CN117467645A

CN117467645A - Xylanase mutant and preparation method and application thereof

Info

Publication number: CN117467645A
Application number: CN202311295733.8A
Authority: CN
Inventors: 梁伟凡; 李阳源; 邓智远; 陈文广; 唐雪梅; 容晓燕; 张智良
Original assignee: Guangdong Vtr Bio Tech Co ltd
Current assignee: Guangdong Vtr Bio Tech Co ltd
Priority date: 2023-10-08
Filing date: 2023-10-08
Publication date: 2024-01-30

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a xylanase mutant and a preparation method and application thereof. The amino acid sequence of the xylanase mutant is shown as SEQ ID NO. 2. The xylanase mutant provided by the invention has excellent heat resistance, and can keep higher enzyme activity after being treated at a high temperature of 90 ℃, and the retention rate of the enzyme activity is improved by 21.7% compared with that of the parent xylanase. Compared with the parent xylanase, the xylanase mutant has the optimal temperature and the optimal pH kept consistent. Meanwhile, the relative enzyme activities of the xylanase mutant are higher than that of the parent xylanase at 37-90 ℃; the relative enzyme activities under different pH conditions are also higher than those of the parent xylanase. The xylanase mutant provided by the invention has strong heat resistance, and can be widely applied to the fields of food, medicine, feed, papermaking and the like.

Description

Xylanase mutant and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a xylanase mutant and a preparation method and application thereof.

Background

Xylanases (EC 3.2.1.8) are a group of complex enzyme systems that degrade xylan in hemicellulose into xylooligosaccharides and xylose, with the main role of beta-1, 4-endo-xylanases. Xylanase has been widely applied to the fields of feed industry, food industry, paper industry and the like, and plays an important role in various fields. If the xylanase is used as a feed enzyme preparation, the xylanase can effectively degrade xylan in feed, degrade anti-nutritional substances, reduce feed viscosity and improve feed utilization efficiency. In the paper making process, xylanase can be used as a substitute for bleached wood pulp chloride, so that the quality and purity of paper are ensured, and meanwhile, the whiteness and brightness of the paper are improved. In the food industry, xylanases can treat fresh vegetables and fruits, especially vegetables and fruits that require improved quality of baked and steamed products; the flour and the yeast can be used in combination instead of the emulsifier, so that the toughness and the elasticity of the dough are improved, and the fresh-keeping period of the product is prolonged; can also be used together with fungal alpha-amylase, lipase or glucose oxidase to produce higher quality products.

However, the use of xylanases is also limited by conditions, as xylanases are subjected to extreme conditions, such as high temperature conditions during processing, such as in feed, food and paper. This has a serious impact on the action of the xylanase and greatly reduces the effective utilization rate of the xylanase. In particular, during pulp bleaching, the material from alkaline washing has a relatively high temperature (> 80 ℃) and during feed pelletization, a high temperature treatment process of 85-90 ℃ is required. Under high temperature reaction conditions, xylanase enzyme activity decreases significantly, even approaching complete inactivation.

Therefore, there is a need to provide a xylanase with good heat resistance, which is not deactivated by high temperature in industrial processing and can perform good enzymolysis.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a xylanase mutant and a preparation method and application thereof. The xylanase mutant provided by the invention has good heat resistance, can keep higher enzyme activity at high temperature, and is beneficial to industrial application.

The invention provides a xylanase mutant.

Specifically, the amino acid sequence of the xylanase mutant is shown as SEQ ID NO. 2.

The amino acid sequence of the xylanase mutant has the following site mutation relative to the parent xylanase (xynCDBFV) shown in SEQ ID NO. 1: a7N, S49E, T C.

SEQ ID NO.1：QSFCSSASHSGQSVKVTGNKVGTIGGVGYELWADSGNN SATFYSDGSFSCTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIVDNWLSPFPPGDWVGNKKHGSFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGTIDISAHFDQWEKLGMTMGKLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD。

SEQ ID NO.2：QSFCSSNSHSGQSVKVTGNKVGTIGGVGYELWADSGNN SATFYSDGSFECTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIVDNWLSPFPPGDWVGNKKHGSFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGCIDISAHFDQWEKLGMTMGKLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD。

In some embodiments of the invention, the xylanase mutant is derived from a rumen fungus Neocallimastix patriciarum.

The invention also provides a nucleic acid molecule comprising a nucleotide fragment as shown in (a) and/or (b):

(a) Nucleotide fragments encoding the xylanase mutants described above;

(b) A nucleotide fragment fully complementary to (a).

The invention also provides a recombinant expression vector comprising the nucleic acid molecule.

The invention also provides a recombinant cell comprising the nucleic acid molecule or the recombinant expression vector.

In some embodiments of the invention, the recombinant cell comprises a bacterial or fungal cell.

In some embodiments of the invention, the fungal cell is a yeast cell or a filamentous fungal cell.

In some embodiments of the invention, the yeast cell is a pichia cell.

The invention also provides a preparation method of the xylanase mutant.

Specifically, the preparation method of the xylanase mutant comprises the following steps:

(1) Culturing the recombinant cells;

(2) Inducing the recombinant cells to express the xylanase mutant.

The invention also provides application of the xylanase mutant.

In particular to application of the xylanase mutant in preparing a xylan degrading agent.

In particular to application of the xylanase mutant in preparing foods, medicines, feeds, textiles, detergents or paper products.

For example, the hydrolysate obtained by hydrolyzing xylose or xylooligosaccharide by using the xylanase mutant can be used as a thickener, a fat substitute or an antifreeze food additive in the food industry; in the pharmaceutical industry, the xylanase mutants are used in combination with other substances, which are capable of delaying the release of pharmaceutical ingredients. In addition, the hydrolysate obtained by hydrolyzing xylose or xylooligosaccharide by the xylanase mutant can be further converted into liquid fuel, single cell protein, solvent or low-calorie sweetener and the like.

Compared with the prior art, the invention has the beneficial effects that:

(1) The xylanase mutant provided by the invention has more excellent heat resistance, can keep higher enzyme activity after being treated at a high temperature of 90 ℃, and has an enzyme activity retention rate improved by 21.7% compared with that of a parent xylanase. Compared with the parent xylanase, the xylanase mutant has the optimal temperature and the optimal pH kept consistent. Meanwhile, the relative enzyme activities of the xylanase mutant are higher than that of the parent xylanase at 37-90 ℃; the relative enzyme activities under different pH conditions are also higher than those of the parent xylanase.

(2) The xylanase mutant provided by the invention has strong heat resistance, and can be widely applied to the fields of food, medicine, feed, papermaking and the like.

Drawings

FIG. 1 is a graph of the relative enzyme activities of xylanase mutants and parent xylanases at different temperatures;

FIG. 2 is a graph of the relative enzyme activity results of xylanase mutants and parent xylanases at different pH values.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.

The biological materials, reagents or devices used in the examples below are commercially available as they are or may be obtained by methods known in the art unless otherwise specified. The molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.

One embodiment of the invention provides a xylanase mutant, which is obtained by carrying out multiple mutation and high-throughput screening on the basis of a parent xylanase (xynCDBFV) gene (Genebank: KP 691331.1) of parent rumen fungi Neoc allimastix patriciarum, and then obtaining the xylanase mutant with improved heat-resistant stability through expression, wherein the amino acid sequence of the xylanase mutant is shown as SEQ ID No. 2. The xylanase mutants have xylanase activity, including but not limited to having hydrolase activity, e.g., capable of hydrolyzing glycosidic linkages present in xylan, such as catalytic hydrolysis of internal beta-1, 4-xylosidic linkages; and has higher enzyme activity than the parent xylanase shown in SEQ ID NO.1 after high temperature treatment (90 ℃).

The vector containing the polynucleotide sequence for encoding the xylanase mutant is introduced into recombinant cells, and the recombinant cells are induced to express the xylanase mutant, so that the vector can be used for processing food, medicines, feeds, nutritional additives, textiles, detergents, paper products and other fields.

The embodiment of the invention also provides a preparation method of the xylanase mutant, which comprises the following steps:

(a) Constructing a recombinant expression vector comprising a gene encoding the xylanase mutant of the invention;

(b) Introducing a recombinant expression vector into a recombinant cell;

(c) Inducing recombinant cells comprising the recombinant expression vector to express the xylanase.

The embodiment of the invention also provides a production method of xylanase with improved heat resistance stability, which comprises the following steps:

culturing said recombinant strain under suitable conditions to produce a xylanase mutant;

purifying to obtain the generated xylanase mutant;

and optionally processing the resulting xylanase mutant.

The xylanase mutants are secreted into the nutrient medium and can be recovered directly from the medium. If the expressed xylanase mutant is not secreted into the nutrient medium, it can be recovered from the cell lysate.

Xylanase mutants can be expressed in a variety of expression systems and appropriate downstream processing and purification steps must be selected accordingly. In some embodiments of the invention, the xylanase mutants may be expressed in a bacterial host and the protein secreted into the periplasm or extracellular space. Culture of the expression organisms was prepared in appropriate volumes according to standard fermentation methods. In preferred embodiments, the cells are grown in a fermenter, and optionally growth conditions such as pH, temperature, oxygen, and/or nutrient supply are controlled. The first step of purification involves separating cells from the supernatant using one or more of several techniques such as sedimentation, microfiltration, centrifugation or flocculation. In a preferred embodiment, a suitable method is microfiltration. If expressed in cells, the cells are treated to release the protein from the intracellular space. These treatments may include pressurization, enzymatic, osmotic shock, freezing, sonication, or other treatments to produce a cell extract, which may or may not be subjected to further purification.

In some embodiments of the invention, the xylanase mutants are secreted into the supernatant after induction culture, and further protein purification from the supernatant or concentrated supernatant may be performed using one or more of several methods including: extraction or fractionation methods such as ammonium sulfate or ethanol or acid precipitation, or chromatography methods including, but not limited to, ion exchange, hydrophobic interactions, hydroxyapatite, particle size fractionation by gel filtration, phosphocellulose or lectin chromatography, and affinity chromatography, or any combination thereof. In some preferred methods, the affinity tag protein is purified by metal chelator affinity chromatography to obtain the target protein in high purity. In other preferred embodiments, the target protein is obtained in high purity by HPLC purification.

In a further embodiment of the invention, the fermentation cell suspension comprising the expressed xylanase mutant is dried as a whole using methods including, but not limited to, fluid bed drying, conveyor drying, spray drying or drum drying or any combination thereof.

The xylanase mutant provided by the embodiment of the invention can be used for various industrial purposes. In one aspect, the xylanase mutants provided herein are used to degrade cellulosic or xylan-containing materials. The invention also relates to a process for producing a fermentation product comprising: (a) Saccharifying cellulosic or xylan-containing material in the presence of a xylanase mutant of the invention; (b) Fermenting the saccharified cellulosic or xylan-containing material with one or more fermenting microorganisms to produce a fermentation product. In another aspect, the water-soluble arabinoxylans form highly viscous liquids, thereby impeding contact of digestive enzymes with nutrient substrates, resulting in hindered nutrient absorption. Insoluble non-starch polysaccharides physically hinder the binding of enzymes to nutrient substrates. The xylanase mutant provided by the invention can obviously reduce the size of xylan molecules, thereby improving the feed performance and eliminating or reducing the anti-nutritional effect caused by the increase of viscosity.

The following describes more specific examples.

The experimental materials, reagents, and xylanase assay used in the examples below were as follows:

1. strains and vectors: strains containing xylanase xynCDBFV gene and expression plasmid, E.coli strain Top10, pichia X33, vector pPICZαA, vector pGAPzαA, antibiotic Zeocin were purchased from Invitrogen company.

2. Enzyme and kit:ultra-fidelity 2 XMaster Mix PCR polymerase, restriction endonuclease and the like are purchased from NEB company, and the plasmid extraction kit and the purification kit are purchased from Shanghai engineeringCompanies.

3. Culture medium

The E.coli medium was LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). Ampicillin with a final concentration of 100 mug/mL is added to LB+Amp culture medium, and Zeocin with a final concentration of 25 mug/mL is added to LB+Zeo culture medium;

the yeast medium was YPD medium (1% yeast extract, 2% peptone, 2% glucose). Yeast screening medium was YPD+Zeo medium (YPD+Zeo medium was YPD medium supplemented with Zeocin at a final concentration of 100. Mu.g/mL);

yeast induction medium BMGY (1% yeast extract, 2% peptone, 1.34% ynb,0.00004% biotin,1% glycerol (v/v)) and BMMY (divided by 0.5% methanol instead of glycerol, the rest of the ingredients are identical to BMGY);

recombinant yeast fermentation basal salt culture medium: 5% of diammonium phosphate, 0.5% of monopotassium phosphate, 1.5% of magnesium sulfate heptahydrate, 1.95% of potassium sulfate, 0.1% of calcium sulfate and 0.03% of defoamer. 4.35mL of PTM1 per liter after high pressure, wherein PTM1 (trace salt solution): copper sulfate 0.6% and potassium iodide 0.018%. Manganese sulfate monohydrate 0.3%, sodium molybdate dihydrate 0.02%, boric acid 0.002%, cobalt chloride hexahydrate 0.05%, zinc chloride 2%, ferric sulfate heptahydrate 6.5%, concentrated sulfuric acid 0.5% and biotin 0.02%.

