CN112195168A - Thermophilic chitinase Chi304 mutant and preparation method and application thereof - Google Patents
Thermophilic chitinase Chi304 mutant and preparation method and application thereof Download PDFInfo
- Publication number
- CN112195168A CN112195168A CN202011414747.3A CN202011414747A CN112195168A CN 112195168 A CN112195168 A CN 112195168A CN 202011414747 A CN202011414747 A CN 202011414747A CN 112195168 A CN112195168 A CN 112195168A
- Authority
- CN
- China
- Prior art keywords
- chi304
- mutant
- chitinase
- thermophilic
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2442—Chitinase (3.2.1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- 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)
- C12Y302/01014—Chitinase (3.2.1.14)
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a thermophilic chitinase Chi304 mutant and a preparation method and application thereof. According to the invention, a series of single-point mutants of chitinase Chi304 with an amino acid sequence shown in SEQ ID No.1 are designed through gene consensus, and finally, a single-point mutant Y166F with improved enzyme activity and thermal stability and a single-point mutant D421E with improved expression quantity, optimal temperature, thermal stability and substrate binding capacity are obtained through successful screening. In addition, the substrate conversion capacity of the two single-point mutants of Y166F and D421E is obviously improved compared with that of the wild type. The invention lays a theoretical foundation for the industrial high-temperature enzymatic degradation of chitin and creates conditions for the industrial application of chitinase.
Description
Technical Field
The invention relates to a chitinase mutant, in particular to a single-point mutant of thermophilic chitinase Chi304, and also relates to a preparation method of the thermophilic chitinase Chi304 mutant and application of the mutant in chitin degradation, belonging to the field of chitinase mutants and application.
Background
Chitin, also known as chitin and chitin, is a natural high-molecular polysaccharide polymerized from N-acetylglucosamine through beta-1, 4 glycosidic bonds, is a white or grey white amorphous solid under natural conditions, has a molecular weight of usually 30-3000kDa and an acetyl degree of more than 90%, is widely distributed in shells of marine products, shrimps and crabs, cell walls of fungi and algae, is the most important renewable nitrogen source and carbon source in the growth and reproduction of marine microorganisms, and has the second place content in nature compared with cellulose.
With the development of the artificial marine product culture technology, a large amount of table wastes such as shrimp and crab shells and the like are accumulated to cause global pollution aggravation, and the wastes generated in the processing process can account for 50 percent of the raw materials, wherein nearly 30 percent of the wastes are chitin. Marine invertebrates alone produce up to 1.6 million tons of chitin per year. The degradation products of chitin, such as chitin oligosaccharide, chitosan oligosaccharide and the like, are widely applied in the industries of biological medicine, agriculture, food, cosmetics and the like due to the safe and nontoxic characteristics of the chitin, and have good economic benefits and development prospects. The main method for degrading chitin at present is a chemical degradation method for breaking polysaccharide glycosidic bonds by concentrated acid, and the method causes great burden to enterprises due to high production cost and processing energy consumption besides the defects of environmental pollution and uncontrollable products in the treatment process. The enzymatic degradation is the most efficient and environment-friendly method in the existing chitin degradation methods, so that the method has the advantages of low use cost, greenness, safety and the like, and the subsequent purification treatment steps are simplified due to the high purity of the product.
Chitinase, as a glycoside hydrolase, can effectively hydrolyze beta-1, 4 glycosidic bonds in chitin to generate high-value chitooligosaccharides. Chitinase is widely found in bacteria, fungi, viruses, and animals and plants. The high compact crystal structure of chitin causes difficult degradation, and the production and processing are usually carried out under high temperature conditions to achieve the effect of chitin destructuring, so that the high-temperature chitinase has irreplaceable effect in actual production. Currently, most of the known chitinases are separated from culturable microorganisms by using a traditional method, and due to the limitation of the growth conditions of the microorganisms, the separated enzymes have poor thermal stability and low enzyme activity and expression level, so that the requirements of industrial application cannot be met.
Therefore, thermophilic and efficient chitinase is obtained through efficient screening, the enzymatic performance of the chitinase is improved through means such as rational design, and the like, and the chitinase has extremely high industrial application value.
