CN104263711A - Heat-resistant alkali-resistant xylanase as well as encoding gene and recombinant vector thereof - Google Patents

Abstract

The invention discloses a heat-resistant and alkali-resistant xylanase and its coding gene and recombinant vector, the xylanase is selected from the following (1) or (2) or (3): (1) by SEQ ID NO: The full-length xylanase taXynA that the amino acid sequence shown in 2 forms; (2) the recombinant xylanase nsXynAΔSLH that is made up of the amino acid sequence shown in SEQ ID NO:4; (3) SEQ ID NO:2 or SEQ ID NO:2 or The amino acid sequence shown in ID NO: 4 is a protein derived from (1) or (2) that has undergone substitution and/or deletion and/or addition of one or several amino acid residues and has heat-resistant and alkali-resistant xylanase activity . The xylanase described in the present invention has good xylan degrading activity, 90% of the enzyme activity is retained after being treated at 75°C for 120 minutes, and has strong tolerance in slightly acidic, neutral or alkaline environments .

Description

A heat-resistant and alkali-resistant xylanase, its coding gene and recombinant vector

technical field

The invention relates to the field of genetic engineering, in particular to a xylanase XynAΔSLH with higher optimum reaction temperature, temperature stability and alkaline tolerance, and the coding gene of the xylanase and its expression in Bacillus subtilis high-yield expression strategy.

Background technique

Lignocellulosic biomass is a major representative of biomass energy, which is abundant in the earth. Two types of polysaccharides, cellulose and hemicellulose, are the main components of lignocellulosic biomass, the product of plant photosynthesis, and one of the most abundant renewable resources in nature. Among them, xylan is the main component of hemicellulose complex in plant cells, widely distributed in plant cell walls, and is the most abundant polysaccharide in nature after cellulose and starch, accounting for about 100% of plant carbohydrates. One-third of the total amount of compounds is a huge, renewable biomass energy.

Xylanase is a general term for a class of enzymes that can degrade xylan into xylooligosaccharides or xylomonosaccharides, and xylanase in a narrow sense refers to endoxylanases, which are two main chain One of the hydrolytic enzymes, the most important enzyme in the process of xylan degradation, acts on the glycoside chain of the xylan main chain from the inside of the xylan main chain through endolysis, and randomly breaks the main xylan chain skeleton, thereby generating xylooligosaccharides and very few xylose monosaccharides, reducing the degree of polymerization of xylan (Collins, Gerday et al.2005).

Xylanase can be widely used in food, energy, paper making, feed, medicine, textile and other industries. For example, xylan cannot be digested and absorbed in the digestive tract of animals, and hinders the digestion and utilization of other nutrients. It has strong anti-nutritional properties, which limits the application of many cereal feeds in the feed industry and animal husbandry production. By adding xylanase to the feed, the feed can be digested and absorbed more easily and fully, and the nutritional value of the feed can be effectively improved (Collins, Gerday et al. 2005).

Industrial production often needs to be carried out under high temperature or alkaline conditions, and in general, proteases are often unstable and easily inactivated when encountering high temperature or alkaline environments, which greatly limits the use of many enzyme preparations in industrial production. Applications. Therefore, heat-resistant and alkali-resistant xylanase has great application prospects in industrial production.

Contents of the invention

The object of the present invention is to provide a macromolecule heat-resistant and alkali-resistant xylanase xylanase and its coding gene.

The object of the invention is achieved through the following technical solutions:

A heat-resistant and alkali-resistant xylanase selected from the following (1) or (2) or (3):

(1) full-length xylanase taXynA consisting of the amino acid sequence shown in SEQ ID NO:2;

(2) recombinant xylanase nsXynAΔSLH consisting of the amino acid sequence shown in SEQ ID NO:4;

(3) Substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 and having heat-resistant and alkali-resistant xylanase activity Proteins derived from (1) or (2).

Among them, SEQ ID NO: 2 in the sequence table consists of 1432 amino acids with a molecular weight of about 157.5kD; SEQ ID NO: 4 consists of 1015 amino acids with a molecular weight of about 112.53kD;

In order to facilitate the purification of the xylanase in (2), a 6×His tag is attached to the C-terminus of the protein composed of the amino acid sequence shown in SEQ ID NO:4 in the sequence listing.

