CN111549016B

CN111549016B - Extreme heat-resistant xylanase XYNA and mutant gene, application and preparation method thereof

Info

Publication number: CN111549016B
Application number: CN202010447518.5A
Authority: CN
Inventors: 熊海容; 王晶
Original assignee: South Central University for Nationalities
Current assignee: South Central Minzu University
Priority date: 2020-05-25
Filing date: 2020-05-25
Publication date: 2021-11-26
Anticipated expiration: 2040-05-25
Also published as: CN111549016A

Abstract

The invention belongs to the field of protein engineering and genetic engineering, and particularly relates to extreme heat-resistant xylanase XYNA, a mutant gene thereof, application and a preparation method, wherein the amino acid sequence of the mutant gene is shown as SEQ ID No.1, the mutant gene is shown as SEQ ID No.2, and the extreme heat-resistant xylanase XYNA is efficiently expressed in a pichia pastoris expression system, and has wide application prospects in the fields of feed additives, health-care foods, papermaking, washing, brewing, spinning, medicines and the like.

Description

Extreme heat-resistant xylanase XYNA and mutant gene, application and preparation method thereof

Technical Field

The invention belongs to the field of protein engineering and genetic engineering, and particularly relates to extreme heat-resistant xylanase XYNA and a mutant gene, application and a preparation method thereof.

Background

Xylan is the most important hemicellulose in plant cell walls, accounts for about 35% of the dry weight of plant cells, and is the polysaccharide with the most abundant content except cellulose in nature. Xylan is a hybrid polysaccharide formed by a main chain formed by polymerization of xylose through beta-1, 4-glycosidic bonds and a plurality of side chain groups, is an abundant biomass resource, and can be degraded into xylooligosaccharide and xylose which are urgently needed in the international market under the action of xylanase. However, a large part of xylanase in nature is not effectively utilized, and a great waste of the resource is caused.

Microbial xylanases (EC 3.2.1.8) are important industrial enzymes that randomly catalyze the hydrolysis of beta-1, 4-D-xylosidic bonds within xylan to xylooligosaccharides. Xylo-oligosaccharides as substrates can be further degraded by other xylanases such as beta-D-xylosidase, alpha-L-arabinofuranosidase, D-glucuronidase, etc. (Khandepaker R, Numan MT. bifunctional and the said gene functional use in biotechnology J Ind microbial biotechnology 2008,35: 635-644.). Xylanases are mainly classified into families 10 and 11 of glycoside hydrolases based on significant differences in functional primary and tertiary structure and model, however xylanases with xylanolytic activity have also been found in enzymes of families 5, 7, 8, 16, 26, 30, 43, 52 and 62 of glycoside hydrolases (http:// www.cazy.org/fam/ac _ gh. html) (Collins T, Gerday C, Feller G. Xylanase, xylanase family and exophagic xylanases. FEMS Microbiol Rev 2005,29: 3-23.). The GH 10 family xylanase has a large relative molecular weight (>30000), a complex structure, and usually consists of multiple structural domains, and a Catalytic Domain (CD) is a main component of the xylanase and bears the hydrolysis characteristic of the xylanase. Although they vary greatly in the number and composition of amino acids, their catalytic structures are very close in size, mainly structures that occur repeatedly in α -helices and α -sheets, and belong to a family, called the (α/β) 8-sheet structure, because of structural similarity to TIM, where glutamate and aspartate at specific positions have a large influence on the catalytic properties. The enzyme also contains domains with non-catalytic activity, such as polysaccharide substrate binding domain, thermal stability domain, and a plurality of catalytic domains, etc., which endow the enzyme with the functions of decomposing soluble xylan, insoluble xylanase and other substrates, etc.

The application of xylanase originated from its use in animal feed processing in 1980 and was subsequently gradually applied in the fields of paper making, food, pharmacy, brewing, textile and biofuel, etc. At present, the enzyme is mainly applied to industries such as pulping and papermaking, feed, food and the like, and the application position in modern industry is more and more obvious. Xylanase is one of the key enzymes in the process of producing alcohol by using non-starch raw materials. With the development of bioenergy industry, xylanases have been applied in a wider field (Fawzi EM. high viscous purified xylanase from Rhizomucor miehei NRRL 3169, Ann Microbiol 2010,60: 363-368.). In the industrial production process, extreme environments such as high temperature and the like often exist, the high temperature environment can accelerate the enzymatic reaction, improve the flow property of liquid materials, prevent harmful microorganisms from growing and propagating in the process and the like. The common intermediate-temperature xylanase can generate structural change at high temperature, thereby greatly losing activity. To prevent this heat inactivation, some chemicals are added to protect the enzyme, which not only increases the production cost but also adversely affects the product quality. The heat-resistant xylanase produced by Thermotoga maritima MSB8 is used for hydrolysis, so that the problem that the added enzyme is quickly inactivated in a high-temperature environment can be fundamentally solved.

