CN111187764A - Deep-sea-derived chitosanase CSN5, and coding gene and application thereof - Google Patents
Deep-sea-derived chitosanase CSN5, and coding gene and application thereof Download PDFInfo
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Abstract
The invention relates to a chitosanase CSN5, a coding gene and application thereof, belongs to the technical field of biological engineering, and provides a chitosanase CSN5 amino acid sequence, a nucleotide sequence for coding the chitosanase and application thereof, a recombinant plasmid CSN5/pET-28a containing the gene, a genetic engineering strain containing the recombinant plasmid and application of the genetic engineering strain. The chitosanase CSN5 of the invention has excellent enzymological characteristics: the optimum temperature is 30 ℃, and the activity is higher at 20-40 ℃; the optimum pH is 7, the enzyme activity is higher under the condition of pH 6-7, and the activity can be maintained above 60%. The reaction conditions are mild, and the method has good industrial application prospect in the preparation of chitosan oligosaccharide by an enzyme method.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to deep-sea sludge-derived chitosanase CSN5, and a coding gene and application thereof.
Background
Chitosan is a deacetylated chitin product widely existing in nature, and a large number of researches show that the chitosan has wide application in the fields of chemical industry, medicine, food, agriculture and the like. Compared with chitosan, the degradation product of chitosan oligosaccharide has small molecular weight, is soluble in water, has excellent biological activity, is very easy to be absorbed by human body, is the only basic oligosaccharide with positive charge in nature discovered at present, and has great development value.
At present, the method for preparing chitosan oligosaccharide by degrading chitosan mainly comprises an enzymatic degradation method, a physical and chemical degradation method, wherein the enzymatic degradation method is easier to control due to the advantages of mild reaction conditions, strong specificity, relatively uniform and high safety of products, less environmental pollution and the like, and the enzyme is widely concerned, can catalyze β -1, 4-glycosidic bond for hydrolyzing chitosan, is an enzyme for specifically hydrolyzing chitosan, is widely distributed in biological groups such as bacteria, fungi, viruses and plants, plays an important role in degrading chitosan polymer and preparing chitosan oligosaccharide, and therefore, new methods are required to be continuously tried to obtain novel chitosan enzyme with excellent properties so as to meet the increasing demand.
Microorganisms in various environments are an important source of new enzymes and bioactive molecules, but in the earth of microbial diversification, less than 1% of microorganisms can be obtained by using culturable technologies, which has become a limiting factor in the development and utilization of microbial resources. In contrast, the majority of non-culturable microorganisms (> 99%) represent a huge gene bank for biotechnological development applications. As a culture-independent technique, metagenomics screens and discovers new functional genes from libraries by extracting genomic DNA of all microorganisms in a specific environment, constructing a genomic library. The method of the metagenomics is successfully used for developing various industrial enzymes, and avoids the process of microorganism separation culture, so that the development and excavation of novel chitosanase resources by using the method of the metagenomics have wide development prospect.
Disclosure of Invention
The technical problem to be solved by the invention is to provide chitosanase CSN5, a coding gene and application thereof, wherein the chitosanase gene CSN5 is from a deep sea sludge metagenome.
The invention is realized by the following technical scheme:
a chitosanase CSN5, the amino acid sequence of which is shown in SEQ ID NO. 1.
The invention also provides a gene CSN5 for coding the chitosanase CSN5, and the nucleotide sequence of the gene is shown as SEQ ID No. 2.
Another objective of the invention is to provide a recombinant plasmid CSN5/pET-28a, wherein the recombinant plasmid CSN5/pET-28a contains the gene CSN5 of the chitosanase CSN 5.
The invention also provides a genetic engineering strain containing the recombinant plasmid csn5/pET-28 a.
Furthermore, the expression vector of the recombinant plasmid csn5/pET-28a is preferably pET28 a.
Furthermore, the genetic engineering strain is obtained by transforming Escherichia coli E.coli BL21(DE3) by the recombinant plasmid csn5/pET-28 a.
The invention also provides application of the chitosan CSN5 in catalyzing chitosan to produce chitosan oligosaccharide.