The xylanase in (1), (2) or (3) above can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression. The coding gene of xylanase in (2) and (3) can be by the codon that deletes one or several amino acid residues in the DNA sequence in the sequence SEQID NO:1 or NO:3, and/or carry out a or missense mutations of a few base pairs.

The coding gene of the above-mentioned xylanase is selected from the following (1) or (2) or (3) or (4):

(1) its nucleotide sequence encodes the protein molecule described in claim 1;

(2) its nucleotide sequence is the sequence shown in SEQ ID NO:1 or SEQ ID NO:3;

(3) Its nucleotide sequence is a derivative sequence obtained by substituting and/or deleting and/or adding one or several nucleotides to the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3;

(4) A nucleotide sequence having more than 90% homology with the DNA sequence defined in (1) and encoding the xylanase.

Recombinant vector containing the above-mentioned coding gene: the expression vector pHTHis and the gene of claim 2 are subjected to double digestion with restriction endonucleases BamH I and Pst I respectively, and the digested products are purified and ligated, and transformed into E.coli BL21 (DE3) Competent cells. Positive clones were screened by colony PCR and sequencing; positive clones were inoculated into LB medium containing ampicillin resistance and cultured overnight to extract plasmids to obtain recombinant vectors.

The preparation of the expression vector pHTHis: the gene fragment of SEQ ID NO: 9 and the plasmid pHT01 were double digested with BamH I and Aat II, respectively purified and ligated, transformed into E.coli DH5α competent cells, passed through the colony Positive clones were screened out by PCR and sequencing; the positive clones were inoculated into LB medium containing ampicillin resistance and cultured overnight, and then the plasmid was extracted, which was the expression vector pHTHis.

The cloning/expression region of the expression vector pHTHis is shown in FIG. 1 .

Recombinant bacteria containing the above-mentioned coding gene: transfer the recombinant vector to Bacillus subtilis by chemical transformation method, and then screen with chloramphenicol LB plate to obtain the xylanase recombinant bacteria.

The xylanase involved in the invention is derived from Thermoanaerobacterium aotearoense SCUT27, and has better heat resistance.

The coding gene of the above-mentioned xylanase also belongs to the protection scope of the present invention.

Recombinant vectors, recombinant bacteria, transgenic cell lines and expression cassettes containing the above coding genes all belong to the protection scope of the present invention.

Experiments have proved that the xylanase expression vector of the present invention is conducive to the high-efficiency expression and purification of large molecular weight xylanase, the expressed xylanase accounts for about 15% of the total protein of the bacteria, and the purification efficiency is as high as 57%. Live for 248.8U/mg. The expression vector has the characteristics of high expression amount, high recovery rate, high catalytic activity, etc., and is beneficial to the industrial production of xylanase preparations.

The optimum reaction temperature of the xylanase of the present invention is 80°C, and the optimum reaction pH is 6.5; there is almost no loss of enzyme activity when treated at 70°C for 2 hours, and it can still retain about 2 hours at 75°C. 90% of the remaining enzyme activity has strong tolerance in slightly acidic, neutral and alkaline environments. The xylanase of the present invention has heat-resistant and alkali-resistant properties, can be well applied to decompose xylanase in hemicellulose, and produce functional oligosaccharides, and is suitable for many non-strong acid industries with high temperature requirements environment, has great potential for popularization and application in industrial production.

Description of drawings

Figure 1 is the sequence of the cloning/expression region of the pHTHis vector.

Fig. 2 is the agarose gel electrophoresis diagram of the recombinant xylanase gene PCR product of the present invention. M, DNA molecular weight standard; 1, PCR product of recombinant xylanase gene nsXynA.

Fig. 3 is an SDS-PAGE diagram of samples collected during the recombinant xylanase purification process of the present invention. M, protein molecular weight standard; 1, broken supernatant (crude enzyme solution) of resuspended bacteria; 2, broken precipitate of resuspended bacteria; 3, nickel column purification through liquid; 4, the first elution of 68mM imidazole buffer 5, the protein sample contained in the second protein peak eluted with 68mM imidazole buffer; 6, the protein sample eluted with 116mM imidazole buffer; 7, the protein sample eluted with 500mM imidazole buffer .

Fig. 4 is a graph showing the optimal reaction pH of the recombinant xylanase of the present invention.

Fig. 5 is a graph showing the optimal reaction temperature of the recombinant xylanase of the present invention.