Disclosure of Invention
One of the purposes of the invention is to provide a thermophilic chitinase Chi304 mutant;
the second purpose of the invention is to apply the thermophilic chitinase Chi304 mutant and the coding gene thereof to the degradation of chitin;
in order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
as a specific embodiment, the invention provides a thermophilic chitinase Chi304 mutant, which is obtained by carrying out single-site mutation on any one of Y71K, L133T, F135I, Y166F, Y172F, W181Y, A220E, G262N, F396Y or D421E on an amino acid sequence shown in SEQ ID No. 1; preferably, a mutant obtained by subjecting the amino acid sequence shown in SEQ ID No.1 to single-site mutation of any one of L133T, Y166F, Y172F, A220E or D421E; more preferably, the amino acid sequence shown in SEQ ID No.1 is subjected to single-site mutation of Y166F or D421E amino acid to obtain the mutant.
The amino acid single-site mutation 'Y71K' refers to the mutation of the 71 th amino acid of thermophilic chitinase Chi304 with the amino acid sequence shown in SEQ ID NO.1 from tyrosine (Y) to lysine (K); the expression of the remaining single-site mutations is analogized.
The coding gene of the thermophilic chitinase Chi304 mutant also belongs to the protection scope of the invention.
The invention also discloses a recombinant expression vector or a recombinant host cell containing the coding gene of the thermophilic chitinase Chi304 mutant; wherein, the recombinant expression vector can be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector.
The invention further provides a method for preparing any one of the thermophilic chitinase Chi304 mutants, which comprises the following steps:
(1) the coding gene of the thermophilic chitinase Chi304 mutant can be operably connected with an expression regulation element to construct a recombinant expression vector;
(2) transforming the recombinant expression vector into a host cell, culturing the host cell, inducing and expressing the recombinant protein, and purifying to obtain the recombinant protein.
The invention also discloses the thermophilic chitinase Chi304 mutant, the coding gene of the thermophilic chitinase Chi304 mutant and the application of the recombinant expression vector containing the coding gene in degrading chitin.
Detailed description of the invention
Design of Chi304 single-point mutant, construction and induced expression of single-point mutant
Design of Chi304 single-point mutant
Chi304 is thermophilic chitinase screened from marine metagenome in earlier stage of laboratory, in order to further improve the comprehensive property of the thermophilic chitinase, key amino acid sites in the thermophilic chitinase are mutated by a gene consensus method, firstly, a Chi304 amino acid sequence is utilized to perform Blast comparison on the National Center for Biotechnology Information (NCBI) of the United states, and the downloaded e value is less than 10-4The sequences are subjected to homologous alignment by using multi-sequence alignment software ClustalW, the frequency of occurrence of corresponding amino acids at each amino acid site is calculated by using a PIRD website (http:// www.elabcaas.cn/bird/premuse. html) in the laboratory of the inventor, and finally, the point with the highest frequency of occurrence of the amino acid site is selected for carrying out single-point mutation on Chi 304. Finally selecting Y71K, L133T, F135I, Y166F, Y172F, W181Y, A220E, G262N, F396Y andthe ten single point mutations D421E were subjected to subsequent experiments.
Construction and induced expression of Chi304 single-point mutant
The amino acid at the corresponding position is mutated by a two-step PCR method, after the sequencing is correct, the mutant is transferred into an escherichia coli expression strain BL21 (DE3) for induced expression, the expressed protein is purified by a Ni column, and the protein expression condition is detected by SDS-PAGE. According to the expression result, the 10 single-point mutant proteins are better expressed, the target bands are single, and the size of the target bands is consistent with that of the wild Chi304 protein and is 70.95 KDa. The protein expression levels of Y71K, G262N and D421E are improved compared with that of the wild Chi 304.
Enzymatic performance determination test of two Chi304 single-point mutants
Enzyme activity detection of Chi304 single-point mutant
After the Chi304 mutant protein is purified by a Ni column and quantified, the enzyme activity of the Chi304 mutant protein is measured by a DNS method. The results show that: compared with Chi304 enzyme activity, the enzyme activity of the four mutants of L133T, Y166F, Y172F and A220E is respectively improved by 6.59 percent, 12.14 percent, 4.59 percent and 7.39 percent. The enzyme activities of the mutants Y71K, F396Y and G262N are not obviously changed. The enzyme activities of the three mutants of F135I, W181Y and D421E are reduced, wherein the enzyme activity of W181Y is the lowest and is reduced to 85.56% of Chi 304.