The invention also provides application of the genetic engineering strain in catalyzing chitosan to produce chitosan oligosaccharide.
Compared with the prior art, the invention has the beneficial effects that:
the chitosanase gene CSN5 is extracted from deep sea sludge, and the amino acid sequence of the coded chitosanase CSN5 has the highest similarity with the peptidoglycan binding protein derived from Geobactraceae bacteria in GenBank, and is 60%. The invention simultaneously constructs the recombinant plasmid CSN5/pET-28a of the gene CSN5 of the chitosanase CSN5 and a genetic engineering strain, and the produced chitosanase CSN5 has excellent enzymological characteristics: the optimum temperature is 30 ℃, and the activity is higher at 20-40 ℃; the optimum pH is 7, the enzyme activity is higher under the condition of pH 6-7, and the activity can be maintained above 60%. The reaction conditions are mild, and the method has good industrial application prospect in the preparation of chitosan oligosaccharide by an enzyme method.
Drawings
FIG. 1: the SDS-PAGE electrophoresis picture of the pure enzyme CSN5 of the chitosanase, wherein M is Marker.
FIG. 2: effect of temperature on the enzymatic activity of purified chitosanase CSN 5.
FIG. 3: influence of pH on the enzymatic Activity of the purified chitosanase CSN 5.
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. Other embodiments obtained by persons skilled in the art based on the embodiments of the present invention without creative efforts also belong to the protection scope of the present invention.
The invention provides chitosanase CSN5, a gene CSN5 for coding the chitosanase CSN5 and application thereof, a recombinant plasmid CSN5/pET-28a containing the gene, a gene engineering strain containing the recombinant plasmid and application of the gene engineering strain.
Example 1 acquisition of a Gene encoding deep-sea chitosanase CSN5
The method for obtaining the chitosanase CSN5 coding gene comprises the following steps:
(1) constructing deep sea sludge metagenome library
The sample for constructing the metagenomic library is from deep sea sludge according to Meta-G-nameTMDNA Isolation Kit (Epicentre) instructions extract and purify metagenomic sample DNA.
Taking a proper amount of purified DNA, and adopting CopyControl as a vectorTMFosmid Library Production Kitwith pCC1FOSTMVector (epicentre), linked to it, transfer the ligation mix into the host strain EPI300TM-T1R e. Then spread on a plate (containing 12.5mg/L chloramphenicol), cultured overnight at 37 ℃ and then the colony on the plate was washed with sterile LB, added with glycerol (20%, v/v) and stored at-80 ℃.
(2) Screening for Positive clones of chitosanase
The preserved bacterial liquid is coated on a culture medium (containing 12.5mg/L chloramphenicol) containing chitosan (0.1%) according to a proper dilution ratio, after the culture medium is cultured at a constant temperature of 37 ℃ for 24-48h, the change of the culture medium around a bacterial colony is observed, a strain capable of forming a clear transparent ring is selected, the streaking is repeated for activity confirmation, and the obtained positive strain is named as 1-CSN 5.
(3) Construction of subclone library and screening of Positive subclones
The plasmid of the screened positive clone 1-CSN5 is extracted according to an alkaline lysis method, and is fragmented by restriction enzyme Sau3AI, separated by agarose electrophoresis, and a DNA fragment with the size of 2-5kb is cut and recovered, then is connected with a plasmid pBluescript II SK (+) fragment cut by restriction enzyme BamHI and is transferred into escherichia coli DH5 α to construct a subclone library, the screening method is consistent with the method, a transparent circle around a colony is observed for selection, and is repeatedly confirmed by scribing, and the obtained equal positive subclone strain is named as 2-CSN 5.
(4) Gene sequencing and analysis of Gene sequence of chitosanase CSN5
Sequencing the screened positive subclone strain CSN5, and performing online analysis on the sequencing result by using NCBI/ORFFinder to obtain a complete open reading frame with the full length of 1116bp, and naming the complete open reading frame as CSN5, wherein the detailed sequence of the complete open reading frame is shown as SEQ ID NO. 2. The length of the encoded protein is 371 amino acid residues, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 1. The amino acid sequence was subjected to homology search in GenBank, and a hypothetical protein derived from Blastocatella sp was found to be similar to the amino acid sequence by up to 62%.
