRRAAQDSAFQQAQNDERDRVYFDPAVRQGKADGIGVLGQFTYYDAIVMHGDGGDSTSFSSIRGRALAKAEPPAQGGNEVTYLNAFLDARVWAMRQEEAHSDTSRVDTAQRVFLTKGNLNLDPPLDWKVYGDSYHIG
SEQ NO.2
S.avermitilis chitosanase gene sequence
gcacccgt cggcctggac gacccggcga agaaagagat cgccatgaag ctcgtgtcca gcgcggagaa ctcctcgctggactggaagg cccagtacaa gtacatcgag gacatcggcg acggccgcgg ctacaccgccgggatcatcg gcttctgctc cggcaccggc gacatgctcg acctcgtcga gctctacacc cagcgcaagc cggggaacgt cctggccacg tatctgcccg ccctgcgcaa cgtcaacggcggcgactcgc accagggcct ggacccgggc ttccccggcg actggcgccg cgcggcccag gactcggcgt tccagcaggc ccagaacgac gaacgcgacc gcgtctactt cgacccggcc gtccggcagg ggaaggcgga cggtatcggc gtactcggac agttcacgta ctacgacgccatcgtcatgc acggggacgg cggtgacagc accagcttca gcagcatccg cgggcgcgccctggccaagg ccgagccgcc ggcgcagggc ggcaacgagg tgacgtacct gaacgccttcctcgacgccc gggtctgggc gatgcggcag gaggaggccc actcggacac cagccgggtc gacaccgccc agcgggtctt cctgacgaag ggcaacctga acctggatcc gccactggac tggaaggtgt acggggacag ctaccacatc ggctga
SEQ NO.3
Mutant chitosanase protein sequences
AGVGLDDPAKKEIAMKLVSSAENSSLDWKAQYKYIEDIGDGRGYTAGIIGFCS
GTGDMLDLVELYTQRKPGNVLATYLPALRNVNGGDSHQGLDPGFPGDWRRA
AQDSAFQQAQNDERDRVYFDPAVRQGKADGIGVLGQFTYYDAIVMHGDGG
DSTSFSSIRGRALAKAEPPAQGGNEVTYLNAFLDARVWAMRQEEAHSDTSRV
DTAQRVFLTKGNLNLDPPLDWKVYGDSYHIG
SEQ NO.4
Mutant chitosanase gene sequences
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme and the concept of the present invention, and should be covered by the scope of the present invention.
Sequence listing
<120> a chitosanase mutant with improved N-terminal mutant enzyme stability and application thereof
<141> 2022-04-12
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 237
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
Ala Pro Val Gly Leu Asp Asp Pro Ala Lys Lys Glu Ile Ala Met Lys
1 5 10 15
Leu Val Ser Ser Ala Glu Asn Ser Ser Leu Asp Trp Lys Ala Gln Tyr
20 25 30
Lys Tyr Ile Glu Asp Ile Gly Asp Gly Arg Gly Tyr Thr Ala Gly Ile
35 40 45
Ile Gly Phe Cys Ser Gly Thr Gly Asp Met Leu Asp Leu Val Glu Leu
50 55 60
Tyr Thr Gln Arg Lys Pro Gly Asn Val Leu Ala Thr Tyr Leu Pro Ala
65 70 75 80
Leu Arg Asn Val Asn Gly Gly Asp Ser His Gln Gly Leu Asp Pro Gly
85 90 95
Phe Pro Gly Asp Trp Arg Arg Ala Ala Gln Asp Ser Ala Phe Gln Gln
100 105 110
Ala Gln Asn Asp Glu Arg Asp Arg Val Tyr Phe Asp Pro Ala Val Arg
115 120 125
Gln Gly Lys Ala Asp Gly Ile Gly Val Leu Gly Gln Phe Thr Tyr Tyr
130 135 140
Asp Ala Ile Val Met His Gly Asp Gly Gly Asp Ser Thr Ser Phe Ser
145 150 155 160
Ser Ile Arg Gly Arg Ala Leu Ala Lys Ala Glu Pro Pro Ala Gln Gly
165 170 175
Gly Asn Glu Val Thr Tyr Leu Asn Ala Phe Leu Asp Ala Arg Val Trp
180 185 190
Ala Met Arg Gln Glu Glu Ala His Ser Asp Thr Ser Arg Val Asp Thr
195 200 205
Ala Gln Arg Val Phe Leu Thr Lys Gly Asn Leu Asn Leu Asp Pro Pro
210 215 220
Leu Asp Trp Lys Val Tyr Gly Asp Ser Tyr His Ile Gly
225 230 235
<210> 2
<211> 714
<212> DNA/RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
gcacccgtcg gcctggacga cccggcgaag aaagagatcg ccatgaagct cgtgtccagc 60
gcggagaact cctcgctgga ctggaaggcc cagtacaagt acatcgagga catcggcgac 120
ggccgcggct acaccgccgg gatcatcggc ttctgctccg gcaccggcga catgctcgac 180
ctcgtcgagc tctacaccca gcgcaagccg gggaacgtcc tggccacgta tctgcccgcc 240
ctgcgcaacg tcaacggcgg cgactcgcac cagggcctgg acccgggctt ccccggcgac 300
tggcgccgcg cggcccagga ctcggcgttc cagcaggccc agaacgacga acgcgaccgc 360
gtctacttcg acccggccgt ccggcagggg aaggcggacg gtatcggcgt actcggacag 420
ttcacgtact acgacgccat cgtcatgcac ggggacggcg gtgacagcac cagcttcagc 480
agcatccgcg ggcgcgccct ggccaaggcc gagccgccgg cgcagggcgg caacgaggtg 540
acgtacctga acgccttcct cgacgcccgg gtctgggcga tgcggcagga ggaggcccac 600
tcggacacca gccgggtcga caccgcccag cgggtcttcc tgacgaaggg caacctgaac 660
ctggatccgc cactggactg gaaggtgtac ggggacagct accacatcgg ctga 714
<210> 3
<211> 237
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
Ala Gly Val Gly Leu Asp Asp Pro Ala Lys Lys Glu Ile Ala Met Lys
1 5 10 15
Leu Val Ser Ser Ala Glu Asn Ser Ser Leu Asp Trp Lys Ala Gln Tyr
20 25 30
Lys Tyr Ile Glu Asp Ile Gly Asp Gly Arg Gly Tyr Thr Ala Gly Ile
35 40 45
Ile Gly Phe Cys Ser Gly Thr Gly Asp Met Leu Asp Leu Val Glu Leu
50 55 60
Tyr Thr Gln Arg Lys Pro Gly Asn Val Leu Ala Thr Tyr Leu Pro Ala
65 70 75 80
Leu Arg Asn Val Asn Gly Gly Asp Ser His Gln Gly Leu Asp Pro Gly
85 90 95
Phe Pro Gly Asp Trp Arg Arg Ala Ala Gln Asp Ser Ala Phe Gln Gln
100 105 110
Ala Gln Asn Asp Glu Arg Asp Arg Val Tyr Phe Asp Pro Ala Val Arg
115 120 125
Gln Gly Lys Ala Asp Gly Ile Gly Val Leu Gly Gln Phe Thr Tyr Tyr
130 135 140
Asp Ala Ile Val Met His Gly Asp Gly Gly Asp Ser Thr Ser Phe Ser
145 150 155 160
Ser Ile Arg Gly Arg Ala Leu Ala Lys Ala Glu Pro Pro Ala Gln Gly
165 170 175
Gly Asn Glu Val Thr Tyr Leu Asn Ala Phe Leu Asp Ala Arg Val Trp
180 185 190
Ala Met Arg Gln Glu Glu Ala His Ser Asp Thr Ser Arg Val Asp Thr
195 200 205
Ala Gln Arg Val Phe Leu Thr Lys Gly Asn Leu Asn Leu Asp Pro Pro
210 215 220
Leu Asp Trp Lys Val Tyr Gly Asp Ser Tyr His Ile Gly
225 230 235
<210> 4
<211> 714
<212> DNA/RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
gcaggcgtcg gcctggacga cccggcgaag aaagagatcg ccatgaagct cgtgtccagc 60
gcggagaact cctcgctgga ctggaaggcc cagtacaagt acatcgagga catcggcgac 120
ggccgcggct acaccgccgg gatcatcggc ttctgctccg gcaccggcga catgctcgac 180
ctcgtcgagc tctacaccca gcgcaagccg gggaacgtcc tggccacgta tctgcccgcc 240
ctgcgcaacg tcaacggcgg cgactcgcac cagggcctgg acccgggctt ccccggcgac 300
tggcgccgcg cggcccagga ctcggcgttc cagcaggccc agaacgacga acgcgaccgc 360
gtctacttcg acccggccgt ccggcagggg aaggcggacg gtatcggcgt actcggacag 420
ttcacgtact acgacgccat cgtcatgcac ggggacggcg gtgacagcac cagcttcagc 480
agcatccgcg ggcgcgccct ggccaaggcc gagccgccgg cgcagggcgg caacgaggtg 540
acgtacctga acgccttcct cgacgcccgg gtctgggcga tgcggcagga ggaggcccac 600
tcggacacca gccgggtcga caccgcccag cgggtcttcc tgacgaaggg caacctgaac 660
ctggatccgc cactggactg gaaggtgtac ggggacagct accacatcgg ctga 714

Claims (4)

1. A chitosan enzyme mutant with improved stability of N-terminal mutant enzyme is characterized in that: the amino acid sequence of the mutant is SEQ ID NO.3.
2. A gene, characterized in that: the gene codes the chitosan enzyme mutant of claim 1, and the nucleotide sequence of the gene is SEQ ID NO.4.
3. A recombinant plasmid, characterized in that: comprising the gene of claim 2.
4. A host cell, characterized in that: comprising the gene of claim 2 or the recombinant plasmid of claim 3.
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