(II) detecting optimum temperature and temperature stability of Chi304 and mutant
Enzyme activity determination is carried out on Chi304 and mutants (Y166F and D421E) at different temperatures (70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃ and 95 ℃), and the result shows that the optimal temperature of Chi304 and mutant Y166F is 85 ℃, while the optimal temperature of mutant D421E is obviously changed compared with Chi304 and is increased from 85 ℃ to 90 ℃. And then respectively carrying out heat preservation treatment on Chi304 and the mutant at the temperature of 90 ℃ and then detecting the enzyme activity of the mutant. The result shows that within 30 min of heat preservation treatment, the relative enzyme activities of Y166F and D421E are both higher than that of Chi304, and the residual enzyme activity is higher than 50%, so that good thermal stability is shown.
(III) Chi304 and mutant optimum pH and pH value stability detection
The optimum pH values of the mutants (Y166F and D421E) and Chi304 are detected, and the two mutants are found to be consistent with the optimum pH value of Chi304 and both exert the maximum enzyme activity in a glycine-sodium hydroxide buffer solution with the pH of 9.0.
When pH stability is detected, after the solution is processed on ice for 1 hour, the stability of the pH values of Y166F and D421E is not changed greatly, and good enzyme activity can be still kept between pH 7.5 and 10.0.
(IV) Chi304 and detection of kinetic parameters of mutant enzymatic reaction
By detecting the kinetic parameters of the enzymatic reaction of Chi304 and the mutants (Y166F and D421E), Chi304 was found to haveK m,k catAndk cat / K mthe values were 1.01 mg. multidot.mL-1、954.47 min-1、943.18 mL·mg-1·min-1. And D421EK mValues lower than Chi304 for good substrate binding, Y166F and D421Ek catHigher than Chi304 indicates an increase in conversion efficiency. Furthermore, of D421Ek cat / K mThe value was also improved by 30% compared to Chi304 (table 1).
In conclusion, the single-point mutant of chitinase Chi304 is designed through gene consensus, and the single-point mutant Y166F with improved enzyme activity and thermal stability and the single-point mutant D421E with improved expression quantity, optimal temperature, thermal stability and substrate binding capacity are successfully screened. In addition, the substrate conversion capacity of Y166F and D421E is obviously improved compared with that of the wild type. The invention lays a theoretical foundation for the industrial high-temperature enzymatic degradation of chitin and creates conditions for the industrial application of chitinase.
Definitions of terms to which the invention relates
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "polynucleotide" or "nucleotide" means a deoxyribonucleotide, deoxyribonucleoside, ribonucleoside, or ribonucleotide in either single-or double-stranded formRibonucleotides and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al,Nucleic Acid Res.5081 (1991); the results of Ohtsuka et al,J. Biol. Chem. 260: 2605 and 2608 (1985); and Cassol et al (1992); Rossolini et al,MolCell. Probes8: 91-98(1994))。
the terms "mutation" and "mutant" have their usual meanings herein, and refer to a genetic, naturally occurring or introduced change in a nucleic acid or polypeptide sequence, which has the same meaning as is commonly known to those of skill in the art.
The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome.
The term "transformation" refers to the process by which eukaryotic cells acquire a new genetic marker due to the incorporation of foreign DNA.
Drawings
FIG. 1 SDS-PAGE analysis of Chi304 and its mutants.
FIG. 2 detection of Chi304 and its mutant enzyme activity.
FIG. 3 detection notes of optimal temperature and temperature stability of Chi304 and its mutants: a: detecting the optimum temperature; b: and (5) detecting the temperature stability.
FIG. 4 shows the optimal pH and pH stability detection results of Chi304 and its mutants; a: detecting the most suitable pH value; b: and (5) detecting the pH stability.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 design of Chi304 Single-Point mutants, construction and inducible expression of Single-Point mutants
Design of Chi304 single-point mutant
Chi304 is thermophilic chitinase (the amino acid sequence of which is shown in SEQ ID No. 1) screened from marine metagenome in earlier stage of the laboratory of the inventor, and in order to further improve the comprehensive property of the thermophilic chitinase, the key amino acid sites of the thermophilic chitinase are mutated by a gene consensus method, and the specific method is as follows:
firstly, a Chi304 amino acid sequence is utilized to carry out Blast comparison on the National Center for Biotechnology Information (NCBI) and download an e value less than 10-4The sequences are subjected to homologous alignment by using multi-sequence alignment software ClustalW, the frequency of occurrence of corresponding amino acids at each amino acid site is calculated by using a PIRD website (http:// www.elabcaas.cn/bird/premuse. html) in the laboratory of the inventor, and finally, the point with the highest frequency of occurrence of the amino acid site is selected for carrying out single-point mutation on Chi 304. Finally, ten single-point mutations of Y71K, L133T, F135I, Y166F, Y172F, W181Y, A220E, G262N, F396Y and D421E are selected for subsequent experiments.
