CN117925577A

CN117925577A - Method for improving xylanase activity, xylanase and application thereof

Info

Publication number: CN117925577A
Application number: CN202410309935.1A
Authority: CN
Inventors: 徐柯; 杨洵; 曾慧玲; 郑星; 林敏�; 王劲; 周正富
Original assignee: Jintang Baixinwei Technology Co ltd
Current assignee: Jintang Baixinwei Technology Co ltd
Priority date: 2024-03-19
Filing date: 2024-03-19
Publication date: 2024-04-26
Anticipated expiration: 2044-03-19
Also published as: CN117925577B

Abstract

The present disclosure relates to the field of biotechnology, and in particular, to a method of increasing xylanase activity and xylanase and application thereof, and more particularly, to a method of increasing xylanase activity, a protein having xylanase activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant into which the gene is transformed, a method of preparing xylanase, and application of xylanase in catalyzing degradation of xylan compounds at low temperature. The method realizes the amino acid optimization of key sites of xylanase, and lays a foundation for improving the application effect of xylanase in the fields of industrial production of foods, feeds, papermaking, detergents, medicines and the like and aquaculture.

Description

Method for improving xylanase activity, xylanase and application thereof

Technical Field

The present disclosure relates to the field of biotechnology, and in particular, to a method for improving xylanase activity, a protein having xylanase activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant transformed with the gene, a method for preparing xylanase, and an application of xylanase in catalyzing degradation of a xylan compound at a low temperature.

Background

Xylan is a heterogeneous polysaccharide widely present in plant cell walls, and is one of the main components constituting hemicellulose in plant cell walls. A class of hydrolytic enzymes capable of degrading xylan into xylo-oligosaccharides or xylose is broadly referred to as xylanases and mainly comprises beta-1, 4-endo-xylanases, beta-xylosidases and the like. Xylanases find wide application in a variety of industries, including dough making, pulp bleaching, animal feed digestion, and also in pharmaceutical carriers, as well as in cosmetic additives, and the like.

Most of the xylanases reported so far are from fungi or bacteria, which have the highest activity at 40-60 ℃ under neutral or slightly acidic conditions, and therefore these xylanases are popular in industry.

However, in the production of low-temperature dough fermentation, aquatic feeds and the like, the xylanase is difficult to have stable enzyme activity in a medium-low temperature scene of 15-30 ℃, and some xylanases even lose activity, so that the production application requirements are difficult to meet.

Disclosure of Invention

In order to further meet the demands of practical applications, the present disclosure provides a method for improving xylanase activity, a protein having xylanase activity, a gene encoding the protein, a recombinant vector into which the gene is inserted, a transformant transformed with the gene, a method for preparing xylanase, and application of xylanase in catalyzing degradation of xylan compounds at low temperature.

In order to achieve the above object, a first aspect of the present disclosure provides a method for improving xylanase activity, the method comprising mutating one of amino acid residues to be mutated of a wild-type xylanase, wherein the amino acid sequence of the wild-type xylanase is shown as SEQ ID NO.2, and the amino acid residue to be mutated is a lysine residue at position 106; the mutation is modified into a substitution of an amino acid residue; the mutant engineering includes: the lysine residue at position 106 was mutated to valine residue.

A second aspect of the present disclosure provides a protein having xylanase activity, which is derived by substituting 1 amino acid for the amino acid sequence of wild-type xylanase shown as SEQ ID No. 2; and the amino acid sequence of the protein is shown as SEQ ID NO. 1.

In a third aspect the present disclosure provides a gene encoding the protein according to the second aspect, said gene being a DNA molecule having the nucleotide sequence shown in SEQ ID NO. 3.

A fourth aspect of the present disclosure provides a recombinant vector which is a recombinant expression vector into which the gene according to the third aspect is inserted.

Alternatively, the nucleotide sequence of the recombinant vector is shown as SEQ ID NO. 4.

A fifth aspect of the present disclosure provides a transformant whose host is a genetically engineered bacterium; the gene introduced into the transformant includes the gene described in the third aspect, or the recombinant vector introduced into the transformant includes the recombinant vector described in the fourth aspect.