(5) Cloning and recombinant expression of chitosanase CSN5 gene
According to the sequence analysis result, a primer with an enzyme cutting site is designed to amplify the whole gene of the chitosanase CSN5, and the sequence of the primer is as follows:
CSN5 BF: 5'cgcggatccatggggtttagctca 3' (the underlined part is the BamHI cleavage site);
CSN5 SR: 5'cgagctctcatatcgtctccttta 3' (the SacI cleavage site is underlined).
And performing PCR amplification on the gene of the chitosanase CSN5 by using the primers, performing double enzyme digestion on the product by using restriction enzymes BamHI and SacI, purifying the enzyme digestion product, connecting the purified enzyme digestion product with a prokaryotic expression vector pET-28a (+) subjected to the same double enzyme digestion to obtain a recombinant expression plasmid CSN5/pET-28a containing the gene CSN5, transferring the plasmid into escherichia coli DH5 α, and selecting a transformant for sequencing verification.
The sequencing result shows that the sequence of the inserted fragment of the recombinant plasmid is completely consistent with the nucleic acid sequence of SEQ ID NO. 2, and the recombinant Escherichia coli strain CSN5/BL21(DE3) is obtained.
Example 2 expression and purification of recombinant chitosanase CSN5
The recombinant plasmid CSN5/pET-28a in example 1 was transformed into E.coli BL21(DE3) according to a conventional method to obtain a genetically engineered strain producing chitosanase CSN 5.
Inoculating the above genetically engineered strain CSN5/BL21(DE3) into LB culture medium, and culturing at constant temperature of 37 deg.C to OD600When the concentration reaches 0.6-0.8, IPTG is added for induction (the final concentration is 0.1mmol/L), and then the culture is continued for 20h at the temperature of 20 ℃. Centrifuging to collect thallus (6000 × g, 10min), washing thallus twice with normal saline, and adding NPI-0 buffer solution (50mM Na)2HPO40.3M NaCl, pH8.0) to prepare a suspension. And (3) carrying out thallus crushing in an ice-water bath by using ultrasonic crushing (the power is 200-400W, the work time is 3s, the gap time is 5s, and the ultrasonic treatment time is 15 min). After the ultrasonic treatment, the supernatant (10000 × g, 25min) was collected by centrifugation, which was the crude enzyme solution of chitosanase CSN 5.
The recombinant chitosanase CSN5 was purified by Ni affinity chromatography column, and the resulting crude chitosanase CSN5 was filtered through a 0.25 μm membrane and applied to a Ni-NTA column (1mL, Qiagen) equilibrated with NPI-0 at a flow rate of 1 mL/min. Then sequentially using NPI-10(50mM Na)2HPO40.3M NaCl, 10mM imidazole, pH8) and NPI-200(50mM Na)2HPO40.3M NaCl, 200mM imidazole, pH8) and the desired protein, and desalting and concentrating the resulting eluate containing the desired protein by ultrafiltration to obtain the purified chitosanase CSN5, all at 4 ℃. The protein purity was checked by SDS-PAGE electrophoresis, and the result is shown in FIG. 1, giving the electrophoretically pure chitosanase CSN5, having a molecular weight of about 40 kDa.
Example 3 enzymatic Properties testing of recombinant chitosanase CSN5
(1) Activity assay of purified chitosanase CSN5
The activity determination method is carried out by measuring the amount of reducing sugar products by using a DNS method, and specifically comprises the following steps: the reaction mixture containing sodium phosphate buffer (50mM, pH 7), colloidal chitosan (1%, w/v; 85% DDA) and the appropriate amount of chitosanase was incubated at 30 ℃ for 20min and then the reaction was quenched in boiling water for 10 min. By measuring OD with GlcN as standard520Determining the amount of reducing sugars in the supernatant. One unit (U) of the chitosan enzyme activity was defined as the amount of enzyme that released 1. mu. mol reducing sugar per minute under the above assay conditions. All experiments were performed in triplicate and the mean and standard deviation values were used for analysis. The measured activity of chitosanase CSN5 was 1.8U/mg.