Construction and induced expression of Chi304 single-point mutant
The amino acid at the corresponding position is mutated by a two-step PCR method, after the sequencing is correct, the mutant is transferred into an escherichia coli expression strain BL21 (DE3) for induced expression, the expressed protein is purified by a Ni column, and the protein expression condition is detected by SDS-PAGE.
According to the expression result, the 10 single-point mutant proteins are better expressed, the target bands are single, and the size of the target bands is consistent with that of the wild Chi304 protein and is 70.95 KDa. In addition, the protein expression levels of Y71K, G262N and D421E are improved compared with that of the wild Chi304 (FIG. 1).
Test example 1 enzymatic Performance assay of Chi304 Single-Point mutants
Enzyme activity detection of Chi304 single-point mutant
After the Chi304 mutant protein is purified by a Ni column and quantified, the enzyme activity of the Chi304 mutant protein is measured by a DNS method. The results show that: compared with Chi304 enzyme activity, the enzyme activity of the four mutants of L133T, Y166F, Y172F and A220E is respectively improved by 6.59 percent, 12.14 percent, 4.59 percent and 7.39 percent. The enzyme activities of the mutants Y71K, F396Y and G262N are not obviously changed. The enzyme activities of three mutants of F135I, W181Y and D421E are reduced, wherein the enzyme activity of W181Y is the lowest and is reduced to 85.56% of Chi304 (figure 2). The results of protein expression and enzyme activity are combined to determine that the mutants Y166F and D421E are main research objects.
(II) detecting optimum temperature and temperature stability of Chi304 and mutant
Enzyme activity determination is carried out on Chi304 and mutants (Y166F and D421E) under different temperature conditions (70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃ and 95 ℃), and the result shows that the optimal temperature of Chi304 and mutant Y166F is 85 ℃, while the optimal temperature of mutant D421E is obviously changed compared with Chi304 and is increased from 85 ℃ to 90 ℃ (FIG. 3A). And then respectively carrying out heat preservation treatment on Chi304 and the mutant at 90 ℃ for 15 min, 30 min, 45 min, 60 min, 75 min and 90 min, and then detecting the enzyme activity. The result shows that within 30 min of heat preservation treatment, the relative enzyme activities of Y166F and D421E are both higher than that of Chi304, and the residual enzyme activity is higher than 50%, so that good thermal stability is shown (FIG. 3B).
(III) Chi304 and mutant optimum pH and pH value stability detection
The optimum pH values of the mutants (Y166F and D421E) and Chi304 were tested, and it was found that both mutants were consistent with the optimum pH value of Chi304 and exhibited the maximum enzyme activity in glycine-sodium hydroxide buffer solution of pH9.0 (FIG. 4A).
When pH stability is tested, the stability of the pH values of Y166F and D421E is not changed greatly after the buffer solutions with different pH values are treated on ice for 1h, and good enzyme activity can still be kept between pH 7.5 and 10.0 (figure 4B).
(IV) Chi304 and detection of kinetic parameters of mutant enzymatic reaction
By detecting the kinetic parameters of the enzymatic reaction of Chi304 and the mutants (Y166F and D421E), Chi304 was found to haveK m,k catAndk cat / K mthe values were 1.01 mg. multidot.mL-1、954.47 min-1、943.18 mL·mg-1·min-1. And D421EK mValues lower than Chi304 for good substrate binding, Y166F and D421Ek catHigher than Chi304 indicates an increase in conversion efficiency. Furthermore, of D421Ek cat / K mThe value was also improved by 30% compared to Chi304 (table 1).
In conclusion, the single-point mutant of chitinase Chi304 is designed through gene consensus, and the single-point mutant Y166F with improved enzyme activity and thermal stability and the single-point mutant D421E with improved expression quantity, optimal temperature, thermal stability and substrate binding capacity are successfully screened. In addition, the substrate conversion capacity of Y166F and D421E is obviously improved compared with that of the wild type.