The xylanase derived from bacteria has higher thermal stability than xylanase derived from fungi, and pichia pastoris has the potential of efficient secretion, correct folding protein and extremely high cell concentration culture and is often used as an expression system for producing exogenous protein on a large scale, and xylanase genes derived from bacteria are applied to pichia pastoris capable of being cultured at high density after sequence optimization, so that the xylanase is more suitable for industrial production. Xylanase 1VBR derived from Thermotoga maritima MSB8 has excellent enzymological properties (Winterhalter C, Liebl W. two extreme thermostable xylans of the hyperthermophilic bacteria Thermoyoga maritima MSB8. apple Environ Microbiol.1995,61(5):1810-1815.), extreme thermostability, and can maintain higher enzyme activity under wide pH conditions.

The error-prone PCR technique is an evolution of the normal PCR technique, which is a PCR technique that makes DNA more susceptible to mismatching during the course of replication amplification, and is also called mismatch PCR or error-prone PCR. Error-prone PCR generally utilizes low-fidelity Taq DNA polymerase and some means such as changing PCR reaction systems to reduce the fidelity of DNA replication in the PCR process and increase the mismatch rate, thereby obtaining a DNA sequence or gene different from the original one.

Disclosure of Invention

The invention aims to obtain a xylanase mutant XYNA with higher heat resistance by mutating a xylanase 1VBR gene sequence through error-prone PCR, screening after expression and enzyme characteristic identification, efficiently expressing in a pichia pastoris expression system, and applying the obtained extremely heat-resistant xylanase XYNA to the fields of feed additives, health-care food, papermaking, washing, brewing, spinning, medicines and the like.

The invention provides extreme heat-resistant xylanase XYNA, and the amino acid sequence of the extreme heat-resistant xylanase XYNA is shown in SEQ ID No. 1.

Furthermore, the optimum reaction temperature of the extremely heat-resistant xylanase XYNA is 100 ℃.

A mutant gene of extreme heat-resistant xylanase XYNA, wherein the mutant gene codes the extreme heat-resistant xylanase XYNA, and the gene sequence of the mutant gene is shown as SEQ ID NO. 2.

And an expression vector comprising the extremely thermostable xylanase XYNA coding sequence.

Wherein the expression vector is pPIC 9K-XYNA.

And a recombinant strain comprising the extremely thermostable xylanase XYNA coding sequence.

Wherein, the recombinant strain is recombinant escherichia coli or recombinant yeast.

And the application of the extremely heat-resistant xylanase XYNA in feed additives, health-care food, papermaking, washing, brewing, spinning or medicines.

A preparation method of a gene sequence of extreme heat-resistant xylanase XYNA comprises the steps of taking a pET-22b (+) -1VBR plasmid as a template, using Taq enzyme, setting the concentrations of Mg2+ and Mn2+ in a PCR system to be 2.5mM and 0.8mM respectively, setting the primer sequences to be 5'-CGCCATGGATTCTCAGAATGTATC-3' and 5'-TCGCGACTCGAGTTTTCTTTCTTC-3', carrying out two rounds of error-prone PCR, and screening to obtain a mutant gene sequence of the xylanase XYNA, wherein the mutation positions of the mutant gene sequence are D592G, V632A, K789E and I837M.

The method for preparing the extreme heat-resistant xylanase XYNA comprises the steps of transforming host cells by using the expression vector to obtain a recombinant strain, and culturing the recombinant strain to express the extreme heat-resistant xylanase XYNA.

The xylanase mutant XYNA with high heat resistance is provided, is efficiently expressed in a pichia pastoris expression system, and has wide application prospects in the fields of feed additives, health-care food, papermaking, washing, brewing, spinning, medicines and the like.

In addition, the xylanase 1VBR gene KR078269 is synthesized by the whole gene, and comprises a terminator sequence and enzyme cutting site sequences at two ends of the terminator sequence. Error-prone PCR mutation is carried out on the gene sequence to obtain the gene sequence of the xylanase XYNA. The whole gene synthetic xylanase gene is inserted into an expression vector pPIC9K through double enzyme digestion and connection to construct a recombinant expression plasmid pPIC 9K-XYNA. The recombinant expression plasmid is transformed into escherichia coli DH5 alpha competent cells, and a positive clone strain is screened out by a PCR verification method. The protein content of the xylanase XYNA secreted by the gene engineering strain GS115-XYNA for producing the xylanase XYNA provided by the invention in a fermentation liquid can reach an electrophoresis pure level, and almost no purification is needed.

Drawings

FIG. 1 is an SDS-PAGE analysis of xylanase mutants XYNA;

FIG. 2 is a thermal death curve (pH 5.5, 100 ℃) of xylanase mutant XYNA;

FIG. 3 shows the optimum temperature (pH 5.5, 10 min) of xylanase mutant XYNA.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.

