CN114657194B - Chitinase Chi6115 and encoding gene and application thereof - Google Patents
Chitinase Chi6115 and encoding gene and application thereof Download PDFInfo
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Abstract
The invention discloses chitinase Chi6115 and a coding gene and application thereof. The enzyme sequence is shown in SEQ ID NO:2. based on metagenome sequencing data of a deep sea hydrothermal sulfide sample, the chitinase Chi6115 gene sequence is obtained by an artificial synthesis method, enzyme cutting sites BamHI and XhoI are added, the gene is cloned into a pET-28a (+) expression vector, induced expression is carried out in E.coli BL21, and recombinant chitinase Chi6115 protein with higher purity is obtained by purification. The optimum temperature of the enzyme is 55 ℃, and the optimum pH is 6.0; the enzyme activity is higher in the range of pH 5.0-6.5 at 45-60 ℃, and the enzyme activity is maintained to be more than 85%; the enzyme not only can hydrolyze colloid chitin, but also can hydrolyze insoluble chitin substrate, and has important application value in the aspects of chitin resource development and utilization and chitosan oligosaccharide preparation.
Description
Technical Field
The invention relates to chitinase Chi6115 derived from deep sea hydrothermal sulfide, and a coding gene and application thereof, and belongs to the technical field of bioengineering.
Background
Chitin is the most abundant renewable natural organic high molecular polymer in marine environment, and the reserve of chitin is the second place in nature, and is only second to cellulose, widely existing in shells of crustaceans, insect exoskeletons, cell walls of fungi and algae plants, and the yield of chitin produced by organisms every year can reach 100-1000 hundred million tons. Chitin is a polymer formed by covalent linkage of N-acetyl-D-glucosamine molecules through beta- (1-4) glycosidic bonds, and can be decomposed by hydrolysis of beta- (1-4) glycosidic bonds by microbial chitinase, and finally oligosaccharide, chitosan or N-acetylglucosamine are generated.
Chitin has biological properties of resisting bacteria and viruses, promoting wound healing, reducing cholesterol, stopping bleeding, resisting oxidation and the like, but the high molecular chitin has high viscosity and poor solubility, so that the application of the chitin is limited. The chitosan oligosaccharide prepared by chemical or enzymatic hydrolysis has good water solubility and reduced viscosity, and has good antibacterial, antioxidant, hypertension reducing, and immunoregulatory effects. The chemical method for preparing the chitosan oligosaccharide often requires extreme reaction conditions to generate useless byproducts, and the microbial enzyme method for producing the chitosan oligosaccharide has mild reaction conditions, is friendly to the environment, has good specificity and controllable reaction process, thereby being an effective method for producing the chitosan oligosaccharide by replacing the chemical method.
Many organisms, including bacteria, archaea, fungi, viruses, algae, plants and animals, are capable of synthesizing chitinases, which are generally classified as endochitinases and exochitinases. Endochitinase hydrolyzes chitin randomly inside the chitin molecule to form soluble low molecular weight chitooligosaccharides, while exochitinase releases chitobiose from the non-reducing end of the chitin chain. Most of the chitinases found at present only hydrolyze chitin pretreated with acid, whereas hydrolases capable of directly hydrolyzing chitin without pretreatment have been reported to a small extent. Before chitin hydrolysis, chitin powder (insoluble chitin) needs to be pretreated with inorganic acid such as hydrochloric acid, phosphoric acid, etc., to loosen the natural chitin structure and facilitate hydrolysis.
The deep sea hot liquid region is rich in various crustaceans such as shrimps and crabs and rich in microorganism resources, and degradation and regeneration of chitin play a very important role in maintaining the balance of the special marine ecosystem of the deep sea hot liquid region. The novel high-efficiency chitinase is excavated from the deep sea hydrothermal region, so that good genes and enzyme resources can be provided for effective development and utilization of chitin resources.
Disclosure of Invention
The invention mainly aims at overcoming the defects of the prior art and provides chitinase Chi6115 and a coding gene and application thereof.
The nucleotide sequence encoding the enzyme is as follows: (SEQ ID NO: 1)
The amino acid sequence of the enzyme is as follows: (SEQ ID NO: 2)
The invention also aims at providing a preparation method of chitinase Chi6115, which comprises the following steps:
(1) The chitinase chi6115 gene is synthesized, and the nucleotide sequence of the chitinase chi6115 gene is shown as SEQ ID NO. 1.
(2) Construction of recombinant plasmids
Two enzyme cutting sites of BamHI and XhoI are respectively introduced into the 5 'and 3' ends of the artificially synthesized chitinase chi6115 gene sequence, the DNA fragment of the chitinase chi6115 gene with the BamHI and XhoI enzyme cutting sites is connected to an expression vector pET-28a (+) which is subjected to the same restriction enzyme cutting after being subjected to restriction enzyme cutting, the connection product is transformed into an escherichia coli Escherichia coli TOP receptor strain, recombinant plasmids containing the chitinase chi6115 gene are screened, and double enzyme cutting and sequencing verification are carried out.
(3) Expression and purification of chitinase Chi6115
The recombinant plasmid containing chitinase Chi6115 gene is transformed into E.coli BL21 receptor strain, IPTG is used for inducing chitinase Chi6115 protein expression, and then the recombinant protein is purified.
Preferably, in step (3), the chitinase Chi6115 protein expression is induced with IPTG.
Preferably, in step (3), the recombinant protein is purified using Ni-Sepharose affinity chromatography (Ni Sepharose TM6Fast Flow kit).
The invention provides chitinase Chi6115, wherein the optimal temperature of the chitinase is 55 ℃, and the optimal pH value is 6.0; the enzyme activity is higher in the range of pH 5.0-6.5 at 45-60 ℃, and the enzyme activity is maintained to be more than 85%; the enzyme not only can hydrolyze colloid chitin, but also can hydrolyze insoluble chitin substrate, and has important application value in the aspect of chitin resource utilization.
Drawings
FIG. 1 shows SDS-PAGE electrophoresis of the purified enzyme proteins;
FIG. 2 shows the effect of different temperatures on enzyme activity;
FIG. 3 is a graph showing the effect of different pH on enzyme activity;
FIG. 4 is an analysis of enzyme hydrolysates.
Detailed Description
Example 1 preparation of chitinase Chi6115
The method comprises the following steps:
experimental materials
Coli E.coil TOP10 and E.coil BL21 strain (from ThermoFisher corporation), expression vector pET-28a (+) (from Novagen corporation); restriction enzymes BamHI and XhoI (available from full formula gold Co.); t4 ligase (available from Takara Co.); LB medium (1% peptone, 0.5% yeast extract, 1% NaCl per liter); binding buffer (phosphate buffer: 20mM phosphate, pH 7.4); rinsing buffer (500 mM NaCl, 10-50 mM imidazole, 20mM phosphate, pH 7.4); elution buffer (500 mM NaCl,500mM imidazole, 20mM phosphate, pH 7.4); 3, 5-dinitrosalicylic acid (DNS) (available from Shanghai, inc.); kanamycin (purchased from Shanghai); ni Sepharose TM6Fast Flow kit (from Takyo corporation); 30kDa ultrafiltration tube (from Takyo corporation); standard of chitosan and chitosan (available from Miragen); standards of chitosan, chitosan pentasaccharide, and chitosan hexasaccharide (available from Megazyme company); powdered chitin (purchased from Sigma). The LB medium containing kanamycin used in the present invention had a kanamycin concentration of 50. Mu.g/mL.
Experimental procedure
(1) Acquisition of chitinase chi6115 Gene
The deep sea hydrothermal sulfide sample of this example was obtained from the hydrothermal region of depth 2900m of the American Karsch ridge of North west India, and based on the metagenome sequencing data of the deep sea hydrothermal sulfide sample, a chitinase gene, named chi6115, was obtained by analysis, and the nucleotide sequence thereof was the sequence shown in SEQ ID NO: 1. And (3) carrying out full-sequence synthesis on the chitinase chi6115 gene to obtain the chitinase chi6115 gene.