Sequence listing
<110> institute of biotechnology of Chinese academy of agricultural sciences
<120> thermophilic chitinase Chi304 mutant and preparation method and application thereof
<130> BJ-2002-201104A-L
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 644
<212> PRT
<213> Marine metagenome (Artificial sequence)
<400> 1
Met Lys Arg Ser Phe Leu Cys Val Leu Leu Ala Gly Gly Leu Leu Leu
1 5 10 15
Ile Gly Gly Cys Leu Val Pro Leu Gly Pro Ser Glu Arg Glu Gly Gly
20 25 30
Glu Ser Ala Arg Ala Ala Ala Gly Gly Leu Pro Tyr His Ile Val Val
35 40 45
Tyr Phe Pro Glu Trp Gly Ile Tyr Ala Gly His Asn Tyr Tyr Tyr Pro
50 55 60
Glu His Val Pro Phe Glu Tyr Ile Thr His Leu Asn Tyr Ala Phe Leu
65 70 75 80
Glu Ile Lys Lys Val Gly Thr Asn Gln Phe Gln Leu Gly Ile Ile Asp
85 90 95
Ser Trp Ala Ser Leu Gln Lys Ala Tyr Gly Thr Trp Asp Gln Thr Glu
100 105 110
Arg Met Gly Asn Ile Arg Glu Leu Lys Tyr Gln Arg Asp Leu Arg Asn
115 120 125
Pro Asn Val Lys Leu Leu Phe Ser Val Gly Gly Trp Ser Arg Ser Gly
130 135 140
Tyr Phe Ser Glu Met Ala Tyr Thr Pro Glu Gly Arg Ala Ser Phe Ile
145 150 155 160
Gln Ser Cys Ile Glu Tyr Leu Arg Thr Tyr Gly Tyr Asp Gly Ile Asp
165 170 175
Ile Asp Trp Glu Trp Pro Gly Val Tyr Arg Ala Pro Ser Asn Ser Pro
180 185 190
Pro Gly Asp Gln Gly Asn Pro Val Tyr Gly Thr Pro Glu Glu Asp Lys
195 200 205
Arg Asn Phe Thr Leu Leu Leu Lys Glu Leu Arg Ala Ala Leu Asp Gln
210 215 220
Ala Gly Gln Gln Asp Gly Lys His Tyr Leu Leu Thr Val Ala Val Gly
225 230 235 240
Ala Gly Lys Asp Lys Ile Asp Met Thr Glu Pro Asn Val Tyr His Gln
245 250 255
Tyr Val Asp Tyr Ile Gly Leu Met Thr Tyr Asp Phe His Gly Gly Trp
260 265 270
Glu Asn Val Thr Asn His Gln Ser Pro Leu Tyr Pro Asn Pro Asn Asp
275 280 285
Pro Ser Glu Tyr Arg Asp Thr Tyr Asn Ile Asp Trp Val Val Lys Tyr
290 295 300
Phe Ser Gly Gln Tyr Ser Val Pro Lys Glu Lys Leu Val Val Gly Leu
305 310 315 320
Pro Tyr Tyr Ser Arg Gly Trp Lys Gly Val Asp Pro Asn Thr Gly Ile
325 330 335
Asn Gly Leu Phe Ala Gln Ala Gln Gly Ala Ala Asp Thr Gly Ala Trp
340 345 350
Asp Gly His Thr Ala Pro Tyr Tyr Gln Thr Arg Ala Trp Gly Gln Gly
355 360 365
Ala Asp Gly Phe Ile Arg Tyr Trp Asp Asp Thr Ala Lys Val Pro Trp
370 375 380
Leu Tyr Asn Pro Ser Thr Gly Tyr Met Trp Thr Phe Glu Asp Leu Glu
385 390 395 400
Ser Ile Thr Ile Lys Ala Asn Tyr Leu Lys Gln Asn Gly Tyr Gly Gly
405 410 415
Phe Leu Ile Trp Asp Ile Thr Gly Asp Tyr Pro Asn Val Ala Asn Gly
420 425 430
Glu Lys Gly Ala Glu Leu Thr Lys Ala Ile Tyr Glu Leu Phe Gly Gly
435 440 445
Asp Ser Val Pro Ser Pro Thr Pro Thr Pro Ser Thr Thr Pro Thr Pro
450 455 460
Ser Pro Thr Pro Thr Ala Thr Pro Ser Pro Thr Pro Thr Pro Thr Pro
465 470 475 480
Thr Ser Thr Pro Thr Ser Thr Pro Ser Pro Thr Pro Thr Pro Thr Val
485 490 495
Thr Pro Thr Pro Thr Pro Thr Gly Thr Ala Tyr Pro Val Trp Asp Pro
500 505 510
Ser Gln Val Tyr Val Gly Gly Asp Arg Val Tyr Trp Asn Gly His Asn
515 520 525
Trp Glu Ala Arg Trp Trp Thr Lys Gly Glu Glu Pro Gly Thr Thr Gly
530 535 540
Glu Trp Gly Val Trp Lys Asp Leu Gly Pro Ala Glu Gly Ala Thr Pro
545 550 555 560