Fig. 6 is a graph showing the pH stability of the recombinant xylanase of the present invention.

Fig. 7 is the residual enzyme activity curve of the recombinant xylanase of the present invention after incubation at 70, 75 and 80°C for different time.

Fig. 8 is the remaining enzyme activity curve of the recombinant xylanase of the present invention incubated at 75-80°C for 30 minutes.

Detailed ways

Unless otherwise specified, the experimental methods used in the following examples are conventional methods, such as the methods described in "Molecular Cloning Experiment Guide" (third edition).

Example 1

Construction of Intracellular Expression Vector pHTHis in Bacillus subtilis

Using the Escherichia coli-Bacillus subtilis shuttle plasmid pHT01 (MOBITEC company) as the base carrier, with primer 1 (SEQ ID NO:5), primer 2 (SEQ ID NO:6), without adding template, carry out PCR amplification to obtain a A 58nt fragment (SEQ ID NO: 9) containing a hexahistidine tag coding sequence. The PCR reaction conditions were: denaturation at 98°C for 10 sec, annealing at 53°C for 5 sec, and extension at 72°C for 5 sec. Both the fragment and the plasmid pHT01 were digested with BamH I and Aat II, purified and ligated, transformed into E.coli DH5α competent cells, and positive clones were screened by colony PCR and sequencing. The positive clones were inoculated into LB medium containing ampicillin resistance and cultured overnight, and then the plasmids were extracted as the cloning expression vector pHTHis for subsequent experiments. The sequence of the cloning/expression region is shown in Figure 1.

Primer 1: 5'-TGTACGGATCCGGTGGTTCTCTGCAGCATCA-3' (SEQ ID NO: 5)

Primer 2: 5'-CCGATGACGTCTTAATGATGATGATGATGATGCTGCAGAGAACCAC-3' (SEQ ID NO: 6)

Example 2

Cloning of Xylanase Encoding Gene xynA

The xynA amino acid sequence (SEQ ID NO: 2) of the xylanase gene was compared by Blast. It can be seen that the enzyme is a macromolecular protease composed of multiple domains, and the end of the C-terminal contains three consecutive SLH domains and a link Peptide, related to anchoring on the cell surface, has no significant impact on its enzymatic properties, so it is removed to improve the expression efficiency of the enzyme, and finally the amino acid sequence of the xylanase used in the present invention is determined as SEQ ID NO: 4, The corresponding coding gene sequence is SEQ ID NO:3.

Using the genome of Thermoanaerobacterium aotearoense SCUT27 as a template, carry out PCR amplification of the xylanase coding gene (SEQ ID NO: 7) and primer 4 (SEQ ID NO: 8) NO: 3). The PCR reaction conditions are: denaturation at 98°C for 10 sec, annealing at 56°C for 5 sec, and extension at 72°C for 3 min. The sequences of primers 3 and 4 and the reaction system are as follows. The results of agarose gel electrophoresis of PCR products are shown in Figure 2.

Primer 3: 5'-CGTTCGGATCCATGGACGATACTAATACAAATCTGG-3' (SEQ ID NO: 7)

Primer 4: 5'-AATACCTGCAGAGAAGGTTTACCTGTAAGCATCA-3' (SEQ ID NO: 8)

After the purification of the xylanase gene fragment obtained by PCR amplification, the plasmid pHTHis constructed in Example 1 was double-digested with restriction endonucleases BamH I and Pst I respectively, and the digested products were purified and ligated, and transformed into E .coli BL21(DE3) competent cells, positive clones were screened by colony PCR and sequencing. The positive clone was inoculated into LB medium containing ampicillin resistance and cultured overnight, and then the plasmid was extracted, which was the expression vector pHTHis-nsXynAΔSLH of the recombinant xylanase. The vector was transferred to Bacillus subtilis (Bacillus subtilis) 1012wt (purchased from MOBITEC) by chemical transformation method, and screened with chloramphenicol LB plates with a final concentration of 5 μg/mL to obtain the recombinant xylanase intracellular Expression strain B. subtilis 1012wt/pHTHis-nsXynAΔSLH. The preparation and transformation methods of Bacillus subtilis competent cells refer to the Spizizen method (Anagnostopoulos and Spizizen 1961).

