CN103898079B - Temperature neutral xylanase XYN11B and gene and application in one - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular, the invention relates to a mesophilic neutral xylanase XYN11B and its gene and application. The present invention provides a new medium-temperature neutral xylanase XYN11B, which has the amino acid sequence shown in SEQ ID NO.1 or 2, and the present invention also provides a gene encoding the above-mentioned medium-temperature neutral xylanase XYN11B , the nucleotide sequence of which is shown in SEQ ID NO.4 or 5, as well as the recombinant vector and recombinant strain containing the gene and its application. The optimal pH of the xylanase XYN11B of the present invention is 6.0, and it has relatively high enzyme activity at pH 5.0-7.0; its optimal temperature is 50°C, and it has an enzyme activity of more than 80% of the optimal temperature at 40°C , at 30°C, it has an enzyme activity above 45% of the optimum temperature.

Description

A mesophilic neutral xylanase XYN11B and its gene and application

technical field

The present invention relates to the field of genetic engineering, in particular, the present invention relates to a mesophilic neutral xylanase XYN11B and its gene and application.

Background technique

Hemicellulose is one of the main components in the plant cell wall, mainly distributed in the secondary wall, and its content is second only to cellulose, accounting for about 30% to 35% of the dry weight of the cell. Among them, xylan is one of the main components of plant hemicellulose, and it is also an important renewable resource in nature besides cellulose. Xylan widely exists in agricultural by-products such as corncobs, wheat bran, rice bran, straw, bagasse, etc., but this important renewable resource has been difficult to be effectively utilized. The complete degradation of xylan requires a multi-step enzymatic reaction and the synergistic action of multiple hydrolases, β-1,4-endo-xylanase (endo-1,4-β-xylanase, E.C.3.2.1.8) and β - Xylosidase is the most critical hydrolase. Based on amino acid sequence homology, xylanases are classified into glycoside hydrolase families 10, 11, 39, 43, 52, 62 and 67 (Henrissat B, et al., 1993). For nearly half a century, xylanase has attracted more and more attention due to its wide application in feed, food, pulp and paper industry and energy fields.

The arabinoxylan in the fruit juice production process will increase the viscosity of the juice, reduce the speed of ultrafiltration concentration, increase the production process and time, and also have a certain impact on the product flavor; in terms of bread baking, after wheat and rye are processed into powder, the The non-starch polysaccharide is pentosan (the main component is arabinoxylan), of which the water-extractable arabinoxylan accounts for about 20% to 30%, and the water-insoluble arabinoxylan accounts for about 70% to 80%. Non-leachable pentosans have a positive effect on bread, while non-leachable pentosans are detrimental to bread quality. In addition, xylan belongs to non-starch polysaccharide (non-starch polysaccharide, NSP), which is a major anti-nutritional factor in plant feed, and the content of cereal feed is relatively high; its high viscosity and water-holding performance significantly change digestion The physical characteristics of the food and the physiological activity of the intestinal tract reduce the digestibility of the animal's feed, and a large amount of nutrients that are not absorbed by the animal accumulate in the intestinal tract of the animal, creating a good environment for the reproduction of intestinal microorganisms. Some harmful microorganisms The metabolites produced by mass reproduction change the intestinal pH environment and affect the function of intestinal digestive enzymes (Marquardt et al., 1979; White et al., 1983).

The screening of mesophilic xylanases derived from microorganisms is a hotspot in xylanase research, but most xylanases generally have low enzyme activity at around 37°C, which cannot well meet the physiological requirements of animals. condition. The xylanase XYN11B provided by the invention is the 11th family xylanase, which specifically acts on xylan substrates and has relatively high activity on water-insoluble arabinoxylan. Its optimum pH is 6.0, and it has high enzyme activity at pH 5.0-7.0; and its optimum temperature is 50°C, and it has an optimum temperature of more than 80% of the enzyme activity at 40°C. Enzyme activity at temperatures above 45%. Compared with other xylanases, the xylanase XYN11B provided by the present invention can play a good role in hydrolysis at the temperature of the gastrointestinal tract (30-40°C), and has a good effect on food and animal feed Application prospect.

Contents of the invention

The object of the present invention is to provide a kind of middle temperature neutral xylanase which can be used for food and animal feed.

Another object of the present invention is to provide a gene encoding the above-mentioned mesophilic neutral xylanase.

Another object of the present invention is to provide a recombinant vector comprising the above gene.

Another object of the present invention is to provide recombinant strains containing the above genes.