Ser Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Ala Thr Pro
565 570 575
Thr Pro Thr Ser Thr Met Thr Pro Thr Pro Thr Ala Ser Pro Thr Pro
580 585 590
Thr Pro Ser Gly Gly Pro Ala Glu Trp Gln Ala Tyr Thr Ala Tyr Ala
595 600 605
Val Gly Asp Arg Val Ser Tyr Gly Gly Val Val Tyr Arg Cys Leu Gln
610 615 620
Ala His Thr Ser Leu Pro Gly Trp Glu Pro Pro Asn Ala Pro Ala Leu
625 630 635 640
Trp Gln Ala Glu
<210> 2
<211> 1932
<212> DNA
<213> Marine metagenome (Artificial sequence)
<400> 2
atgaagcgta gcttcctgtg cgttctgctg gcgggtggcc tgctgctgat cggtggctgc 60
ctggttccgc tgggtccgag cgagcgtgaa ggtggcgaga gcgcgcgtgc ggcggcgggt 120
ggcctgccgt accacatcgt ggtttatttc ccggaatggg gcatttacgc gggtcacaac 180
tactattacc cggagcacgt tccgttcgaa tacatcaccc acctgaacta tgcgtttctg 240
gagattaaga aagtgggcac caaccagttt caactgggta tcattgacag ctgggcgagc 300
ctgcagaaag cgtacggcac ctgggatcaa accgagcgta tgggtaacat tcgtgaactg 360
aagtatcagc gtgacctgcg taacccgaac gtgaaactgc tgttcagcgt tggtggctgg 420
agccgtagcg gctactttag cgagatggcg tataccccgg aaggtcgtgc gagcttcatc 480
caaagctgca ttgagtacct gcgtacctat ggttacgatg gcatcgacat tgattgggaa 540
tggccgggcg tttatcgtgc gccgagcaac agcccgccgg gtgaccaggg taacccggtg 600
tatggtaccc cggaggaaga taagcgtaac tttaccctgc tgctgaaaga actgcgtgcg 660
gcgctggacc aagcgggtca gcaagatggc aagcactacc tgctgaccgt ggcggttggt 720
gcgggcaagg acaaaatcga tatgaccgaa ccgaacgtgt atcaccagta cgttgactat 780
attggtctga tgacctacga tttccacggt ggctgggaga acgttaccaa ccaccaaagc 840
ccgctgtatc cgaacccgaa cgacccgagc gaataccgtg acacctataa catcgattgg 900
gtggttaagt actttagcgg ccagtatagc gttccgaagg agaaactggt ggttggtctg 960
ccgtattaca gccgtggttg gaaaggcgtg gatccgaaca ccggtatcaa cggtctgttt 1020
gcgcaggcgc agggtgcggc ggacaccggc gcgtgggatg gtcacaccgc gccgtattac 1080
caaacccgtg cgtggggtca gggtgcggac ggctttattc gttactggga cgataccgcg 1140
aaagtgccgt ggctgtacaa cccgagcacc ggttatatgt ggaccttcga ggatctggaa 1200
agcatcacca ttaaggcgaa ctacctgaaa cagaacggct atggtggctt tctgatctgg 1260
gacattaccg gtgattaccc gaacgttgcg aacggcgaga agggcgcgga actgaccaaa 1320
gcgatctatg aactgtttgg tggcgacagc gtgccgagcc cgaccccgac cccgagcacc 1380
accccgaccc cgtctcctac cccgaccgcg accccgtctc ccaccccgac cccgaccccg 1440
accagcaccc cgaccagcac cccgtctcca accccgaccc cgactgtgac cccgaccccg 1500
accccgacgg gtaccgcgta cccggtttgg gacccgagcc aggtgtacgt tggtggcgat 1560
cgtgtgtatt ggaacggcca caactgggag gcgcgttggt ggaccaaggg cgaggaaccg 1620
ggtaccaccg gcgagtgggg tgtttggaaa gacctgggcc cggcggaagg tgcgaccccg 1680
tctccgaccc cgaccccgac cccgaccccg accccgactg ctaccccgac cccgaccagc 1740
acgatgaccc cgaccccgac cgcttctcct accccgaccc cgagcggtgg cccggcggaa 1800
tggcaagcgt acaccgcgta tgcggtgggc gatcgtgtta gctacggtgg cgtggtttat 1860
cgttgcctgc aggcgcacac cagcctgccg ggttgggagc cgccgaacgc gccggcgctg 1920
tggcaagcgg aa 1932
Claims (10)
1. A thermophilic chitinase Chi304 mutant is characterized in that the mutant is obtained by carrying out any one amino acid single-site mutation of Y71K, L133T, F135I, Y166F, Y172F, W181Y, A220E, G262N, F396Y or D421E on an amino acid sequence shown in SEQ ID No. 1.