A sixth aspect of the present disclosure provides a method of preparing a xylanase, the method comprising: inoculating the transformant of the fifth aspect into a culture medium for culturing, and obtaining a cultured material.

The seventh aspect of the present disclosure is a xylanase comprising a protein of the second aspect for low temperature catalysis of degradation of a xylan compound.

Optionally, the method comprises the following steps: contacting a xylan compound with the xylanase under enzymatic reaction conditions; the conditions of the enzymatic reaction include: the temperature is 10-30deg.C, pH is 5-8, and the time is 5-60min; the xylan compound is arabinoxylan and/or beech xylan.

Through the technical scheme, the disclosure provides a method for improving xylanase activity, a protein with xylanase activity, a gene for encoding the protein, a recombinant vector inserted with the gene, a transformant transformed with the gene, a method for preparing xylanase and application of xylanase in low-temperature catalysis of degradation of xylan compounds. The method realizes the amino acid optimization of key sites of xylanase, so that the protein with xylanase activity has good low-temperature adaptability, can catalyze the degradation of xylan at the temperature of 10-30 ℃, and lays a foundation for improving the application effect of xylanase in the fields of industrial production of foods, feeds, papermaking, detergents, medicines and the like and aquaculture.

Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.

Drawings

The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:

FIG. 1 is a diagram of a purification gel of wild-type xylanase DmgXylA and mutant K106V. Wherein M is marker;1 is xylanase DmgXylA;2 is mutant K106V.

FIG. 2 is a graph of analysis of degradation products of wild-type xylanase DmgXylA. Wherein M is a mixed sugar Marker; x1 is a xylo-oligosaccharide; x2 is xylobiose; x3 is xylotriose; xylotetraose at position X4; x5 is wood five ponds; dmgXylA is the product of hydrolysis of xylan.

FIG. 3 is a graph showing an optimal temperature analysis of the wild-type xylanase DmgXylA.

FIG. 4 is a graph showing the analysis of the temperature stability of wild-type xylanase DmgXylA.

FIG. 5 is a graph of the optimum pH analysis for wild-type xylanase DmgXylA.

FIG. 6 is a graph of pH stability analysis of wild-type xylanase DmgXylA.

FIG. 7 is the enzymatic activity of wild-type xylanase DmgXylA and mutant K106V.

FIG. 8 is a phylogenetic tree analysis of wild-type xylanase DmgXylA.

Detailed Description

Specific embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.

The first aspect of the present disclosure provides a method for improving xylanase activity, the method comprising mutating one of amino acid residues to be mutated of a wild-type xylanase, wherein the amino acid sequence of the wild-type xylanase is shown as SEQ ID NO.2, and the amino acid residue to be mutated is lysine residue at position 106; the mutation is modified into a substitution of an amino acid residue; the mutant engineering includes: the lysine residue at position 106 was mutated to valine residue.

In the present disclosure, the inventors of the present disclosure found that the enzyme activity of xylanase of mutant strain K106V was increased by 2.67-fold compared to wild-type xylanase by mutating lysine residue at position 106 of wild-type xylanase to valine residue at 30 ℃ at ph=7. Furthermore, the xylanase of the mutant strain K106V still has better enzyme activity at the temperature of 10-30 ℃.

In the present disclosure, the genetically engineered bacterium may be a wild-type genetically engineered bacterium or an artificially modified genetically engineered bacterium, for example, at least one of competent cells of escherichia coli BL21 (DE 3), bacillus subtilis, pichia pastoris, saccharomyces cerevisiae, and filamentous fungi.

Among them, the medium and the culture conditions may be any of known various suitable choices. The cultured material contains the protein with xylanase activity provided in the second aspect of the disclosure, so that the protein has xylanase enzyme activity, can be directly used as a xylanase composition according to the requirement, and can be purified according to the requirement for reuse.

The present disclosure is further illustrated by the following examples, but the present disclosure is not limited thereby.

The examples of the present disclosure are not to be construed as specific experimental conditions, either as conventional conditions well known to those skilled in the art or as suggested by the manufacturer.

Example 1

The embodiment is to construct recombinant escherichia coli engineering strain for expressing dmgXylA genes and mutants.

The expression plasmid pET28a is a commercial product of German merck company in the present disclosure; coli BL21 (DE 3): is a commercial product of Beijing nuozan company; site-directed mutagenesis kits were purchased from Biyun Tian Biotechnology Co.