Example 3

Expression and purification of recombinant xylanase

(1) Expression of recombinase

Transfer 10 mL of overnight culture solution of Bacillus subtilis B. subtilis 1012wt containing recombinant plasmid pHTHis-nsXynAΔSLH to 1L LB (containing 5 μg/mL chloramphenicol) medium, culture at 250 rpm at 37°C, when the OD ₆₀₀ of the culture solution reaches about 0.5 , add IPTG to a final concentration of 0.1 mM, induce culture for 10 h, collect the cells by centrifugation, break the cells with lysozyme and/or ultrasonic breaker, and collect the supernatant by centrifugation to obtain the crude enzyme solution. SDS-PAGE shows ( FIG. 3 ), that the expression level of the recombinant xylanase accounts for about 15% of the total bacterial protein in this example.

(2) Purification of recombinant enzyme

The target protein was purified by nickel column affinity chromatography. First equilibrate Ni-NTA (1×5cm) with equilibration buffer (20mM, pH 7.5 Tris-HCl buffer, 0.5M NaCl, 20mM imidazole); pass the above-mentioned crude enzyme solution through the equilibrated Ni column at a flow rate of 3mL/min After loading the sample, continue to flush to equilibrium with the equilibrium buffer at the same flow rate, increase the flow rate to 5mL/min, and continue to flush to equilibrium. Elution was performed with buffers with different imidazole concentrations (68, 116 and 500 mM) and corresponding protein samples were collected. The SDS-PAGE results of the protein samples collected during the purification process are shown in Figure 3.

The experimental results showed that the recovery rate of xylanase purified from recombinant bacteria B.subtilis1012wt/pTHHis-nsXynAΔSLH by nickel column affinity chromatography was as high as 57%, and 19.2mg of pure xylanase was finally obtained from 1L fermentation broth.

Example 4

Enzyme Activity Determination of Recombinant Xylanase

Xylanase can degrade xylan into oligosaccharides, and the enzyme activity of xylanase can be detected by measuring the amount of reducing sugar produced after the catalytic reaction by DNS method (MILLER 1959). The specific steps are as follows: add 90 μL 1% beech xylan solution to 190 μL 100 mM, pH 6.5 Bis-Tris-HCl buffer solution, add 20 μL of appropriately diluted crude enzyme solution or purified enzyme solution, react at 80 ° C for 5 minutes, and immediately add 200 μ L of DNS solution, boiled water bath for 3 minutes, centrifuged after cooling in ice water, measured the absorption value at 540nm, and used D-xylose as the standard during the whole reaction process. Enzyme activity unit (U) is defined as: under the above reaction conditions, the amount of enzyme required to produce 1 μmol of reducing sugar equivalent to D-xylose per minute is 1 enzyme activity unit.

It has been determined that the specific enzyme activity of the xylanase obtained in Example 3 using beech wood xylan as a substrate reaches 248.8 U/mg.

Example 5

Detection of Enzymatic Properties of Recombinant Xylanase

The above-mentioned purified enzyme solution was subjected to the following property tests respectively.

1. Determination of the optimum reaction pH

The purified enzyme solution was reacted in buffer solutions with different pH values, and the relative enzyme activity was determined. The method for detecting enzyme activity is the same as the method for detecting enzyme activity described in Example 3, except that the buffer system used is different.

The selected pH range and buffer system are as follows: 0.1M acetic acid-potassium acetate buffer pH 3.5-6.0, 0.1M Bis-Tris-HCl buffer pH 6.0-7.0, 0.1M Tris-HCl buffer pH 7.0-9.0. By comparing the enzyme activity difference at different pH values, the optimum reaction pH value of the enzyme can be known. Relative enzyme activity is defined as: the percentage of enzyme activity and maximum enzyme activity at different pH values. The results showed that the optimal reaction pH value of the recombinant xylanase of the present invention was 6.5 (the results are shown in Figure 4).

2. Determination of the optimum reaction temperature

Purified enzyme solution is tested for enzyme activity at different temperatures (50, 55, 60, 65, 70, 75, 80, 85 and 90°C) at the optimum reaction pH value, and the relative enzyme activity can be used to know the maximum activity of the enzyme. Suitable reaction temperature. The method for detecting the enzyme activity is the same as the enzyme activity detection method described in Example 3 except that the reaction temperature is different. The results showed that the optimal reaction temperature of the recombinant xylanase of the present invention was 80° C. (the results are shown in FIG. 5 ).

