CN112195168B - Thermophilic chitinase Chi304 mutant and preparation method and application thereof - Google Patents
Thermophilic chitinase Chi304 mutant and preparation method and application thereof Download PDFInfo
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- 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)
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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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)
- C12Y302/01014—Chitinase (3.2.1.14)
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, 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. 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 deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. 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: 2605-2608 (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 show good substrate binding energyForce, 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
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Claims (8)
1. A thermophilic chitinase Chi304 mutant is characterized in that the mutant is obtained by carrying out Y166F single-site mutation on an amino acid sequence shown in SEQ ID No. 1.
2. The gene encoding the Chi304 thermophilic chitinase mutant of claim 1.
3. A recombinant expression vector comprising the coding gene of claim 2.
4. A host cell comprising the recombinant expression vector of claim 3.
5. A method for preparing the thermophilic chitinase Chi304 mutant as claimed in claim 1, which comprises:
(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.
6. The use of the thermophilic chitinase Chi304 mutant of claim 1 for degrading chitin.
7. The use of the gene encoding the thermophilic chitinase Chi304 mutant as claimed in claim 2 for degrading chitin.
8. Use of the recombinant expression vector of claim 3 for degrading chitin.
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| 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 |
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| 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 |
| US8377650B2 (en) * | 2006-03-17 | 2013-02-19 | The Norwegian University Of Life Sciences (Umb) | Method of enhancing degradation of chitin |
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| 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 |
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