(2) Effect of temperature on the enzymatic Activity of purified chitosanase CSN5
The resulting chitosanase CSN5 was diluted with a sodium phosphate buffer (100mM, pH3.5) and then subjected to enzymatic reactions at 20-60 ℃ respectively. The results are shown in FIG. 2, where the highest value of the enzyme activity was taken as 100%. The result shows that the optimum temperature of the purified chitosanase CSN5 is 30 ℃, and the purified chitosanase CSN5 has higher activity at 20-40 ℃.
(3) Effect of pH on the enzymatic Activity of purified chitosanase CSN5
The resulting pure chitosanase CSN5 enzyme was diluted with 100mM buffer (citrate buffer, pH 4-6; phosphate buffer, pH 6-8) at pH 4-8, respectively, and then subjected to enzymatic reaction at 30 ℃. The results are shown in FIG. 3, where the highest value of the enzyme activity was taken as 100%. The result shows that the optimum pH of the recombinant chitosanase CSN5 is 7, the enzyme activity is higher under the condition of pH 6-7, and the activity can be maintained by more than 60%.
SEQ ID NO:1
MGFSSSEIAAAAAIVRVFETGTPAGNYSEVAVLNDGAGISYGISQFTHRSGSL AEVVSRYLASGGVAGRDVFASRLPMLRDRSATAIAKLTADRDFRRALAAAGHAREMREVQEAVAFERYMLPAIRACEGSGFRLPLSLAVIYDSMTHGSYNKIRDRVRVSPAGKGDFEKSWITAYVRERDAWLGSIPRLRSTRYRTRFFKSRIAAGRWGLELPINVHGALITNEILGISPDASGKSGTAAGPNHPPQTMQNEAPQTPTTQPAENPQARPPVSANEVLGRVERGFERAAESFDRVERIGLGAANRTDRAKSLWATVAGTVWQTLWAAVGLLAGLPREVWLVVAVIAAVFTLAYLYRQITLGRLRELKETI
SEQ ID NO:2
atggggtttagctcatcggagatcgccgcggcggccgcgatcgtccgcgtgtttgagacgggcacgccggcgggcaactacagcgaggtcgcggtgctgaacgacggagccggaatttcatacggcatttcgcagttcacccatcgctcgggttcgctggctgaggtcgtttcgcgttacctcgcgagcggcggcgtcgcgggccgagatgtgttcgcatcgcggctgccgatgcttcgggatcgctcggcaacggccatcgcaaagctcacggcggatcgcgactttcgccgagcacttgccgctgcgggccacgcccgcgagatgcgcgaggtgcaggaggcggtcgcctttgagcggtatatgctcccggcgatccgtgcgtgcgagggctcgggctttcggctgccgctctcgctggccgtcatttacgactcgatgacacacggttcttacaacaagatccgcgaccgcgtgcgggtctcgcctgcaggcaagggcgactttgaaaagagctggatcacggcatacgtccgcgagcgggacgcctggctcggttcgatcccgcggctgcgttcgacgcgatatcggacgcgctttttcaagagccgcatcgcggccgggcgttggggcctcgagcttccgatcaacgttcacggcgctctcattacaaacgaaattcttggcatctcgccggacgcatccggcaagagcggaacggcggccgggccgaaccacccgccacaaacaatgcagaacgaagcacctcaaacacccaccacacaacccgccgaaaaccctcaggcccggccgcctgtttcggcaaatgaggtgctcggccgcgttgagcggggctttgaacgtgcggcggaaagctttgaccgcgtggagcgcatcgggctcggggcggcgaaccgcaccgaccgggcgaagtcgctatgggcaacggtcgcgggcacggtttggcaaacgctttgggcggcggtcggccttctcgccggattgccgcgagaggtctggctggtcgtggcggtcatcgccgccgtcttcacactcgcctatctttaccgccagatcacgctcgggcggctgcgggaactaaaggagacgatatga。
Sequence listing
<110> research institute for aquatic products in yellow sea of China institute for aquatic science
<120> deep-sea-derived chitosanase CSN5, and coding gene and application thereof
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>371