2. The thermophilic chitinase Chi304 mutant according to claim 1, characterized in that the mutant is obtained by subjecting the amino acid sequence shown in SEQ ID No.1 to single-site mutation of any one of the amino acid residues L133T, Y166F, Y172F, A220E or D421E.
3. The thermophilic chitinase Chi304 mutant according to claim 2, characterized in that the mutant is obtained by performing amino acid single-site mutation of Y166F or D421E on the amino acid sequence shown in SEQ ID No. 1.
4. The gene encoding the Chi304 mutant thermophilic chitinase of any one of claims 1-3.
5. A recombinant expression vector comprising the coding gene of claim 4.
6. A host cell comprising the recombinant expression vector of claim 5.
7. A method for preparing the thermophilic chitinase Chi304 mutant as claimed in any one of claims 1-3, comprising:
(1) the coding gene of the thermophilic chitinase Chi304 mutant can be operably connected with an expression regulation element to construct a recombinant expression vector;
(2) transforming the recombinant expression vector into a host cell, culturing the host cell, inducing and expressing the recombinant protein, and purifying to obtain the recombinant protein.
8. Use of the thermophilic chitinase Chi304 mutant as claimed in any of claims 1-3 for degrading chitin.
9. The use of the gene encoding the thermophilic chitinase Chi304 mutant as claimed in claim 4 for degrading chitin.
10. Use of the recombinant expression vector of claim 5 for degrading chitin.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110225439.4A CN112899257B (en) | 2020-12-07 | 2020-12-07 | Thermophilic chitinase Chi304 mutant and application thereof in degradation of chitin |
CN202011414747.3A CN112195168B (en) | 2020-12-07 | 2020-12-07 | Thermophilic chitinase Chi304 mutant and preparation method and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011414747.3A CN112195168B (en) | 2020-12-07 | 2020-12-07 | Thermophilic chitinase Chi304 mutant and preparation method and application thereof |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110225439.4A Division CN112899257B (en) | 2020-12-07 | 2020-12-07 | Thermophilic chitinase Chi304 mutant and application thereof in degradation of chitin |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112195168A true CN112195168A (en) | 2021-01-08 |
CN112195168B CN112195168B (en) | 2021-03-19 |
Family
ID=74033930
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110225439.4A Active CN112899257B (en) | 2020-12-07 | 2020-12-07 | Thermophilic chitinase Chi304 mutant and application thereof in degradation of chitin |
CN202011414747.3A Active CN112195168B (en) | 2020-12-07 | 2020-12-07 | Thermophilic chitinase Chi304 mutant and preparation method and application thereof |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110225439.4A Active CN112899257B (en) | 2020-12-07 | 2020-12-07 | Thermophilic chitinase Chi304 mutant and application thereof in degradation of chitin |
Country Status (1)
Country | Link |
---|---|
CN (2) | CN112899257B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116536289A (en) * | 2023-06-21 | 2023-08-04 | 中国农业科学院生物技术研究所 | Chitinase with lysozyme activity, mutant and application thereof |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113249360B (en) * | 2021-07-06 | 2021-10-01 | 深圳润康生态环境股份有限公司 | Chitinase mutant ChiM and application |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070218046A1 (en) * | 2006-03-17 | 2007-09-20 | The Norwegian University Of Life Sciences (Umb) | Method of enhancing degradation of chitin |
CN109576249A (en) * | 2018-12-14 | 2019-04-05 | 大连大学 | A kind of acid-tolerant mutant of low temperature resistant chitinase and its application |
CN109943553A (en) * | 2018-12-14 | 2019-06-28 | 大连大学 | A kind of low temperature resistant mutant of chitinase and its application |
CN110777134A (en) * | 2019-10-31 | 2020-02-11 | 山东大学 | Mutant chitinase and application thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5928928A (en) * | 1995-06-07 | 1999-07-27 | Universiteit Van Amsterdam | Human chitinase, its recombinant production, its use for decomposing chitin, its use in therapy or prophylaxis against infection diseases |