4. Chemical reagent

Xylanase standards were purchased from Sigma and xylan was prepared by extraction from this company, and other reagents were purchased from guangzhou chemical reagent plants.

5. Xylanase assay

The xylanase activity is determined by a national standard' determination of xylanase activity of feed additives-spectrophotometry (GB/T23874-2009). Xylanases degrade xylan into oligosaccharides and monosaccharides, which can undergo chromogenic reactions with 3, 5-dinitrosalicylic acid (DNS) reagents in boiling water bath conditions. The color depth of the reaction solution is in direct proportion to the reducing sugar amount generated by enzymolysis, and the generating amount of the reducing sugar is in direct proportion to the activity of xylanase in the reaction solution. Therefore, the activity of xylanase in the reaction solution can be calculated by measuring the intensity of the color of the reaction solution by spectrophotometry. According to the experimental requirement, different pH values can be adjusted to detect the enzyme activity.

The amount of enzyme required to degrade and release 1. Mu. Mol of reducing sugar per minute from a xylan solution at a concentration of 5mg/mL at 37℃and pH5.5 was one enzyme activity unit U.

Example 1

In this example, xylanase xynCDBFV gene synthesis and vector construction were performed.

The xylanase (xynCDBFV) gene (Genebank: KP 691331.1) of rumen fungus Neocallimastix patriciarum has an amino acid sequence shown in SEQ ID NO. 1.

EcoRI and XbaI cleavage sites are introduced into the 5 'end and the 3' end of xylanase xynCDBFV gene and are connected to a pUC57-amp vector, pUC57-xynCDBFV is inoculated to LBA culture medium for culturing for 24 hours, plasmids are extracted, ecoRI and XbaI are used for cleavage, target gene fragments are recovered by cutting gel, products are purified and recovered, and are connected to an expression vector pPICzαA, so that the expression vector pPICzαA-xynCDBFV is obtained.

Example 2

Error-prone PCR random mutagenesis was performed in this example.

Using the expression vector pPICzαA-xynCDBFV obtained in example 1 as a template, mutations were randomly introduced into xylanase xynCDBFV gene by error-prone PCR method.

PCR amplification was performed using the following primers:

Xyn-F：5’-gaaaagagaggctgaagctgaattc-3’(SEQ ID NO.3)，

Xyn-R：5’-tgagatgagtttttgttctagacta-3’(SEQ ID NO.4)。

the amplified product is digested by EcoRI and XbaI, the PCR amplification result is detected by agarose electrophoresis, and the target product of PCR amplification is purified and recovered. The template was digested with restriction enzyme DpnI, ligated to expression vector pPIcz. Alpha.A digested with EcoRI and XbaI, and the digested product was transferred into E.coli Top10 competent cells by chemical conversion heat shock, and the recombinant transformant was verified by bacterial liquid PCR, and the plasmid of the transformant was extracted. Linearizing the mutant plasmid with PmeI endonuclease, purifying the linearized plasmid fragment, transferring into pichia X33 competent cells by an electrotransformation method, and screening by YPD+Zeo culture medium to obtain the yeast recombinant transformant.

Example 3

The high-throughput screening of mutant strains with high enzymatic activity was performed in this example.

The yeast recombinant transformants obtained in example 2 were picked up one by one into 24-well plates with toothpicks, 1mL of a culture medium containing BMGY was added to each well, and cultured at 30℃for about 24 hours at 220rpm, and the supernatant was removed by centrifugation. 1.6mL BMMY medium was added to each of the cells for induction culture. After culturing for 24 hours, the supernatant was centrifuged, and 200. Mu.L to 96-well plates were taken out of the supernatant, respectively, for xylanase enzyme activity measurement. And obtaining the yeast recombinant transformant with high enzyme activity through high-flux screening.

Example 4

The present example screens heat-resistant raised xylanase mutants at high throughput.

Taking 100 mu L of supernatant to a 96-hole PCR plate from the mutant which is not reduced compared with the parent xylanase enzyme activity obtained in the example 3, carrying out treatment at 90 ℃ for 5 minutes on a PCR instrument, then carrying out xylanase enzyme activity measurement, and calculating the ratio of the enzyme activity after heat treatment to the enzyme activity of the national standard method, namely the heat-resistant retention rate. Through multiple rounds of screening comparison, xylanase mutants with significantly improved heat-resistant retention relative to the parent are screened.