<212>PRT
<213> chitosanase CSN5(1-CSN5)
<400>1
Met Gly Phe Ser Ser Ser Glu Ile Ala Ala Ala Ala Ala Ile Val Arg
1 5 10 15
Val Phe Glu Thr Gly Thr Pro Ala Gly Asn Tyr Ser Glu Val Ala Val
20 25 30
Leu Asn Asp Gly Ala Gly Ile Ser Tyr Gly Ile Ser Gln Phe Thr His
35 40 45
Arg Ser Gly Ser Leu Ala Glu Val Val Ser Arg Tyr Leu Ala Ser Gly
50 55 60
Gly Val Ala Gly Arg Asp Val Phe Ala SerArg Leu Pro Met Leu Arg
65 70 75 80
Asp Arg Ser Ala Thr Ala Ile Ala Lys Leu Thr Ala Asp Arg Asp Phe
85 90 95
Arg Arg Ala Leu Ala Ala Ala Gly His Ala Arg Glu Met Arg Glu Val
100 105 110
Gln Glu Ala Val Ala Phe Glu Arg Tyr Met Leu Pro Ala Ile Arg Ala
115 120 125
Cys Glu Gly Ser Gly Phe Arg Leu Pro Leu Ser Leu Ala Val Ile Tyr
130 135 140
Asp Ser Met Thr His Gly Ser Tyr Asn Lys Ile Arg Asp Arg Val Arg
145 150 155 160
Val Ser Pro Ala Gly Lys Gly Asp Phe Glu Lys Ser Trp Ile Thr Ala
165 170 175
Tyr Val Arg Glu Arg Asp Ala Trp Leu Gly Ser Ile Pro Arg Leu Arg
180 185 190
Ser Thr Arg Tyr Arg Thr Arg Phe Phe Lys Ser Arg Ile Ala Ala Gly
195 200 205
Arg Trp Gly Leu Glu Leu Pro Ile Asn Val His Gly Ala Leu Ile Thr
210 215 220
Asn Glu Ile Leu Gly Ile Ser Pro Asp Ala Ser Gly Lys Ser Gly Thr
225 230 235 240
Ala Ala Gly Pro Asn His Pro Pro Gln Thr Met Gln Asn Glu Ala Pro
245 250 255
Gln Thr Pro Thr Thr Gln Pro Ala Glu Asn Pro Gln Ala Arg Pro Pro
260 265 270
Val Ser Ala Asn Glu Val Leu Gly Arg Val Glu Arg Gly Phe Glu Arg
275 280 285
Ala Ala Glu Ser Phe Asp Arg Val Glu Arg Ile Gly Leu Gly Ala Ala
290 295 300
Asn Arg Thr Asp Arg Ala Lys Ser Leu Trp Ala Thr Val Ala Gly Thr
305 310 315 320
Val Trp Gln Thr Leu Trp Ala Ala Val Gly Leu Leu Ala Gly Leu Pro
325 330 335
Arg Glu Val Trp Leu Val Val Ala Val Ile Ala Ala Val Phe Thr Leu
340 345 350
Ala Tyr Leu Tyr Arg Gln Ile Thr Leu Gly Arg Leu Arg Glu Leu Lys
355 360 365
Glu Thr Ile
370
<210>2
<211>1116
<212>DNA
<213> chitosanase CSN5 gene (1-CSN5)
<400>2
atggggttta gctcatcgga gatcgccgcg gcggccgcga tcgtccgcgt gtttgagacg 60
ggcacgccgg cgggcaacta cagcgaggtc gcggtgctga acgacggagc cggaatttca 120
tacggcattt cgcagttcac ccatcgctcg ggttcgctgg ctgaggtcgt ttcgcgttac 180
ctcgcgagcg gcggcgtcgc gggccgagat gtgttcgcat cgcggctgcc gatgcttcgg 240
gatcgctcgg caacggccat cgcaaagctc acggcggatc gcgactttcg ccgagcactt 300
gccgctgcgg gccacgcccg cgagatgcgc gaggtgcagg aggcggtcgc ctttgagcgg 360
tatatgctcc cggcgatccg tgcgtgcgag ggctcgggct ttcggctgcc gctctcgctg 420
gccgtcattt acgactcgat gacacacggt tcttacaaca agatccgcga ccgcgtgcgg 480
gtctcgcctg caggcaaggg cgactttgaa aagagctgga tcacggcata cgtccgcgag 540
cgggacgcct ggctcggttc gatcccgcgg ctgcgttcga cgcgatatcg gacgcgcttt 600
ttcaagagcc gcatcgcggc cgggcgttgg ggcctcgagc ttccgatcaa cgttcacggc 660
gctctcatta caaacgaaat tcttggcatc tcgccggacg catccggcaa gagcggaacg 720
gcggccgggc cgaaccaccc gccacaaaca atgcagaacg aagcacctca aacacccacc 780
acacaacccg ccgaaaaccc tcaggcccgg ccgcctgttt cggcaaatga ggtgctcggc 840
cgcgttgagc ggggctttga acgtgcggcg gaaagctttg accgcgtgga gcgcatcggg 900
ctcggggcgg cgaaccgcac cgaccgggcg aagtcgctat gggcaacggt cgcgggcacg 960
gtttggcaaa cgctttgggc ggcggtcggc cttctcgccg gattgccgcg agaggtctgg 1020
ctggtcgtgg cggtcatcgc cgccgtcttc acactcgcct atctttaccg ccagatcacg 1080
ctcgggcggc tgcgggaact aaaggagacg atatga 1116
<210>3
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
cgcggatcca tggggtttag ctca 24
<210>4
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
cgagctctca tatcgtctcc ttta 24
Claims (7)
1. A chitosanase CSN5 is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.
2. The gene CSN5 for coding the chitosanase CSN5 of claim 1 has a nucleotide sequence shown in SEQ ID No. 2.
3. A recombinant plasmid CSN5/pET-28a, said recombinant plasmid CSN5/pET-28a comprising the gene CSN5 of chitosanase CSN5 of claim 2.
4. A recombinant plasmid csn5/pET-28a as claimed in claim 3, wherein the expression vector of the recombinant plasmid csn5/pET-28a is pET28 a.
5. A genetically engineered strain of recombinant plasmid csn5/pET-28a, obtained by transforming E.coli BL21(DE3) with the recombinant plasmid csn5/pET-28a according to claim 3.
6. Use of the chitosanase CSN5 of claim 1 in catalyzing chitosan to produce chitosan oligosaccharide.
7. The use of the genetically engineered strain of claim 5 in catalyzing the production of chitosan oligosaccharides.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111607580A (en) * | 2020-06-03 | 2020-09-01 | 青岛农业大学 | Novel chitosanase CHI3, encoding gene thereof and preparation method thereof |
| CN111705048A (en) * | 2020-06-03 | 2020-09-25 | 青岛农业大学 | Novel chitosanase CHI2, encoding gene and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101397552A (en) * | 2008-04-28 | 2009-04-01 | 浙江丰安生物制药有限公司 | High efficiency recombinant expressed chitoanase |
| KR20160052946A (en) * | 2014-10-29 | 2016-05-13 | 주식회사 씨앤지 | Chitosan decomposition enzyme producing microorganism and composition for plant cultivation and method using the same |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111607580A (en) * | 2020-06-03 | 2020-09-01 | 青岛农业大学 | Novel chitosanase CHI3, encoding gene thereof and preparation method thereof |
| CN111705048A (en) * | 2020-06-03 | 2020-09-25 | 青岛农业大学 | Novel chitosanase CHI2, encoding gene and application thereof |
| CN111705048B (en) * | 2020-06-03 | 2021-11-30 | 青岛农业大学 | Novel chitosanase CHI2, encoding gene and application thereof |
| CN111607580B (en) * | 2020-06-03 | 2021-12-03 | 青岛农业大学 | Chitosanase CHI3, encoding gene thereof and preparation method thereof |
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| CN111187764B (en) | 2021-03-26 |
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