Experimental materials:

1) strains and plasmids: escherichia coli (Escherichia coli) DH5 alpha and Pichia pastoris GS115 were gifted to the institute for feed, Chinese academy of agricultural sciences; the pPIC9K secretory expression vector was purchased from Invitrogen.

2) Enzymes and kits: restriction enzyme, Taq enzyme, Pyrobest DNA polymerase, T4 DNA ligase and other tool enzymes purchased from TaKaRa company; DNA purification kits were purchased from Seiki Biotechnology Ltd.

3) Biochemical reagents: g418 was purchased from Invitrogen; protein molecular weight standards were purchased from shanghai institute of biochemistry; IPTG, X-Gal, SDS and carob were purchased from Sigma; TEMED, ammonium persulfate, acrylamide and methylene bisacrylamide are used as traditional Chinese medicine reagents.

50mM disodium phosphate-citric acid buffer: dissolving 7.10g of disodium hydrogen phosphate in 800mL of double distilled water, adjusting the pH value to any value within the range of 4.0-7.5 by using citric acid, and then fixing the volume to 1L.

50mM Tris-HCl buffer: 6.06g of Tris is dissolved in 800mL of double distilled water, the pH value is adjusted to any value within the range of 7.5-9.0 by 1M HCl, and then the volume is adjusted to 1L.

50mM glycine-sodium hydroxide buffer: dissolving 7.50g of glycine in 800mL of double distilled water, adjusting the pH value to any value within the range of 9.0-12 by using 1M of sodium hydroxide solution, and then fixing the volume to 1L.

The experimental procedures in the following examples are conventional unless otherwise specified.

The percentages in the following examples are by mass unless otherwise specified.

Example 1: acquisition of xylanase mutant strain XYNA Gene sequence

According to the sequence alignment, the amino acid sequence of xylanase 1VBR from Thermotoga maritima MSB8 published in PDB has 100% similarity with the predicted xylanase (EHA58720.1) in Thermotoga maritima MSB8 genome sequence (CP 007013.1: 1,869,644 bp). The predicted xylanase gene sequence is a gene sequence (873,589 → 874,572bp) in the Thermotoga maritima MSB8 genome sequence.

Based on the codon usage preference of Pichia pastoris, rare codons in the Pichia pastoris are converted into high-frequency expression codons, and meanwhile, the amino acid sequence of xylanase 1VBR is contrasted to perform codon optimization on the predicted xylanase (EHA58720.1) gene sequence to obtain the optimized xylanase 1VBR gene sequence (NCBI gene number: KR 078269). Using pET-22b (+) -1VBR plasmid as a template, using Taq enzyme, setting the concentrations of Mg2+ and Mn2+ in a PCR system to be 2.5mM and 0.8mM respectively, setting the primer sequences to be 5'-CGCCATGGATTCTCAGAATGTATC-3' and 5'-TCGCGACTCGAGTTTTCTTTCTTC-3', carrying out two rounds of error-prone PCR, and screening to obtain the gene sequence of the xylanase mutant strain XYNA.

Example 2: construction of recombinant expression plasmid pPIC9K-XYNA containing xylanase mutant strain XYNA Gene

Adding a terminator sequence preferred by pichia pastoris at the 3 ' end of xylanase XYNA gene, respectively introducing restriction enzyme EcoR I and Not I sites at the 5 ' end and the 3 ' end, and handing the gene sequence with Wuhan engine scientific creative biotechnology limited company to complete the whole gene synthesis. The optimized xylanase XYNA gene and the secretory expression vector pPIC9K are subjected to double enzyme digestion by using restriction enzymes EcoR I and Not I, and then are connected by using ligase to construct a recombinant expression plasmid pPIC 9K-XYNA. The recombinant expression plasmid is transformed into escherichia coli DH5 alpha competent cells, and a positive clone strain pPIC9K-XYNA-DH5 alpha is screened out by a PCR verification method.

Example 3: construction of Pichia pastoris gene engineering strain for efficient secretory expression of xylanase XYNA

The strain pPIC9K-XYNA-DH5 alpha is activated and cultured by adopting LB liquid culture medium, and the recombinant plasmid pPIC9K-XYNA is extracted. The recombinant plasmid was linearized with the restriction enzyme Bgl II and the digested product was recovered. Pichia pastoris GS115 competent cells were prepared with reference to the EasySelect Pichia Expression Kit. And (3) gently and uniformly mixing about 10 mu g of linearized plasmid and 80 mu L of competent cells, placing the mixture on ice for 15min, transferring the mixture into a precooled 0.2cm electric rotating cup, immediately adding 1mL of precooled 1mol/L sorbitol after 1500V electric shock is finished, standing the mixture in an incubator at 30 ℃ for 1h, coating the mixture on an MD (MD) plate, and performing inversion culture at 30 ℃ for about 48h until a transformant appears.