Another object of the present invention is to provide a genetic engineering method for preparing the above-mentioned mesophilic neutral xylanase.

Another object of the present invention is to provide the application of the above-mentioned neutral neutral xylanase.

The present invention isolates a new mesophilic neutral xylanase XYN11B from Humicola insolens.

The present invention provides a medium-temperature and medium-alkaline xylanase XYN11B, the amino base sequence of which is shown in SEQ ID NO.1.

SEQ ID NO.1:

MVAFSSLFLGASIAATALAAPGELPGMHLNKRQTYTQSATGTHDGYYFSFWTDGGPNVRYTNEAGGQYSVTWSGNGNWVGGKGWNPGAARTINYTGTYQPNGNSYLAVYGWTRNPLVEYYVVENFGTYDPSTGAQRLGSINVDGSTYNVYRTRRTNAPSIEGTRSFDQYWSVRVNKRVGGSVDMNAHFNAWRQAGLTLGTHDYQIVATEGYFSSGSARINVGGGSTGGGNNGGGNNGGNPGGNPGGNPGGNPGGNCSPRWGQCGGQGWNGPTCCESGTTCRQQNQWYSQCL

Wherein, the enzyme includes 291 amino acids, and the N-terminal 19 amino acids are its predicted signal peptide sequence "mvafsslflgasiaatala" (SEQ ID NO.3).

Therefore, the theoretical molecular weight of the mature mesophilic mesophilic xylanase XYN11B is 29.0kDa, and its amino acid sequence is shown in SEQ ID NO.2:

APGELPGMHLNKRQTYTQSATGTHDGYYFSFWTDGGPNVRYTNEAGGQYSVTWSGNGNWVGGKGWNPGAARTINYTGTYQPNGNSYLAVYGWTRNPLVEYYVVENFGTYDPSTGAQRLGSINVDGSTYNVYRTRRTNAPSIEGTRSFDQYWSVRVNKRVGGSVDMNAHFNAWRQAGLTLGTHDYQIVATEGYFSSGSARINVGGGSTGGGNNGGGNNGGNPGGNPGGNPGGNPGGNCSPRWGQCGGQGWNGPTCCESGTTCRQQNQWYSQCL

The xylanase XYN11B of the invention has the characteristics of moderate temperature neutrality, good substrate specificity, high activity at physiological temperature and the like. The present invention has screened an 11th family xylanase produced by HumicolainsolensY1, its optimum pH value is 6.0, and it maintains a relatively high enzyme activity in the range of pH 5.0-7.0; its optimum temperature is 50°C, at At 40°C, it has an enzyme activity of more than 80% of the optimum temperature, and at 30°C, it has an enzyme activity of more than 45% of the optimum temperature.

The present invention provides xyn11B encoding the above-mentioned mesophilic and alkaline xylanase. Specifically, the genome sequence of the gene is shown in SEQ ID NO.4:

atggtcgccttctcgtccctcttcctcggcgcttccatcgccgccacggcgctcgccgcccccggtgagctgcctggcatgcacctgaacaagcgccagacctacacccagagcgctaccggcactcacgacggctactacttctccttctggactgacggtggcccgaacgtccgctacaccaacgaggccggtggtcagtacagtgtgacctggtctggtaacggcaactgggtcggtggcaagggttggaacccgggtgctgctcggtaggttcatcatctaaccgacttctctcaagatcatcactaacatatggcagtaccatcaactacactggtacctaccagcccaacggcaactcgtacctggccgtctacggctggactcgcaaccctctggtcgagtactacgtcgtcgagaacttcggcacctacgatccctcgactggtgcccagcgcctgggcagcatcaacgttgatggctcgacctacaacgtctaccgcacccgccgcaccaacgctccctcgattgagggcacccgctccttcgaccagtactggtccgtccgtgtcaacaagcgcgttggcggctctgttgacatgaacgcccacttcaacgcctggcggcaggccggcctcaccctgggtacccacgactaccagatcgtggctactgagggctacttctccagcggctcggcccgcatcaacgtcggcggcggctccactggcggcggcaacaacggtggcggcaacaacggtggcaacccgggcggcaacccgggcggcaaccctggcggcaaccctggcggcaacgtaagtctattccgcacatgcttcatagaatgaccttaaccaaatactgacaatgggatcctgcagtgctctcctcgctggggccagtgcggtggccagggctggaacggccctacctgctgcgagtccggcaccacctgccgccagcagaaccagtggtactctcagtgcctgtaa

The invention isolates and clones the xylanase gene xyn11B based on the PCR method, and the DNA sequence analysis result shows that the xyn11B structural gene xyn11B of the xylanase XYN11B has a full length of 995 bp. Wherein, the base sequence of the signal peptide is:

ATGGTCGCCTTCTCGTCCCTCTTCCTCGGCGCTTCCATCGCCGCCACGGCGCTCGCC (SEQ ID NO. 6).