WO1998006859A1 (en) * | 1996-08-09 | 1998-02-19 | Human Genome Sciences, Inc. | Human chitinase alpha and chitinase alpha-2 |
-
2020
- 2020-12-07 CN CN202110225439.4A patent/CN112899257B/en active Active
- 2020-12-07 CN CN202011414747.3A patent/CN112195168B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070218046A1 (en) * | 2006-03-17 | 2007-09-20 | The Norwegian University Of Life Sciences (Umb) | Method of enhancing degradation of chitin |
CN109576249A (en) * | 2018-12-14 | 2019-04-05 | 大连大学 | A kind of acid-tolerant mutant of low temperature resistant chitinase and its application |
CN109943553A (en) * | 2018-12-14 | 2019-06-28 | 大连大学 | A kind of low temperature resistant mutant of chitinase and its application |
CN110777134A (en) * | 2019-10-31 | 2020-02-11 | 山东大学 | Mutant chitinase and application thereof |
Non-Patent Citations (1)
Title |
---|
GENPEPT: "WP_013314362.1", 《GENPEPT》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116536289A (en) * | 2023-06-21 | 2023-08-04 | 中国农业科学院生物技术研究所 | Chitinase with lysozyme activity, mutant and application thereof |
CN116536289B (en) * | 2023-06-21 | 2023-09-15 | 中国农业科学院生物技术研究所 | Chitinase with lysozyme activity, mutant and application thereof |
Also Published As
Publication number | Publication date |
---|---|
CN112899257B (en) | 2022-07-12 |
CN112195168B (en) | 2021-03-19 |
CN112899257A (en) | 2021-06-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108285900B (en) | Recombinant alginate lyase and construction method and application thereof | |
JP5468680B2 (en) | Novel alpha-neo agarobiose hydrolase and method for obtaining monosaccharides using the same | |
CN112646794B (en) | Exoinulase mutant MutY119V with improved low-temperature activity | |
CN112831485B (en) | Low-temperature activity improved exoinulase mutant MutDR121EH9 | |
CN112852782B (en) | Low-temperature adaptive improved low Wen Waiqie inulase mutant MutDL121EK5 and application thereof | |
CN112195168B (en) | Thermophilic chitinase Chi304 mutant and preparation method and application thereof | |
CN106414728A (en) | Agarooligosaccharide hydrolase and method for producing 3,6-anhydro-l-galactose and galactose from agarose by using same | |
CN112111472B (en) | Novel beta-xylosidase and preparation thereof | |
CN112725319B (en) | Alginate lyase FaAly7 with polyG substrate specificity and application thereof | |
CN108048430B (en) | Endoglucanase NfEG12A mutant and coding gene and application thereof | |
CN112941089A (en) | Alginate lyase mutant gene, alginate lyase mutant, engineering bacterium containing mutant, construction method and application | |
CN112941052A (en) | Chitosanase OUC-T613 and application thereof | |
KR101062978B1 (en) | Production Optimization and Gene Cloning of Endoglucanases Derived from the New Bacterium Penicillium Pinophilum KMJ601 | |
CN110144341B (en) | Alginate lyase mutant | |
KR20100040438A (en) | A novel agarase and an enzymatic production method of agarooligosaccharide from agarose using the same | |
CN111394374A (en) | Cellulase gene gk2691 for encoding cellulase family GH30 and application thereof | |
CN104087604A (en) | Genetic expression sequence of inulin fructotransferase | |
CN115141841A (en) | Pichia pastoris mutant strain and application thereof in production of alginate lyase | |
CN102245763A (en) | Acidothermus celluloyticus xylanase | |
CN109609485A (en) | A kind of chitin deacetylase and its application | |
JP7011132B2 (en) | New chitosanase CHI1, its coding gene and its use | |
CN111705048B (en) | Novel chitosanase CHI2, encoding gene and application thereof | |
CN111484988B (en) | Bifunctional enzyme with xylanase and feruloyl esterase activities, and coding gene and application thereof | |
CN111607580A (en) | Novel chitosanase CHI3, encoding gene thereof and preparation method thereof | |
CN111471667A (en) | Chitosanase Csn-PT and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |