CN105779420B

CN105779420B - High-temperature-resistant acid arabinofuranosidase AbfaHLB (AbfaHLB), gene and application thereof

Info

Publication number: CN105779420B
Application number: CN201610323552.5A
Authority: CN
Inventors: 吴培均; 李富伟; 罗建杰; 李兆勇
Original assignee: BEIJING CREATE VALUE BIOLOGY Co Ltd
Current assignee: BEIJING CREATE VALUE BIOLOGY Co Ltd
Priority date: 2016-05-16
Filing date: 2016-05-16
Publication date: 2020-02-18
Anticipated expiration: 2036-05-16
Also published as: CN105779420A

Abstract

The invention relates to the field of genetic engineering, in particular to high-temperature-resistant acid arabinofuranosidase AbfaHLB and a gene and application thereof. The invention provides an arabinofuranosidase AbfaHLB derived from Bacillus badius HLB, and an amino acid sequence of the arabinofuranosidase AbfaHLB is shown as SEQ ID NO.1, and the invention provides a coding gene abfa HLB for coding the arabinofuranosidase. The arabinofuranosidase provided by the invention has the following properties: the optimum pH value is 4.5, the optimum temperature is 50 ℃, and the thermal stability is good at 80 ℃. As a novel enzyme preparation, the xylanase can be widely used in feed, food, energy industry and the like.

Description

High-temperature-resistant acid arabinofuranosidase AbfaHLB (AbfaHLB), gene and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to high-temperature-resistant acid arabinofuranosidase AbfaHLB (AbfaHLB), a gene thereof, a recombinant vector containing the gene and application.

Background

Xylans are the most abundant group of polysaccharides in hemicelluloses, widely found in hardwoods (15-30%), softwoods (7-10%) and herbaceous plants (less than 30%), and are chemically cross-linked together, backbone hydrolases, including β -1, 4-xylanase and β -xylosidase, and side chain hydrolases, requiring coenzymes such as α -l-arabinofuranosidase, α -glucuronic acid, acetylxylan esterase, etc., wherein arabinofuranosidases (α -l-arabinofuranosidase, EC 3.2.1.55) can be isolated from polysaccharides containing arabinose residues, such as arabinans, arabinoxylans, etc., non-reducing galactose, etc., to produce a non-reducing galactose molecule, which is derived from a microbial source, after isolation at pH 0.5, at pH 0, at pH 70, after purification by microbial hydrolysis at pH 0.5970.

The arabinofuranosidase can also increase the terpene alcohol concentration during brewing, improve the flavor of wine, increase the clarity of juice, and is used in the juice production industry as one of the hemicellulose degrading enzyme systems, α -L-arabinofuranosidase participates in the reuse of low-cost agricultural product residues, produces monosaccharides that can be used for fermentation to produce fuel ethanol and other by-products.

The industrial production needs enzyme to carry out short-term high-temperature treatment, most of the arabinofuranosidase at present has the optimum temperature of about 50 ℃, but the property of poor thermal stability can not meet the industrial requirements of feed granulation, brewing processing and the like. Therefore, the obtained enzyme with excellent thermal stability can reduce the production cost and meet the requirements of different industries on enzyme properties.

Disclosure of Invention

The invention aims to provide high-temperature-resistant acid arabinofuranosidase which can be efficiently applied.

It is still another object of the present invention to provide a gene encoding the above high temperature acid resistant arabinofuranosidase.

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 a recombinant strain comprising the above gene.

Another purpose of the invention is to provide a genetic engineering method for preparing the high-temperature resistant arabinofuranosidase.

The invention also provides application of the high-temperature resistant arabinofuranosidase.

The invention separates a new high temperature resistant arabinofuranosidase AbfaHLB from Bacillus badius HLB.

The invention provides high-temperature-resistant acid arabinofuranosidase AbfaHLB, the amino acid sequence of which is shown as SEQ ID NO. 1:

wherein, the enzyme gene codes 502 amino acids without signal peptide sequence.

The arabinofuranosidase AbfaHLB disclosed by the invention has good enzyme activity in the acid and neutral ranges and has good thermal stability. The arabinofuranosidase AbfaHLB of the screened Bacillus badius HLB of the invention has the following properties: the optimum pH value is 4.5, the enzyme activity is more than 60% in the range of pH value 2.0-6.0, the pH value stability is good, and after 1 hour of action at 37 ℃, the residual enzyme activity is more than 85% in the range of pH value 2.0-8.0. The activity of the compound has more than 60 percent at the optimum temperature of 50 ℃ and 60 ℃, and the compound has good thermal stability and good stability at 80 ℃.

The invention provides a gene AbfaHLB for coding the high-temperature acid-resistant arabinofuranosidase. Specifically, the genome sequence of the gene is shown as SEQ ID NO. 2:

the coding gene of the arabinofuranosidase AbfaHLB is separated and cloned by a PCR method, and the DNA complete sequence analysis result shows that the full length of the coding gene of the arabinofuranosidase AbfaHLB is 1509 bp.

The theoretical molecular weight of the mature protein is 56.6kDa, and the sequence of the coding gene of the arabinofuranosidase AbfaHLB and the deduced amino acid sequence are subjected to BLAST comparison in GenBank, and the consistency of the gene and the sequence of the arabinofuranosidase from Alicyclobacillus heperidum is 77 percent. The AbfaHLB is shown to be a novel arabinofuranosidase.

The invention also provides a recombinant vector containing the arabinofuranosidase gene abfaHLB, preferably pPIC 9-abfaHLB. The arabinofuranosidase gene of the present invention is inserted between appropriate restriction sites in an expression vector such that its nucleotide sequence is operably linked to an expression control sequence. In a most preferred embodiment of the present invention, it is preferred that the arabinofuranosidase gene of the present invention is inserted between EcoR I and Not I restriction sites on the plasmid pPIC9 to give a recombinant yeast expression plasmid pPIC 9-abfaHLB.

The invention also provides a recombinant strain containing the high-temperature resistant arabinofuranoside gene abfaHLB, wherein the strain is preferably escherichia coli, saccharomycete, bacillus or lactobacillus, and is further preferably pichia pastoris, for example, the recombinant strain GS 115/abfaHLB.

The invention also provides a method for preparing the high-temperature-resistant acid arabinofuranosidase AbfaHLB, which comprises the following steps:

1) transforming host cells by using the recombinant vector to obtain a recombinant strain;

2) culturing the recombinant strain, and inducing the expression of the recombinant arabinofuranosidase;

3) recovering and purifying the expressed arabinofuranosidase AbfaHLB.

Preferably, the host cell is pichia pastoris, and the recombinant expression plasmid is preferably transformed into pichia pastoris GS115 to obtain the recombinant strain GS 115/abfaHLB.

The invention also provides application of the above arabinofuranosidase AbfaHLB. Preferably, the application of the above arabinofuranosidase AbfaHLB in the feed and food industry, and further preferably the application in the feed and food industry for degrading arabinoxylan is provided.

The invention firstly aims to solve the technical problem of overcoming the defects of the prior art and provide a novel arabinofuranosidase which has excellent properties and is suitable for application in feed, wine and food industries. The optimum pH value of the arabinofuranosidase is 4.5, and the arabinofuranosidase has higher enzyme activity at the pH value of 2.0-6.0; the pH stability is good. The high temperature resistance of the material can ensure that the material can be applied to industrial production requiring high temperature environment. The arabinofuranosidase is suitable for feed industry, and can act synergistically with xylanase to effectively eliminate or reduce anti-nutritional effect caused by viscosity increase. In the brewing industry, the soluble and insoluble arabinoxylan can be effectively degraded, the viscosity of wort can be effectively reduced, and the filtering efficiency is improved to clarify beer. In addition, the method is beneficial to increasing the concentration of terpene alcohol in the brewing process and increasing the fragrance in the brewing process of white spirit and sake. Thus, the application of the arabinofuranosidase in the food industry shows its great potential.

The arabinofuranosidase AbfaHLB derived from Bacillus badius HLB has the following properties: the optimum pH value is 4.5, the enzyme activity is more than 60% in the range of pH value 2.0-6.0, the pH value stability is good, and after 1 hour of action at 37 ℃, the residual enzyme activity is more than 80% in the range of pH value 2.0-8.0. The activity of the compound has more than 60 percent at the optimum temperature of 50 ℃ and 60 ℃, and the compound has good thermal stability and good stability at 80 ℃. The characteristics of good pH value, heat stability and the like make the compound have great potential in the application of feed and food industries.

Drawings

FIG. 1 shows the optimum pH of the recombinant thermostable acid arabinofuranosidase of the present invention.

FIG. 2 shows the pH stability of the recombinant thermostable acid arabinofuranosidase of the present invention.

FIG. 3 shows the optimum temperature of the recombinant thermostable acid arabinofuranosidase of the present invention.

FIG. 4 shows the thermostability of the recombinant thermostable acid arabinofuranosidase of the present invention.

Detailed Description

Test materials and reagents

1. Bacterial strain and carrier

The pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.

2. Enzymes and other biochemical reagents

Enzymes and other biochemical reagents: the endonuclease was purchased from TaKaRa, and the ligase was purchased from Invitrogen. p-nitrophenyl-a-L-arabinofuranoside (pNPAf) was purchased from Sigma, and others were made available from general Biochemical Agents.

3. Culture medium

(1) Coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH Natural).

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

(3) BMMY medium: except that 0.5% methanol was used instead of glycerol, the other components were the same as BMGY, pH was natural.

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

Example 1 cloning of arabinofuranosidase Gene abfaHLB in Bacillus badius HLB of Bacillus badius

Extracting Bacillus badius HLB genome DNA:

(1) taking 0.5-2mL of culture bacteria liquid, centrifuging at 10000rpm for 30s, sucking supernatant as much as possible, and collecting thalli;

(2) adding 200 mu L of buffer RB into an EP tube for resuspension, centrifuging at 10000rpm for 30s, and discarding the supernatant;

(3) for gram-positive bacteria: adding 120 μ L lysozyme, mixing, and water-bathing at 37 deg.C for 30-60 min;

(4) centrifuging at 12000rpm for 2min, discarding the supernatant, and then shaking or blowing the cells to resuspend in 180 μ L buffer solution RB;

(5) adding 20 μ L RNase A (25mg/mL) solution, shaking, mixing, and standing at room temperature for 5-10 min;

(6) adding 800 μ L of binding solution CB, adding 100 μ L of isopropanol, immediately whirling, oscillating, and mixing completely, wherein flocculent precipitate may appear;

(7) adding the mixture (including possible precipitate) in the previous step into an adsorption column AC, placing the adsorption column in a collecting tube, centrifuging at 13000rpm for 30-60s, and discarding the waste liquid;

(8) adding 500 μ L inhibitor removing solution IR, centrifuging at 12000rpm for 30s, and discarding waste liquid;

(9) adding 700 mul of rinsing liquid WB at 12000rpm, centrifuging for 30s, and discarding waste liquid;

(10) adding 500 mul of rinsing liquid WB at 12000rpm, centrifuging for 30s, and discarding waste liquid;

(11) placing the adsorption column AC back into an empty collection tube, centrifuging at 13000rpm for 2min, removing rinsing liquid as much as possible, and inhibiting downstream reaction by residual ethanol in the rinsing liquid;

(12) taking out the adsorption column AC, placing into a clean centrifuge tube, adding 100 μ L elution buffer EB in the middle part of the adsorption membrane, standing at room temperature for 3-5min, and centrifuging at 12000rpm for 1 min. Adding the obtained solution into a centrifugal adsorption column again, standing at room temperature for 2min, and centrifuging at 12000rpm for 1 min;

(13) the resulting DNA was stored at-20 ℃.

Obtaining a bacillus-derived arabinofuranosidase AbfaHLB gene sequence from an NCBI gene database, carrying out sequence alignment analysis, and designing synthetic primers P1, P2:

P1:5'-ATGTCTATGGATGTAGATCCACGTTTA-3'；

P2:5'-TACATTTACACGTAAACGAATCCAAGA-3'。

carrying out PCR amplification by using Bacillus badius HLB total DNA of the Bacillus badius as a template. The PCR reaction parameters are as follows: denaturation at 94 deg.C for 5 min; then denaturation at 94 ℃ for 30sec, annealing at 45 ℃ for 30sec, extension at 72 ℃ for 2min, 30 cycles, and heat preservation at 72 ℃ for 10min to obtain a fragment with the size of about 1500bp, recovering the fragment, connecting and transforming the fragment with pEASY-T3 vector, and sending the fragment to Beijing Rui Bo Xin scientific biotechnology Limited for sequencing. A1509 bp gene fragment was obtained by gene sequencing, encoding 502 amino acids and a stop codon.

Example 2 preparation of arabinofuranosidase AbfaHLB

Designing a synthetic expression primer according to the obtained gene sequence of the arabinofuranosidase AbfaHLB:

P3:5'-GATGAATTCATGTCTATGGATGTAGATCCACGTTTA-3'；

P4:5'-GCTGCGGCCGCTACATTTACACGTAAACGAATCCAAGA-3'。

and performing PCR amplification by taking the Bacillus badius HLB total DNA of the Bacillus badius as a template to obtain the arabinofuranosidase AbfaHLB gene with the recombinant enzyme cutting site. Carrying out double enzyme digestion (EcoR I + Not I) on the expression vector pPIC9, carrying out double enzyme digestion (EcoR I + Not I) on the gene abFAHLB (EcoR I + Not I) for coding the arabinofuranosidase, carrying out enzyme digestion to obtain a gene fragment for coding mature arabinofuranosidase, connecting the gene fragment with the expression vector pPIC9, obtaining a recombinant plasmid pPIC-abFAHLB containing Bacillus badius HLB arabinofuranosidase gene abFAHLB, and carrying out electric shock transformation on Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS 115/abFAHLB.

The GS115 strain containing the recombinant plasmid was inoculated into 400mL of BMGY culture medium, cultured at 30 ℃ and 250rpm with shaking for 48 hours, and then centrifuged to collect the cells. Then, 200mL of BMMY medium was used for resuspension, and shaking culture was performed at 30 ℃ and 250 rpm. After 72h induction, the supernatant was collected by centrifugation. And (3) measuring the activity of the arabinofuranosidase, wherein the expression amount of the recombinant arabinofuranosidase is 60U/mL.

Example 3 Activity analysis of recombinant arabinofuranosidase AbfaHLB

Measurement of arabinofuranosidase activity: the amount of the product p-nitrophenol formed by enzymatic hydrolysis of the substrate pNPAf was determined at 405 nm. The reaction steps are as follows: 250 μ L of 2mM pNPAf substrate was mixed with 150 μ L of buffer, 100 μ L of appropriately diluted enzyme solution was added, reaction was carried out at 40 ℃ for 10min, and 1.5mL of 1M Na was added₂CO₃The reaction was stopped and the OD was measured at 405 nm.

Example 4 determination of the Properties of recombinant arabinofuranosidase AbfaHLB

1. Determination of optimum pH and pH stability of recombinant arabinofuranosidase AbfaHLB

The recombinant arabinofuranosidase AbfaHLB purified in example 3 was subjected to enzymatic reactions at different pH to determine its optimum pH. The substrate is prepared by 0.1mol/L citric acid-disodium hydrogen phosphate buffer solution with different pH values, and the activity of the arabinofuranosidase is measured at the temperature of 37 ℃. The results (FIG. 1) show that the recombinant arabinofuranosidase has an optimum pH of 4.5 and a relative enzyme activity of 50% or more at pH 2.0-7.0. The recombinant arabinofuranosidase was treated in the above buffers with various pH values at 37 ℃ for 60min, and the enzyme activity was measured at 37 ℃ in a buffer system of pH4.5 to investigate the pH stability of the recombinant enzyme. The results (FIG. 2) show that the recombinant arabinofuranosidase is stable at pH 2.0-8.0, and the residual enzyme activity after 60min treatment in this pH range is above 85%, indicating that the enzyme has wide pH tolerance.

2. Determination of optimum temperature and thermal stability of recombinant arabinofuranosidase AbfaHLB

The optimal temperature of the recombinant arabinofuranosidase AbfaHLB was determined by performing enzymatic reactions in a citrate-disodium hydrogen phosphate buffer (pH4.5) buffer system at various temperatures. The temperature resistance was determined by treating arabinofuranosidase AbfaHLB at different temperatures for different times and then determining the enzyme activity at 37 ℃. The results of the enzyme reaction optimum temperature measurement (figure 3) show that the optimum temperature is 50 ℃, and the enzyme activity is kept higher at 20-65 ℃. The enzyme thermostability test shows (figure 4), the recombinant arabinofuranosidase AbfaHLB has good thermostability, and can keep more than 90% of enzyme activity when being incubated at 70 ℃ for 2h, and can keep more than 80% of enzyme activity when being incubated at 80 ℃ for 10 min.

3. Effect of different chemical Agents on the Activity of the recombinant arabinofuranosidase AbfaHLB

The enzyme activity was measured at 50 ℃ and pH4.5 by adding different metal ions and chemical reagents in different concentrations to the enzymatic reaction system and investigating the effect on the enzyme activity, the final concentration of each substance was 1mmol/L the results showed that the activity of most of the ions and chemical reagents β -mercaptoethanol and EDTA recombinant arabinofuranosidase did not change significantly at a concentration of 1mmol, but Ag +, Hg2+ almost completely inhibited the activity, while SDS strongly inhibited the activity.

4. Antitrypsin and pepsin capacity detection of recombinant arabinofuranosidase AbfaHLB

Pepsin at pH2.0Gly-HCl buffer at a concentration of 0.1mg/mL and trypsin at pH7.0 citric acid-disodium hydrogen phosphate buffer at a concentration of 0.1mg/mL were added. The purified arabinofuranosidase AbfaHLB and protease were mixed in a 10: after treating the protease in the protease buffer solution for 120min at a ratio of 1(w/w), samples were taken at different times and the residual enzyme activity of the treated enzyme was determined by standard methods. The result shows that the residual relative enzyme activity is 96.9 percent after the arabinofuranosidase AbfaHLB is treated by the pepsin for 120min, and 94.7 percent after the arabinofuranosidase AbfaHLB is treated by the trypsin for 120 min. The fact that the AbfaHLB of the arabinofuranosidase has better capability of resisting hydrolysis of pepsin and trypsin is shown.

Claims

1. A high temperature resistant acid arabinofuranosidase AbfaHLB is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. A high temperature resistant acid arabinofuranosidase gene, abfaHLB, encoding the arabinofuranosidase abfaHLB of claim 1.

3. The high temperature resistant acidic arabinofuranosidase gene abfaHLB of claim 2, wherein the base sequence is as shown in SEQ ID No. 2.

4. A recombinant vector comprising the high temperature resistant acid arabinofuranosidase gene abfaHLB of claim 2 or 3.

5. The recombinant vector according to claim 4, wherein the recombinant vector is pPIC 9-abfaHLB.

6. A recombinant strain comprising the high temperature resistant acid arabinofuranosidase gene abfaHLB of claim 2 or 3.

7. A method for preparing high-temperature-resistant acid arabinofuranosidase AbfaHLB is characterized by comprising the following steps:

1) transforming a host cell with the recombinant vector of claim 4 or 5 to obtain a recombinant strain;

2) culturing the recombinant strain, and inducing the expression of the recombinant arabinofuranosidase AbfaHLB;

3) recovering and purifying the expressed arabinofuranosidase AbfaHLB.

8. Use of the high temperature resistant acid arabinofuranosidase AbfaHLB of claim 1 in feed processing, the brewing industry, fruit juice processing, and energy applications.