The gene sequence of the mature xylanase XYN11B is shown in SEQ ID NO.5.

SEQ ID NO.5

atggtcgccttctcgtccctcttcctcggcgcttccatcgccgccacggcgctcgccgcccccggtgagctgcctggcatgcacctgaacaagcgccagacctacacccagagcgctaccggcactcacgacggctactacttctccttctggactgacggtggcccgaacgtccgctacaccaacgaggccggtggtcagtacagtgtgacctggtctggtaacggcaactgggtcggtggcaagggttggaacccgggtgctgctcgtaccatcaactacactggtacctaccagcccaacggcaactcgtacctggccgtctacggctggactcgcaaccctctggtcgagtactacgtcgtcgagaacttcggcacctacgatccctcgactggtgcccagcgcctgggcagcatcaacgttgatggctcgacctacaacgtctaccgcacccgccgcaccaacgctccctcgattgagggcacccgctccttcgaccagtactggtccgtccgtgtcaacaagcgcgttggcggctctgttgacatgaacgcccacttcaacgcctggcggcaggccggcctcaccctgggtacccacgactaccagatcgtggctactgagggctacttctccagcggctcggcccgcatcaacgtcggcggcggctccactggcggcggcaacaacggtggcggcaacaacggtggcaacccgggcggcaacccgggcggcaaccctggcggcaaccctggcggcaactgctctcctcgctggggccagtgcggtggccagggctggaacggccctacctgctgcgagtccggcaccacctgccgccagcagaaccagtggtactctcagtgcctgtaa

The theoretical molecular weight of the mature protein is 29.0kDa. The xyn11B sequence of the xylanase gene and the deduced amino acid sequence were compared by BLAST in GenBank, and it was confirmed that XYN11B is a new xylanase.

The present invention provides a recombinant vector comprising the above-mentioned medium-temperature and medium-alkaline xylanase gene xyn11B, which is selected as pPIC-xyn11B. The xylanase gene of the present invention is inserted between suitable restriction enzyme cutting sites of the expression vector, so that its nucleotide sequence is operably linked with the expression control sequence. As a most preferred embodiment of the present invention, it is preferred that the xylanase gene of the present invention is inserted between EcoRI and the NotI restriction enzyme site on the plasmid pPIC9, so that the nucleotide sequence is positioned at the AOX1 promoter Downstream of and regulated by it, the recombinant yeast expression plasmid pPIC9-xyn11B was obtained.

The present invention also provides a recombinant strain comprising the above-mentioned medium-temperature and medium-alkaline xylanase gene xyn11B, preferably, the strain is Escherichia coli or yeast, preferably the recombinant strain GS115/xyn11B.

The present invention also provides a method for preparing medium-temperature and medium-alkaline xylanase XYN11B, comprising the following steps:

1) Transforming host cells with the above-mentioned recombinant vectors to obtain recombinant strains;

2) Cultivate the recombinant strain and induce the expression of recombinant xylanase;

3) Recover and purify the expressed xylanase XYN11B.

Wherein, the host cell is preferably Pichia pastoris cell, Saccharomyces cerevisiae cell or S. polymorpha cell, and the recombinant yeast expression plasmid is preferably transformed into Pichia pastoris cell (Pichia pastoris) GS115 to obtain recombinant strain GS115/xyn11B.

The present invention also provides the application of the above-mentioned medium-temperature medium-alkaline xylanase XYN11B.

The first technical problem to be solved by the present invention is to overcome the low enzymatic activity of xylanase derived from microorganisms at low temperature and to provide a new xylanase with excellent properties and suitable for application in food and animal feed. Carbohydrase XYN11B. The optimum pH of the xylanase XYN11B of the present invention is 6.0, and it has high enzyme activity at pH 5.0-7.0; it has high enzyme activity at lower temperature (30-40°C), and has poor thermal stability . It can be well used in fruit juice production and bread baking in the food industry to improve production efficiency and flavor; it can also be used in animal feed, especially ruminant feed, to exert hydrolysis in animal physiological environment and improve feed utilization .

Description of drawings

Figure 1 Optimum pH of recombinant xylanase.