Single colonies were picked and inoculated in the order of the numbers on MD plates containing 0.25, 0.5, 1.0, 2.0, 3.0 and 4.0mg/mL G418, respectively, and cultured by inversion at 30 ℃ for about 48h until single colonies appeared. The recombinant strain with the strongest resistance is selected and inoculated in a BMGY medium containing 3mL of the corresponding number, and is subjected to shake cultivation at 30 ℃ and 200rpm for about 48 hours until the OD600 reaches 1.8-6.0. The cells were collected, resuspended in 1mL BMMY medium, and induced in 0.5% methanol for about 48 h.

Collecting supernatant, and detecting enzyme activity by using a DNS method to screen a positive recombinant strain GS115-XYNA with higher enzyme yield.

Example 4: property analysis of xylanase mutant strain XYNA

FIG. 1 is an SDS-PAGE analysis of xylanase mutants XYNA, wherein M is a molecular weight marker.

1) Determination of xylanase XYNA thermal lethality curve: diluting the xylanase solution with a buffer solution with pH 5.5, placing the diluted xylanase solution in a water bath at 100 ℃, respectively treating for different times, detecting the activity of the xylanase under the optimal reaction condition, and calculating the half-life period of the xylanase according to the activity. The result shows that the mutant enzyme can still keep more than 50 percent of relative enzyme activity after being treated for 3 hours at 100 ℃ under the condition of the optimal pH value, and the heat resistance of the mutant enzyme is better than that of the original enzyme 1 VBR. FIG. 2 shows the thermal death curve (pH 5.5, 100 ℃) of xylanase mutant XYNA, wherein 1VBR is represented by ● and XYNA is represented by ■.

2) Determination of xylanase XYNA optimum temperature: reacting the enzyme with 0.5% xylan solution at different temperatures for 15min at pH 5.5, adding 2.5mL DNS reagent, boiling water bath for 5min, cooling to room temperature, and adding water to reach volume of 12.5 mL. OD540 was detected in a spectrophotometer. The relative residual enzyme activity under other conditions is calculated by taking the highest enzyme activity as 100 percent. As a result, the optimum reaction temperature for xylanase XYNA was 100 ℃ or more, and FIG. 3 shows the optimum temperature (pH 5.5, 10 minutes) for xylanase mutant XYNA, wherein 1VBR is represented by ● and XYNA is represented by ■.

SEQ ID NO.3 of the sequence Listing is the original amino acid sequence of the extreme heat-resistant xylanase 1VBR (PDB code: 1VBR, amino acid sequence 517-840); SEQ ID NO.1 is an error-prone PCR mutant XYNA mutated at 4 amino acid positions (amino acid sequence 517-840, mutation positions D592G, V632A, K789E, I837M).

The mutation sites are marked with underlining and bolding below, SEQ ID NO. 1:

SEQ ID NO.4 is the original gene sequence information of the extreme heat-resistant xylanase 1VBR (NCBI gene number: KR078269, base sequence 9-981); SEQ ID NO.2 is the sequencing information of the mutant XYNA gene generated by error-prone PCR: among them, the mutation sites are underlined, and among them, the effective mutation sites are marked by bold face (4 effective mutation sites), and the ineffective mutation sites are marked by italic face and not bold face (3 ineffective mutation sites).

SEQ ID NO.2：

Sequence listing

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Claims

1. An extremely heat-resistant xylanase XYNA, which is characterized in that: the amino acid sequence is shown in SEQ ID NO. 1.

2. An extremely thermostable xylanase XYNA according to claim 1, characterized in that: the optimal reaction temperature of the extremely heat-resistant xylanase XYNA is 100 ℃.

3. A mutant gene of extreme heat-resistant xylanase XYNA, which is characterized in that: the mutant gene codes the extreme heat-resistant xylanase XYNA of claim 1, and the gene sequence is shown in SEQ ID NO. 2.

4. An expression vector comprising the extreme thermostable xylanase XYNA coding sequence of claim 1.

5. The expression vector of claim 4, wherein: the expression vector is pPIC 9K-XYNA.

6. A recombinant strain comprising the extremely thermostable xylanase XYNA coding sequence of claim 1.

7. The recombinant strain of claim 6, wherein: the recombinant strain is recombinant escherichia coli or recombinant yeast.

8. Use of the extremely thermostable xylanase XYNA according to claim 1 in feed additives, food, paper, washing, brewing, textile or medicine.

9.A method for preparing extreme heat-resistant xylanase XYNA, which is characterized by comprising the following steps: transforming a host cell by using the expression vector of claim 4 to obtain a recombinant strain, and culturing the recombinant strain to express the extremely heat-resistant xylanase XYNA.