(2) Construction of recombinant plasmids
Respectively introducing two enzyme cutting sites of BamHI and XhoI at the 5 'and 3' ends of an artificially synthesized chitinase chi6115 gene sequence, carrying out gel recovery after the DNA fragments of the artificially synthesized chitinase chi6115 gene with the BamHI and XhoI enzyme cutting sites are subjected to restriction enzyme digestion by restriction enzymes BamHI and XhoI, carrying out enzyme digestion and gel recovery on a pET-28a (+) vector by using restriction enzymes BamHI and XhoI, and connecting the DNA fragments of the enzyme-cut chi6115 gene with the pET-28a (+) vector by using T4 ligase; the ligation product was mixed with recipient E.coli Top10 competence, placed on ice for 30min, heat-shocked at 42℃for 90s, then added with 400. Mu.L LB liquid medium, resuscitated at 37℃for 45min at 160rpm, spread on LB solid medium containing 50. Mu.g/mL kanamycin, cultured overnight at 37℃to screen recombinant plasmids, and double-digested and sequencing-verified recombinant plasmids were performed to obtain recombinant plasmids containing chitinase chi6115 gene.
(3) Induction expression of genes and preparation of crude enzyme solution
And (3) transforming the recombinant plasmid containing the chitinase chi6115 gene into E.coli BL21 to obtain the recombinant strain containing the chitinase chi6115 gene. The recombinant strain was inoculated in 5mL of LB liquid medium containing 50. Mu.g/mL kanamycin for overnight culture, and inoculated in 500mL of LB liquid medium containing kanamycin at 1% of the inoculation amount, and cultured at 37℃and 200rpm to OD 600 About 0.6, IPTG (isopropyl-. Beta. -D-thiogalactoside) was added to the mixture so that the final concentration was 1mmol/L, the mixture was induced, the expression was induced overnight at 18℃and 200rpm, and the mixture was centrifuged at 5000rpm for 10 minutes to collect cells. Washing the thalli for 2 times by using a binding buffer solution, suspending the thalli in 30mL of the binding buffer solution, adding a proper amount of lysozyme and protease inhibitor, standing for 2 hours at 4 ℃, performing ultrasonic crushing for 30 minutes at the power of 300W and the working/gap time of 3s/5s, centrifuging for 10 minutes at the temperature of 4 ℃ at 6000rpm, and collecting the supernatant to obtain crude enzyme liquid.
(4) Purification of enzymes
The prepared crude enzyme solution is added into a pre-balanced His purification column Ni Sepharose 6Fast Flow, uniformly mixed for 3 hours at 4 ℃, and then the waste liquid is discarded. Thereafter, 1 time of rinsing with 20mL of rinsing buffer containing 10mM imidazole, 1 time of rinsing with 20mL of rinsing buffer containing 20mM imidazole, and 1 time of rinsing with 10mL of rinsing buffer containing 50mM imidazole were performed. After the rinsing, the eluate was eluted three times with an elution buffer containing 500mM imidazole (the first elution volume was 2mL, the second elution volume was 3mL, and the third elution volume was 2 mL), and the eluate was collected in batches, and the purity of the eluate was detected by SDS-PAGE, and the result was shown in FIG. 1. Imidazole and high-concentration NaCl are removed by a 30kDa ultrafiltration tube, and a purified enzyme solution is obtained.
Example 2
Enzymatic Properties of chitinase Chi6115
(1) Chitinase activity assay
Colloid chitin preparation: 5g of chitin powder is slowly added into 50mL of pre-cooled concentrated hydrochloric acid, stirred uniformly and then kept stand at 4 ℃ for 24h. Then the mixture is slowly added into 500mL of pre-cooled deionized water to be stirred uniformly, and chitin sediment is collected after centrifugation for 10min at 5000g at 4 ℃. Repeatedly re-suspending the precipitate with deionized water, centrifuging, collecting, removing excessive hydrochloric acid on colloid chitin until pH is about 6.5, and preserving at 4deg.C.
The reducing sugar content was determined by DNS. 25. Mu.L of enzyme solution and 100. Mu.L of 1% colloidal chitin substrate were mixed and reacted at 55℃for 2 hours. Adding 200 μL DNS reagent after the reaction, boiling for 5min, immediately cooling in ice water, centrifuging briefly, collecting supernatant, and measuring OD 540 Values (inactivated enzyme solution was used as a control), and the amount of reducing sugar and the enzyme activity were calculated according to a standard curve. Definition of enzyme activity unit: under the above assay conditions, the amount of enzyme required to produce 1. Mu. Mol of reducing sugar per minute of hydrocolloid chitin substrate is defined as one enzyme activity unit (U).
(2) Enzyme action temperature
The enzyme activity was measured at 20-70℃using 1% of colloidal chitin prepared with 50mM sodium phosphate buffer (pH 6.0) as a substrate, and the relative enzyme activities at different temperatures were calculated with the maximum enzyme activity being 100%. The result shows that the optimal reaction temperature of the enzyme is 55 ℃, the enzyme activity is higher in the range of 45-60 ℃, and the relative enzyme activity is maintained to be more than 85%; the relative enzyme activity is maintained to be more than 50% in the range of 35-60 ℃. The results are shown in FIG. 2.
(3) pH of enzyme action
The enzyme activity was measured at 55℃using 1% of colloidal chitin prepared from different buffers (50 mM sodium acetate buffer, pH 4.0-6.0; 50mM sodium phosphate buffer, pH 6.0-8.5; 50mM Tris-HCl buffer, pH 6.5-8.5; 50mM glycine-sodium hydroxide buffer, pH 8.5-9.5) as a substrate, and the relative activities of the enzyme at different pH's were calculated with the maximum enzyme activity being 100%. The result shows that the optimal pH of the enzyme is 6.0, the enzyme activity is higher within the pH range of 5.0-6.5, and the enzyme activity is maintained to be more than 85 percent; the enzyme activity is maintained to be more than 50% within the pH range of 5.0-7.5; in the pH range of 6.0-7.0, sodium phosphate buffer is superior to sodium acetate buffer and Tris-hydrochloric acid buffer. The results are shown in FIG. 3.
(4) Hydrolysis product analysis
Chitinase Chi6115 hydrolysate was analyzed by Thin Layer Chromatography (TLC).
mu.L of the enzyme solution was added to 100. Mu.L of 50mM sodium phosphate buffer (pH 6.0) containing 1% of the substrate (powdered chitin/colloidal chitin), and after mixing, the mixture was allowed to react overnight at 55℃and the overnight reaction mixture was centrifuged at 10000rpm for 5 minutes to obtain a supernatant containing the hydrolysate, and the hydrolysate of chitinase Chi6115 was detected by TLC. The developing agent is n-butanol: methanol: 25% ammonia: water (5:4:2:1, v/v/v/v), the color reagent being aniline: diphenylamine: acetone: 85% phosphoric acid (1:1:50:7.5, w/v/v/v). The result shows that the enzyme not only can hydrolyze colloid chitin, but also can hydrolyze powder chitin, and shows that the enzyme can hydrolyze insoluble chitin substrates, thereby having important application value in the aspect of chitin resource utilization; the main product of the enzymatic hydrolysis of powdered chitin and colloidal chitin is chitosan. The results are shown in FIG. 4.
Sequence listing
<110> university of Huaqiao
<120> third sea institute of Nature resources
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2001
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atgatcttca gccgcaccgc aaatcgcagc catgttaatt ggtttatttt tctggtgatc 60
gcactggcaa tggccacctg gatgggcctg caggcactgg catttagtag cgcagcacct 120
tttagtaatg ccgccgcaca gggtgcatgt gtgaatccgg catgggatgt taatgccatt 180
tatgttgccg atgatctggt gagtcataat ggtcatgaat ggcgcgccaa atggtggacc 240
cagggtgaag aaccgggtac caccggcgaa tggggcgtgt gggaagatcg tggcatttgc 300
agtggtggta atccgaccgc cacacctacc accgttccgc cgaccgccac acctacaagt 360
gtgagcccga ccaatacacc tgcacctacc gcaaccagcg cacctggtgc atgcagcgca 420
ccggcatggg acgcaagtgc catttatgtg gcagatgatc tggttagtca taatgatcat 480
gaatggcgtg caaaatggtg gacacagggc gaagaaccgg gcaccaccgg cgagtggggt 540
gtgtgggaag accgcggcat ttgctcaggt ggtaatccta ccgccacacc tacggcagtg 600
ccgccgaccg ctaccgctac cagcgtgcct ccgaccagca cacctgcacc tacagcaaca 660
cctggtggcc cgacacctac acctaccgcc acacctggcc cgggcggtgc aaaagaagtg 720
gtggcctatt ttgcacagtg gggtatttat ggccgcaatt atcatgtgaa aaatattgtt 780
accagcggta gcgccgatac cgtgaccgtg attaattatg gttttggcca tattaaggac 840
ggtcagtgta ttatggttac ccagccgaat gttatggatg cctgggccga ttatcagaaa 900
ggctatagtg ccgatcagag tgtggatggt gtggcagatg catgggatca gccgctgaaa 960
ggtaatttta atcagctgaa aaagctgaaa cagatgtatc cgaatattaa agtgctgatt 1020
agtatcggtg gttggacctg gagtgaaggc tttagtgatg ccgcactgac acctcagagc 1080
cgcgcagcag cagttcagag ctgcattgat atttatctgc gtggcaatct gccggtggcc 1140
gaaggtgcag gtggcctggg tgcagcctat ggcgtgtttg atggtattga tattgattgg 1200
gaatggccgg ccgcaccggg tcatgaacat aatgtgtatc gtccggaaga tacccagaat 1260
tttaccctgc tgctgcagga atttcgcaat cagctggatg cactggaagc agaaaccggt 1320
cgcgattatc tgctgaccat tgcaggcccg gccggcgttg ataaatatga aaaaattcag 1380
ctggaccaga ttcatccgta tctggattgg ctgagcatta tgagttatga tatgcatggt 1440
gcatggagta atattaccgg tcataatgca ccgctgtata ccagcccgga taatccgctg 1500
ggttatccgg ccaataccta tagtgttgat gcagcagttc aagcatatct ggatgccagt 1560
gttccggcaa ataaaattgt tgccggcctg ccgttttatg gtcgcggctg gaccggcgtg 1620
ccgaatggtg gtaccaatgg tctgtatcag atgggtagtg gcgcagcacc gggcgcctat 1680
gaagcaggtg tggaagatta taaagttctg aaaaccctgg gctatccgca gtatcgcgat 1740
ccgattaccg aaacaccttg gctgtatgat ggcaatacct tttggagcta tgatgatccg 1800
accagcattg ccaataaagt ggcatatatt aataaccgcg gtctgcgtgg cgcaatggtg 1860
tggagtctgg atggtgatga tgccaatggc agcctgatgg caaccattgg cgataatatt 1920
caggcaagta gcgcaagcgg tggcgatggt ctgccgccgg aagaacgtgg tgcaattttt 1980
ctgccgattg cccatcgttg a 2001
<210> 2
<211> 666
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Ile Phe Ser Arg Thr Ala Asn Arg Ser His Val Asn Trp Phe Ile
1 5 10 15
Phe Leu Val Ile Ala Leu Ala Met Ala Thr Trp Met Gly Leu Gln Ala
20 25 30
Leu Ala Phe Ser Ser Ala Ala Pro Phe Ser Asn Ala Ala Ala Gln Gly
35 40 45
Ala Cys Val Asn Pro Ala Trp Asp Val Asn Ala Ile Tyr Val Ala Asp
50 55 60
Asp Leu Val Ser His Asn Gly His Glu Trp Arg Ala Lys Trp Trp Thr
65 70 75 80
Gln Gly Glu Glu Pro Gly Thr Thr Gly Glu Trp Gly Val Trp Glu Asp
85 90 95
Arg Gly Ile Cys Ser Gly Gly Asn Pro Thr Ala Thr Pro Thr Thr Val
100 105 110
Pro Pro Thr Ala Thr Pro Thr Ser Val Ser Pro Thr Asn Thr Pro Ala
115 120 125
Pro Thr Ala Thr Ser Ala Pro Gly Ala Cys Ser Ala Pro Ala Trp Asp
130 135 140
Ala Ser Ala Ile Tyr Val Ala Asp Asp Leu Val Ser His Asn Asp His
145 150 155 160
Glu Trp Arg Ala Lys Trp Trp Thr Gln Gly Glu Glu Pro Gly Thr Thr
165 170 175
Gly Glu Trp Gly Val Trp Glu Asp Arg Gly Ile Cys Ser Gly Gly Asn
180 185 190
Pro Thr Ala Thr Pro Thr Ala Val Pro Pro Thr Ala Thr Ala Thr Ser
195 200 205
Val Pro Pro Thr Ser Thr Pro Ala Pro Thr Ala Thr Pro Gly Gly Pro
210 215 220
Thr Pro Thr Pro Thr Ala Thr Pro Gly Pro Gly Gly Ala Lys Glu Val
225 230 235 240
Val Ala Tyr Phe Ala Gln Trp Gly Ile Tyr Gly Arg Asn Tyr His Val
245 250 255
Lys Asn Ile Val Thr Ser Gly Ser Ala Asp Thr Val Thr Val Ile Asn
260 265 270
Tyr Gly Phe Gly His Ile Lys Asp Gly Gln Cys Ile Met Val Thr Gln
275 280 285
Pro Asn Val Met Asp Ala Trp Ala Asp Tyr Gln Lys Gly Tyr Ser Ala
290 295 300
Asp Gln Ser Val Asp Gly Val Ala Asp Ala Trp Asp Gln Pro Leu Lys
305 310 315 320
Gly Asn Phe Asn Gln Leu Lys Lys Leu Lys Gln Met Tyr Pro Asn Ile
325 330 335
Lys Val Leu Ile Ser Ile Gly Gly Trp Thr Trp Ser Glu Gly Phe Ser
340 345 350
Asp Ala Ala Leu Thr Pro Gln Ser Arg Ala Ala Ala Val Gln Ser Cys
355 360 365
Ile Asp Ile Tyr Leu Arg Gly Asn Leu Pro Val Ala Glu Gly Ala Gly
370 375 380
Gly Leu Gly Ala Ala Tyr Gly Val Phe Asp Gly Ile Asp Ile Asp Trp
385 390 395 400
Glu Trp Pro Ala Ala Pro Gly His Glu His Asn Val Tyr Arg Pro Glu
405 410 415
Asp Thr Gln Asn Phe Thr Leu Leu Leu Gln Glu Phe Arg Asn Gln Leu
420 425 430
Asp Ala Leu Glu Ala Glu Thr Gly Arg Asp Tyr Leu Leu Thr Ile Ala
435 440 445
Gly Pro Ala Gly Val Asp Lys Tyr Glu Lys Ile Gln Leu Asp Gln Ile
450 455 460
His Pro Tyr Leu Asp Trp Leu Ser Ile Met Ser Tyr Asp Met His Gly
465 470 475 480
Ala Trp Ser Asn Ile Thr Gly His Asn Ala Pro Leu Tyr Thr Ser Pro
485 490 495
Asp Asn Pro Leu Gly Tyr Pro Ala Asn Thr Tyr Ser Val Asp Ala Ala
500 505 510
Val Gln Ala Tyr Leu Asp Ala Ser Val Pro Ala Asn Lys Ile Val Ala
515 520 525
Gly Leu Pro Phe Tyr Gly Arg Gly Trp Thr Gly Val Pro Asn Gly Gly
530 535 540
Thr Asn Gly Leu Tyr Gln Met Gly Ser Gly Ala Ala Pro Gly Ala Tyr
545 550 555 560
Glu Ala Gly Val Glu Asp Tyr Lys Val Leu Lys Thr Leu Gly Tyr Pro
565 570 575
Gln Tyr Arg Asp Pro Ile Thr Glu Thr Pro Trp Leu Tyr Asp Gly Asn
580 585 590
Thr Phe Trp Ser Tyr Asp Asp Pro Thr Ser Ile Ala Asn Lys Val Ala
595 600 605
Tyr Ile Asn Asn Arg Gly Leu Arg Gly Ala Met Val Trp Ser Leu Asp
610 615 620
Gly Asp Asp Ala Asn Gly Ser Leu Met Ala Thr Ile Gly Asp Asn Ile
625 630 635 640
Gln Ala Ser Ser Ala Ser Gly Gly Asp Gly Leu Pro Pro Glu Glu Arg
645 650 655
Gly Ala Ile Phe Leu Pro Ile Ala His Arg
660 665
Claims (9)
1. A gene sequence for expressing chitinase Chi6115 is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.
2. A recombinant vector comprising a gene sequence of claim 1 that expresses chitinase Chi 6115.
3. A recombinant bacterium comprising the recombinant vector of claim 2.
4. Chitinase Chi6115, characterized in that: the protein sequence is shown as SEQ ID NO. 2.
5. Use of chitinase Chi6115 according to claim 4 for the exploitation of chitin resources.
6. Use of chitinase Chi6115 according to claim 4 for the preparation of chitosan oligosaccharides.
7. The preparation method of chitinase Chi6115 comprises the following steps:
(1) Synthesizing a nucleotide sequence shown as SEQ ID NO. 1;
(2) Construction of recombinant plasmids
In the artificial synthesis of chitinasechi6115Introduction of the 5 'and 3' ends of the Gene sequence, respectivelyBamHI andXhoi two cleavage sites withBamHI andXhochitinase of I cleavage sitechi6115The DNA fragment of the gene is subjected to restriction enzymeBamHI andXhoi after enzyme digestion, isLigating to the expression vector pET-28a (+) digested with the same restriction enzyme, and transforming the ligation product into E.coliEscherichia coliTOP10 receptor strains, screening for chitinase-containing strainschi6115Recombinant plasmid of the gene, and carrying out double enzyme digestion and sequencing verification;
(3) Expression and purification of chitinase Chi6115
Will contain chitinasechi6115Transformation of recombinant plasmid of Gene into E.coliE. coli BL21 receptor strain, induced chitinase Chi6115 protein expression, and then purifying recombinant protein.
8. The method of preparing chitinase Chi6115 according to claim 7, wherein in step (3), the expression of chitinase Chi6115 protein is induced with IPTG.
9. The method for preparing chitinase Chi6115 according to claim 7, wherein in the step (3), the recombinant protein is purified by using Ni-Sepharose affinity chromatography.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5993808A (en) * | 1993-08-17 | 1999-11-30 | Mogen International Nv | Chitinase, DNA coding therefor and plants containing same |
| CN109810966A (en) * | 2019-03-28 | 2019-05-28 | 南京工业大学 | Chitinase CmChi6 gene and cloning expression and application thereof |
| CN112553183A (en) * | 2020-12-18 | 2021-03-26 | 南京工业大学 | Glycoside hydrolase CmChi3 and application thereof in degradation of hydrocolloid chitin |
| CN113528490A (en) * | 2021-06-07 | 2021-10-22 | 昆明理工大学 | Chitinase, coding gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5993808A (en) * | 1993-08-17 | 1999-11-30 | Mogen International Nv | Chitinase, DNA coding therefor and plants containing same |
| CN109810966A (en) * | 2019-03-28 | 2019-05-28 | 南京工业大学 | Chitinase CmChi6 gene and cloning expression and application thereof |
| CN112553183A (en) * | 2020-12-18 | 2021-03-26 | 南京工业大学 | Glycoside hydrolase CmChi3 and application thereof in degradation of hydrocolloid chitin |
| CN113528490A (en) * | 2021-06-07 | 2021-10-22 | 昆明理工大学 | Chitinase, coding gene and application thereof |
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