1. Designing a1 pair PCR specific primer according to dmgXylA gene sequences of a Xinjiang desert environment metagenome:

DX-F：5´-CCCCATGGCATGCAAAGTGTGCCCGGCCCAGA-3´；

DX-R：5´-CCCTCGAGGTAAATCCTGAAATGTCCACTCGCATGGAG-3´。

Site-directed mutagenesis primer:

106F：5'-CTACGCCGTAAGCAAGATGCAGCACCAGTCG-3'；

106R：5'-TCTTGCTTACGGCGTAGTTCCACAGGGCA-3'。

2. The target dmgXylA gene sequence was amplified by PCR.

3. After the PCR product is recovered by glue, the PCR product is connected to a pET-28a vector containing a sticky end obtained by NcoI/XhoI double enzyme digestion through recombinase, and an escherichia coli expression vector pET28a-dmgXylA is constructed.

4. The expression vector is transformed into escherichia coli BL21 (DE 3), and the insertion sequence is verified to be correct through PCR and enzyme digestion and sequencing, and the strain is named BL21-dmgXylA.

5. Site-directed mutagenesis is performed according to the selected mutation site Lys106, primers are designed, the primers contain mutation sites, and site-directed mutagenesis kit is used for constructing mutant expression vectors. Inverse PCR was performed using the wild type plasmid (pET 28 a-dmgXylA) as template, and the whole plasmid was amplified.

6. 10 Μl of the mutant product is directly transformed into BL21 (DE 3), then the mutant product is coated into LB solid medium containing Kan, colony PCR verification is carried out on the mutant strain, and the mutant strain with correct sequencing is used for subsequent experiments by bacterial retention and streaking.

Experimental results of this example: as shown in FIG. 8, phylogenetic tree results show that DmgXylA protein has 49.76% similarity to the reported xylanase Xyn8 cloned from the environment, 45.61% similarity to XynSc, 29.04% similarity to the enzyme from Antarctic bacteria PhXylA, and only 7.94% similarity to both XynB and XynA xylanases, indicating that DmgXylA is a newly discovered xylanase. The present disclosure successfully clones dmgXylA bp gene with the size of 1215bp (SEQ ID NO. 5), codes 404 amino acids (SEQ ID NO. 2), successfully constructs a recombinant escherichia coli engineering strain expressing dmgXylA, and simultaneously screens the recombinant escherichia coli engineering strain of dmgXylA mutant with the Lys106 site-directed mutation. By genetic sequencing, the mutant at the Lys106 site was K106V.

Example 2

This example shows the expression and purification of xylanase DmgXylA and mutants.

The experimental materials of this example include:

Recombinant engineering strain: recombinant expression strains of xylanase DmgXylA and xylanase DmgXylA mutants obtained in example 1;

the experimental method of this embodiment includes:

1. inducible expression and purification of recombinant proteins:

(1) Inoculating the strain into 20mL LB liquid medium with antibiotics at an inoculum size of 1%, and culturing overnight at 37 ℃;

(2) The next day, inoculating the bacterial liquid into 500mL LB liquid culture medium added with kanamycin according to the inoculation amount with the initial concentration of OD ₆₀₀ of 0.1, and culturing at 37 ℃ until the bacterial liquid concentration is 0.6-0.8;

(3) IPTG (final concentration 0.3 mmol/L) was added for protein induction expression at 16℃for 20h. IPTG is an inducer that induces recombinant strain expression DmgXylA and mutant proteins and is not a substrate for the catalytic reaction.

2. Purifying recombinant protein by affinity chromatography:

(1) Centrifuging the induced bacterial liquid, collecting bacterial cells at 5000rpm for 10min, and re-suspending the bacterial cells with NTA-0;

(2) Ultrasonic crushing of bacterial liquid: placing the centrifuge tube containing suspended thalli in a beaker of an ice-water mixture, and placing the centrifuge tube in an ultrasonic breaker for ultrasonic treatment for 5-10min, wherein the ultrasonic breaker is set with the following procedures: ultrasonic treatment is carried out for 3s and 5s, and the power is less than 400W;

(3) Centrifuging the sample 12000 xg after ultrasonic crushing for 30 min, respectively collecting supernatant and precipitate of the crushed liquid by using a centrifuge tube, and obtaining the crushed supernatant which is crude enzyme liquid after centrifugation;

(4) Taking out the nickel column, washing the nickel column twice with deionized water after the ethanol flows out, and balancing the column with NTA-0, wherein the flow rate is kept at 1mL/min; the crude enzyme solution hangs the column, penetrates twice, and has the same flow rate;

(5) Gradient elution is carried out by using prepared NTA-10, NTA-20, NTA-50, NTA-100, NTA-150, NTA-200 and NTA-300, protein is detected by using protein detection liquid, and elution peaks are collected; washing the nickel column with 20% ethanol solution, and storing the nickel column in a refrigerator at 4deg.C; and (3) using ultrafiltration centrifugation to replace the buffer solution of the protein solution, so as to remove imidazole in the protein solution, wherein the obtained protein solution is enzyme solution.

Experimental results show that dmgXylA gene and mutant K106V gene can be expressed in a large amount in escherichia coli. When eluted with 50mM,100mM and 150mM imidazole eluates, a single protein band appeared at about 45kDa, which was closer to the predicted protein size for fusion of His-Tag, indicating that DmgXylA and mutant K106V eluted in a buffer containing 50-150mM imidazole. As shown in FIG. 1, the SDS-PAGE electrophoresis of the purified DmgXylA and mutant K106V proteins has clear background, high protein purity and large expression level. The purified protein was subjected to further investigation of enzymatic properties.

Example 3

This example is an enzymatic property analysis of xylanase DmgXylA.

The experimental materials of this example include: the recombinant protein obtained in example 2.

The experimental method of this embodiment includes:

1. Enzyme activity assay:

(1) The hydrolase activity was determined by the dinitrosalicylic acid (DNS) method. The reaction system consisted of 20. Mu.L of a reasonably diluted enzyme solution (diluted 1:200) and 360. Mu.L of beech xylan solution (10 mg/ml), buffer 0.1M Tris-HCl, pH 7.

(2) After incubation in a hot water bath for 10min, the reaction was terminated by adding 600. Mu.L of DNS, and the reaction was allowed to develop in a metal bath at 100℃and after cooling to room temperature, the absorbance was measured at 540 nm. The enzyme activity unit (U) is defined as the amount of enzyme required to break down xylan to 1. Mu. Mol of xylan per minute under certain conditions.

2. Substrate specificity:

(1) Recombinant protein substrate preference was determined using 10mg/ml beech xylan, wheat arabinoxylan, corncob xylan, pullulan, arabinogalactan (larch), D-cellobiose, microcrystalline cellulose, sodium carboxymethyl cellulose.

(2) The hydrolysate sample was prepared by reacting the pure enzyme solution in three substrates of beech xylan, wheat arabinoxylan and corncob xylan at a final concentration of 1% (w/v) for 10min at a pH of 7.0 and a temperature of 70 ℃. The hydrolysates were analysed by Thin Layer Chromatography (TLC).

3. Enzymatic characterization:

(1) The reaction system containing the appropriate amount of diluted recombinant protein (i.e., xylanase DmgXylA) and beech xylan was placed in 0.1M Tris-HCl buffer (pH 6) and reacted at a temperature of 10℃to 90℃for 10min. The recombinant protein was incubated in 0.1M Tris-HCl buffer (pH 7) at 30℃and 50℃and 70℃for 160min, and samples were taken every 20min for temperature stability experiments.

(2) The recombinant protein was assayed for enzyme activity at 70℃in a citric acid-disodium hydrogen phosphate buffer at pH 3.0-8.0 and glycine-sodium hydroxide buffer at pH 9.0-10.0. The pH stability was determined by treating the recombinant protein in various buffers for 1h and calculating the residual enzyme activity.

Experimental results of this example:

The His tag of the recombinant protein was used to purify the protein, as shown in FIG. 1, which was approximately 45kDa in size, consistent with the predicted molecular weight, and single in band. The recombinant protein has strict substrate specificity, and as shown in table 1, the recombinant protein is active only on wheat arabinoxylan and beech xylan, and is inactive on pullulan, amylose, amylopectin, arabinogalactan (larch), D-cellobiose, microcrystalline cellulose and sodium carboxymethyl cellulose. The recombinant protein of the present disclosure is shown to be a xylanase. Analysis of the hydrolysate as shown in figure 2, the recombinant proteolytic xylan products were xylobiose, xylotriose, xylotetraose, xylopentaose.