Figure 2 pH stability of recombinant xylanase.

Figure 3 Optimal temperature of recombinant xylanase.

Fig. 4 Thermostability of recombinant xylanase.

detailed description

Test materials and reagents

1. Bacterial strain and carrier: the present invention is obtained from Humicola insolens Y1. Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.

2. Enzymes and other biochemical reagents: endonucleases were purchased from TaKaRa Company, and ligases were purchased from Invitrogen Company. Birch xylan was purchased from Sigma, and the others were domestic reagents (all of which can be purchased from common biochemical reagent companies).

3. Medium:

(1) HumicolainsolensY1CGMCC4573 medium is potato juice medium: 1000mL potato juice, 10g glucose, 25g agar, natural pH.

(2) Escherichia coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, natural pH).

(3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V).

(4) BMMY medium: replace glycerin with 0.5% methanol, the rest of the ingredients are the same as BMGY, and the pH is natural.

Note: For the molecular biology experiment methods not specifically described in the following examples, all refer to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or follow the kit and product manual.

Example 1 Cloning of HumicolainsolensY1CGMCC4573 Xylanase Encoding Gene xynA

Humicola Y1CGMCC4573 was isolated from forest soil in Hebei Province, and its optimum growth temperature was 45℃ and pH 6.0. After being cultured in PDA medium for 3 days, the colonies were dark brown with gills formed on the edge. The conidiophores are short, solitary, and dark gray in color. Conidia, solitary, spherical or subspherical, brown.

Extraction of Genomic DNA from Humicola insolensY1CGMCC4573:

Filter the mycelium cultured in liquid for 3 days with sterile filter paper, put it into a mortar, add 2mL of extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 120min, mix every 20min Homogenize once and centrifuge at 13000rpm for 10min at 4°C. The supernatant was extracted in phenol/chloroform to remove impurities, and then an equal volume of isopropanol was added to the supernatant. After standing at -20°C for 30 minutes, centrifuge at 13,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuo, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

Degenerate primers P1, P2 were designed and synthesized according to the conserved (WDVVNE and NDY(F)NL(I)EY) sequences of the tenth family xylanase genes

P1: 5'-TGGGAYGTNGTNAAYGARGC-3';

P2: 5'-TAYTCTATRTTRWARTCRTT-3'.

The total DNA of HumicolainsolensY1CGMCC4573 was used as template for PCR amplification. The PCR reaction parameters were: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 45°C for 30 sec, extension at 72°C for 1 min, and after 30 cycles, incubation at 72°C for 10 min. A fragment of about 159bp was obtained, which was recovered and connected with the pEASY-T3 vector and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

According to the gene fragments obtained by sequencing, the genome sequencing information of HumicolainsolensY1CGMCC4573 was analyzed to obtain the full length of the gene. Analysis of xyn11B xyn11B full-length 995bp, containing two intron sequences, Cdna gene full-length 876bp, encoding 291 amino acids and a stop codon. Analysis with SignalP (http://www.cbs.dtu.dk/services/SignalP) showed that the N-terminal 19 amino acids were predicted signal peptides. The theoretical molecular weight of the mature protein encoded by this gene is predicted to be 29.0kDa.

The preparation of embodiment 2 recombinant xylanase

The expression vector pPIC9 was subjected to double digestion (EcoRI+NotI), and the gene xyn11B encoding xylanase was double-digested (EcoRI+NotI) at the same time, and the gene fragment encoding mature xylanase (not including the signal peptide sequence) was cut out ) was connected with the expression vector pPIC9 to obtain the recombinant plasmid pPIC-xyn11B containing HumicolainsolensY1CGMCC4573 xyn11B xyn11B and transform it into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115/xyn11B.

The GS115 strain containing the recombinant plasmid was inoculated in 400mL of BMGY culture medium, shaken at 250rpm at 30°C for 48h, and then centrifuged to collect the bacteria. Then resuspend in 200mL BMMY medium, shake culture at 250rpm at 30°C. After 72 hours of induction, the supernatant was collected by centrifugation. Determination of xylanase activity. The expression level of recombinant xylanase was 18.5U/mL. SDS-PAGE results showed that the recombinant xylanase was expressed in Pichia pastoris.

In the same way, the expression vector of xyn11B gene of xylanase containing signal peptide sequence was constructed and expressed in Pichia pastoris.

Separation and purification to obtain recombinant xylanase.