3. Determination of pH stability

The purified enzyme solution was diluted to a certain number of times with buffer solutions of different pH values, and stored at 70°C for 30 minutes. After the heat treatment, the relative enzyme activity was measured. The method for detecting the enzyme activity is the same as the method for detecting the enzyme activity described in Example 3, and the enzyme activity detection is carried out at the optimum reaction pH and the optimum reaction temperature.

The selected pH range and system are as follows: 0.1M acetic acid-potassium acetate buffer pH 3.5-6.0, 0.1M Bis-Tris-HCl buffer pH 6.0-7.0, 0.1M Tris-HCl buffer pH 7.0-9.0. By comparing the remaining enzyme activities of enzyme solutions stored at different pH values, it can be known that the enzyme is relatively stable under slightly acidic, neutral and alkaline conditions (the results are shown in Figure 6). The enzyme activity of the purified enzyme solution without heat treatment was recorded as 100%.

4. Determination of Thermal Stability

The purified enzyme solution was incubated at different temperatures (70, 75, and 80° C.), and the remaining enzyme activity of the enzyme solution after incubation for different times was measured under the optimum reaction conditions. In addition, the purified enzyme solution was incubated at 75, 76, 77, 78, 79 and 80° C. for 30 min, respectively, and the remaining enzyme activity of the enzyme solution was measured respectively. Wherein, the enzyme activity of the unheated enzyme liquid is recorded as 100%, and the relative enzyme activity of the purified enzyme liquid after heat treatment at different temperatures and different holding times is compared. The results show that the enzyme has almost no loss of enzyme activity after incubation at 70°C for 2 hours, about 90% of the remaining enzyme activity can be retained at 75°C for 2 hours, and there is almost no enzyme activity at 75, 76, and 77°C for 30 minutes. loss of enzyme activity. It can be seen that the recombinant xylanase has good temperature stability (the results are shown in Figure 7 and Figure 8).

Claims (10)

1. an alkali-resistant xylanase, is characterized in that, is selected from following (1) or (2) or (3):

(1) the total length zytase be made up of the aminoacid sequence shown in SEQ ID NO:2;

(2) recombined xylanase be made up of the aminoacid sequence shown in SEQ ID NO:4;

(3) aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 had the protein derivative by (1) or (2) of alkali-resistant xylanase activity through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.

2. the encoding gene of zytase described in claim 1, is characterized in that,

(1) protein molecule described in its nucleotide sequence coded claim 1;

(2) its nucleotide sequence is SEQ ID NO:1 or the sequence shown in SEQ ID NO:3;

(3) its nucleotide sequence is the replacement of nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3 through one or several Nucleotide and/or the derived sequence of disappearance and/or interpolation gained;

(4) DNA sequence dna limited with (1) has more than 90% homology and the nucleotide sequence of described zytase of encoding.

3. the recombinant vectors containing encoding gene described in claim 2.

4. recombinant vectors according to claim 3, it is characterized in that, the gene of expression vector pHTHis and claim 2 is carried out double digestion respectively with restriction enzyme BamH I and Pst I, connect after digestion products purifying, be converted into E.coli BL21 (DE3) competent cell, filter out positive colony by bacterium colony PCR, order-checking; Positive colony is connected in the LB substratum containing amicillin resistance and extracts plasmid after incubated overnight, namely obtains recombinant vectors.

5. recombinant vectors according to claim 4, it is characterized in that, the preparation of described expression vector pHTHis: the gene fragment of SEQ ID NO:9 and plasmid pHT01 are all carried out double digestion with BamH I, Aat II, connect after purifying respectively, be converted into E.coli DH5 α competent cell, filter out positive colony by bacterium colony PCR, order-checking; Again positive colony is connected in the LB substratum containing amicillin resistance and extracts plasmid after incubated overnight, be expression vector pHTHis.

6. recombinant vectors according to claim 4, is characterized in that, the clone/expression region of described expression vector pHTHis as shown in Figure 1.

7. the recombinant bacterium containing encoding gene described in claim 2.

8. recombinant bacterium according to claim 7, it is characterized in that, recombinant vectors described in any one of claim 3 ~ 6 goes in subtilis (Bacillus subtilis) by chemical transformation, screen with paraxin LB flat board again, namely obtain this zytase recombinant bacterium.

9. the transgenic cell line containing encoding gene described in claim 2.

10. the expression cassette containing encoding gene described in claim 2.