Example 5

This example performs a combination mutation and thermal stability analysis.

In examples 3 and 4, double-site or multiple-site combination mutation was performed on the basis of the heat-resistant forward mutation site, and screening was continued using the high throughput method in examples 3 and 4. Through multiple rounds of combination mutation and screening, xylanase mutants with improved thermostability (the sequences are shown as SEQ ID NO. 2) are finally screened, and the relative thermostability retention rate is shown as table 1.

TABLE 1 relative Heat-resistant Retention of xylanase mutants with parent xylanases

Numbering device	Relative heat retention (%)
		Parent xylanase (xynCDBFV)	100.0％
Xylanase mutant (xyn-TH, A7N, S49E, T174C)	121.7％

As can be seen from Table 1, the xylanase mutants (xyn-TH) screened in the examples of the present invention have a relative heat-resistant retention of 21.7% higher than that of the parent xylanase (xynCDBFV).

Example 6

This example investigated the optimal temperature and optimal reaction pH of the parent xylanase (xynCDBFV) and xylanase mutant (xyn-TH).

The enzyme activities of the xylanases were measured at pH5.5 at 40℃at 50℃at 60℃at 70℃at 80℃at 90℃respectively, wherein the relative enzyme activities of the xylanase mutants (xyn-TH) at different temperatures were calculated using the enzyme activities measured at the optimum temperature of the parent xylanase (xynCDBFV) at 60℃as a control.

TABLE 2 relative enzyme Activity of xylanase mutants and parent xylanases at various temperatures

The results are shown in Table 2 and FIG. 1, and the optimum reaction temperature range of the xylanase mutant (xyn-TH) provided by the invention also falls at 60 ℃. As can be seen from Table 2 and FIG. 1, the xylanase mutants (xyn-TH) provided in the examples of the present invention have higher relative enzyme activities than the parent xylanase (xynCDBFV) at each temperature range, especially at high temperatures (70-90 ℃).

Further, the enzyme activities of xylanases were determined at 37℃at pH3.0, pH4.0, pH5.0 and pH5.5, respectively. Wherein, the enzyme activity of xylanase is measured under the condition of pH5.5 as a control, and the relative enzyme activity of xylanase mutant (xyn-TH) under different pH conditions is calculated.

TABLE 3 relative enzyme Activity of xylanase mutants and parent xylanases at various pH' s

The results of the test are shown in Table 3 and FIG. 2, and the optimal reaction pH for the parent xylanase (xynCDBFV) and the xylanase mutant (xyn-TH) is pH5.5. The relative enzyme activities of xylanase mutants (xyn-TH) are higher than that of the parent xylanase (xynCDBFV) under different pH conditions.

In conclusion, the xylanase mutant (Xyn-TH) provided by the invention has obviously improved heat resistance stability relative to the parent xylanase (xynCDBFV). Meanwhile, the optimal reaction temperature and the optimal reaction pH of the xylanase mutant (Xyn-TH) are not changed obviously compared with the parent xylanase (xynCDBFV).

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. The xylanase mutant is characterized in that the amino acid sequence of the xylanase mutant is shown as SEQ ID NO. 2.

2. The xylanase mutant according to claim 1, wherein the xylanase mutant is derived from rumen fungus Neocallimastix patriciarum.

3. A nucleic acid molecule comprising a nucleotide fragment of (a) and/or (b):

(a) A nucleotide fragment encoding the xylanase mutant of any one of claims 1-2;

(b) A nucleotide fragment fully complementary to (a).

4. A recombinant expression vector comprising the nucleic acid molecule of claim 3.

5. A recombinant cell comprising the nucleic acid molecule of claim 3 or the recombinant expression vector of claim 4.

6. The recombinant cell of claim 5, wherein the recombinant cell comprises a bacterium or a fungus.

7. The recombinant cell of claim 6, wherein the fungus is a yeast cell or a filamentous fungal cell.

8. The method for producing a xylanase mutant according to claim 1 or 2, comprising the steps of:

(1) Culturing the recombinant cell of any one of claims 5-7;

(2) Inducing the recombinant cells to express xylanase mutants.

9. Use of a xylanase mutant according to claim 1 or 2 for the preparation of a xylanolytic agent.

10. Use of a xylanase mutant according to claim 1 or 2 for the preparation of a food, pharmaceutical, feed, textile, detergent or paper product.