The activity analysis of embodiment 3 recombinant xylanase

DNS method: The specific method is as follows: at pH 6.0, 80°C, 1 mL of reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of substrate, react for 10 minutes, add 1.5 mL of DNS to terminate the reaction, and boil for 5 minutes. After cooling to room temperature, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute under given conditions.

The property determination of embodiment 4 recombinant xylanase XYNA

Determination of Example 2 to obtain the properties of recombinant xylanase XYN11B

1. The method for determining the optimal pH and pH stability of recombinant xylanase XYN11B is as follows:

The recombinant xylanase purified in Example 2 was subjected to enzymatic reactions at different pHs to determine its optimum pH. Substrate xylan was tested for xylanase activity in 0.1mol/L citric acid-disodium hydrogen phosphate buffer solution with different pH at 50°C. The results ( FIG. 1 ) showed that the optimum pH of the recombinant enzyme XYN11B was 6.0, and had relatively high relative enzyme activity at pH 5.0-7.0. Xylanase was treated at 37°C for 60min in the above-mentioned buffers with different pH, and then the enzyme activity was measured at 80°C in the pH6.0 buffer system to study the pH stability of the enzyme. Result (Fig. 2) shows that xylanase is all very stable between pH6.0-9.0, and remaining enzyme activity is more than 60% after processing 60min in this pH range, and this illustrates that this enzyme has very good in acid range. pH stability.

2. The optimal temperature and thermostability determination methods of xylanase are as follows:

The determination of the optimal temperature of xylanase is carried out in the citric acid-disodium hydrogen phosphate buffer (pH6.0) buffer system and the enzymatic reaction at different temperatures. The temperature resistance was measured by treating xylanase at different temperatures for different times, and then measuring the enzyme activity at 50°C. The measurement results of the optimum temperature of the enzyme reaction (Figure 3) showed that the optimum temperature was 50°C, and the enzyme activity at 40°C was more than 80% of the optimum temperature. Enzyme thermostability test showed (Figure 4).

3. The K _m value assay method of xylanase is as follows:

Using different concentrations of xylan as the substrate, the enzyme activity was measured at 50°C in the citric acid-disodium hydrogen phosphate buffer (pH6.0) buffer system, and the K _m value at 50°C was calculated. It was determined that when using birch xylan as the substrate, the K _m value was 2.2 mg/mL, and the maximum reaction velocity V _max was 462.8 μmol/min·mg.

4. The influence of different metal ion chemical reagents on XYN11B enzyme activity was determined as follows:

Add different concentrations of different metal ions and chemical reagents into the enzymatic reaction system to study their effects on enzyme activity. The final concentration of various substances is 5mmol/L. Enzyme activity was measured at 50°C and pH 6.0. The results showed that most ions and chemical reagents had no effect on the activity of the recombinant xylanase, only Ag ⁺ and SDS inhibited part of its activity.

5. Substrate specificity of recombinant xylanase

In addition to acting on xylan, the enzyme also degrades barley glucan, soluble and insoluble wheat arabinoxylan (Table 2).

Table 2. Analysis of substrate specificity of xylanase XYN11B

Claims (8)

1. A mesophilic neutral xylanase XYN11B, characterized in that its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2. 2. A mesophilic neutral xylanase gene xyn11B, characterized in that it encodes the mesophilic neutral xylanase XYN11B according to claim 1. 3. The neutral neutral xylanase gene xyn11B according to claim 2, characterized in that its base sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5. 4. The recombinant vector comprising the neutral neutral xylanase gene xyn11B of claim 2. 5. comprise the recombinant vector pPIC-xyn11B of the middle-temperature neutral xylanase gene xyn11B described in claim 2, wherein, the expression vector pPIC9 is carried out EcoRI+NotI double digestion, simultaneously nucleotide sequence is as shown in SEQIDNO.5 The medium-temperature neutral xylanase gene xyn11B was subjected to EcoRI+NotI double digestion, and the gene fragment encoding mature xylanase was cut out and connected with the expression vector pPIC9 after digestion to obtain the recombinant vector pPIC-xyn11B. 6. A recombinant strain comprising the mesophilic neutral xylanase gene xyn11B of claim 2. 7. A method for preparing mesophilic neutral xylanase XYN11B, characterized in that it comprises the following steps: 1) Transforming host cells with the recombinant vector of claim 4 to obtain recombinant strains; 2) Cultivate the recombinant strain and induce the expression of recombinant xylanase; 3) recovering and purifying the expressed mesophilic neutral xylanase XYN11B. 8. The application of the mesophilic neutral xylanase XYN11B according to claim 1 in food and animal feed.