TABLE 1 substrate specificity analysis of xylanase DmgXylA

As shown in FIGS. 3-6, it can be seen from FIG. 3 that the protein of the present disclosure having xylanase DmgXylA activity has the highest enzymatic activity at 70℃and about 18100U/mg. As can be seen from fig. 5 and 6, the protein of the present disclosure having xylanase activity has an optimum pH of 6, and retains more than 75% of the enzyme activity at a pH in the range of 5-8. From fig. 4, it can be seen that the protein having xylanase activity of the present disclosure has excellent low temperature enzyme activity DmgXylA, retains more than 80% of the enzyme activity after 160min of treatment at 30 ℃ and retains more than 90% of the enzyme activity after treatment at 10, 20 ℃.

Example 4

This example is a comparison of the enzymatic activities of mutant K106V and xylanase DmgXylA.

The experimental materials of this example include: xylanase DmgXylA and mutant K106V obtained in example 2.

The experimental method of this embodiment includes:

DmgXylA and low temperature enzyme activity assay of mutants:

the reaction system containing a suitable amount of diluted DmgXylA and mutant K106V and beech xylan was placed in 0.1M Tris-HCl buffer (pH 6) and reacted at 30℃for 10min.

Experimental results of this example:

as a result, as shown in fig. 7, it can be seen from fig. 7 that xylanase activity of mutant K106V was increased 2.67-fold when measured at a temperature of 30 ℃ and ph=7. The result shows that the mutation of lysine 106 to valine significantly improves the enzyme activity of xylanase at low temperature.

The method for improving xylanase activity, the protein with xylanase activity, the gene for encoding the protein, the recombinant vector inserted with the gene, the transformant transformed with the gene, the method for preparing xylanase and the application of xylanase in low-temperature catalytic degradation of xylanase compounds lay a foundation for the application of xylanase in the fields of industrial production of foods, feeds, papermaking, detergents, medicines and the like, aquaculture and the like, and meet the requirements of xylanase in industry.

The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the present disclosure is not limited to the specific details of the embodiments described above, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.

In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.

Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.

Claims

1. A method for improving xylanase activity, which comprises mutating one of amino acid residues to be mutated of wild-type xylanase, and is characterized in that the amino acid sequence of the wild-type xylanase is shown as SEQ ID NO.2, and the amino acid residue to be mutated is lysine residue at position 106; the mutation is modified into a substitution of an amino acid residue;

the mutant engineering includes: the lysine residue at position 106 was mutated to valine residue.

2. A protein with xylanase activity, which is characterized in that the protein is derived by substituting 1 amino acid in the amino acid sequence of wild xylanase shown as SEQ ID NO. 2; and the amino acid sequence of the protein is shown as SEQ ID NO. 1.

3. A gene encoding the protein of claim 2, wherein said gene is a DNA molecule having the nucleotide sequence shown in SEQ ID No. 3.

4. A recombinant vector, characterized in that it is a recombinant expression vector, into which the gene of claim 3 is inserted.

5. The recombinant vector according to claim 4, wherein the nucleotide sequence of the recombinant vector is shown in SEQ ID NO. 4.

6. A transformant, characterized in that the host of the transformant is a genetically engineered bacterium; the gene introduced into the transformant includes the gene of claim 3, or the recombinant vector introduced into the transformant includes the recombinant vector of claim 4 or 5.

7. A method of preparing a xylanase, comprising: the transformant according to claim 6 is inoculated into a medium and cultured to obtain a cultured material.

8. Use of a xylanase comprising the protein of claim 2 for low temperature catalysis of degradation of a xylan compound.

9. The use according to claim 8, characterized by comprising the following method: contacting a xylan compound with the xylanase under enzymatic reaction conditions;

the conditions of the enzymatic reaction include: the temperature is 10-30deg.C, pH is 5-8, and the time is 5-60min;

The xylan compound is arabinoxylan and/or beech xylan.