CN107699551B

CN107699551B - Application of beta-glucosidase SPBGL5 in hydrolysis of xylan polysaccharide substances

Info

Publication number: CN107699551B
Application number: CN201710695489.2A
Authority: CN
Inventors: 庞浩; 唐婷婷; 杜丽琴; 汤宏赤; 黄日波; 林丽华; 郭媛; 严少敏
Original assignee: Guangxi Academy of Sciences
Current assignee: Guangxi Academy of Sciences
Priority date: 2017-08-15
Filing date: 2017-08-15
Publication date: 2021-02-05
Anticipated expiration: 2037-08-15
Also published as: CN107699551A

Abstract

The invention belongs to the technical field of enzymatic engineering, and particularly relates to application of beta-glucosidase SPBGL5 in hydrolysis of xylan polysaccharide substances. The invention constructs a recombinant escherichia coli strain containing a beta-glucosidase gene SPBGL5 from sphingomonas ATCC 31461, the gene is subjected to induction expression and nickel affinity chromatography purification in escherichia coli to obtain an expression product, namely beta-glucosidase SPBGL5, the SPBGL5 has beta-xylosidase activity and beta-arabinosidase activity besides the activity of the beta-glucosidase, can independently hydrolyze beech xylan, birch xylan, oat xylan, corncob xylan, wheat arabinoxylan and lichen polysaccharide, and has great application potential in degrading various xylan polysaccharide substances.

Description

Application of beta-glucosidase SPBGL5 in hydrolysis of xylan polysaccharide substances

Technical Field

The invention belongs to the technical field of enzymatic engineering, and particularly relates to application of beta-glucosidase SPBGL5 in hydrolysis of xylan polysaccharide substances.

Background

Beta-glucosidase (EC 3.2.1.21), the alias cellobiase, gentiobiase and amygdalase, broadly refer to a class of enzymes capable of catalyzing the transfer of sugar groups between oxygen nucleophiles (Singhaia R, Patel A K, Sukumuran R K, et al, role and knowledge of beta-glucosidases in the hydrolosis of cellulose for bio-ethanol production [ J ]. Bioresour technique, 2003,127: 500-.

Beta-glucosidase exists widely in nature, and the structure and catalytic properties of beta-glucosidase from different sources are different. The plant is derived from semen Armeniacae amarum, semen glycines, and Ginseng radix; the animal source includes honey and pig liver (Wangzhijiang, Wei hong Fu. beta-glucosidase research [ J ] feed industry, 2006,27(22):20-22.), and the microorganism source includes yeast, streptomycete, aspergillus, Trichoderma koningii, Clostridium fusiformis, etc.

Beta-glucosidase, a key rate-limiting enzyme in cellulase systems, can hydrolyze cellobiose and cellooligosaccharide to glucose (Lynd L R, Weimer P J, Van Zyl W H, et al. microbial cellulose digestion: saccharides and biotechnology [ J ]. Microbiology and Molecular Biology Reviews,2002,66(66):739.), alleviates the inhibition of cellobiose on other two enzyme system components, thereby improving the hydrolysis rate of the whole cellulase system and playing an important role in degrading fuel ethanol produced by cellulose. Beta-glucosidase can also be used as a flavor enzyme to improve the flavor of fruit juice (lie in, Asahu spring, Douying. Aspergillus niger beta-glucosidase Food flavoring application [ J ]. Food and fermentation industry, 2000,26(2):5-6.), can be reacted with cheap total ginsenosides to convert them into rare ginsenosides CK and Rd (spring, Bohai Hao, King Red bud, et al. recombinant thermophilic beta-glucosidase converts rare ginsenosides Rd and CK, advanced chemical bulletins, 2016,37(2):281 and other.), and can also be reacted with soybean isoflavone to generate soybean isoflavone aglycone which is more easily absorbed by human body (pyro Y H, Lee T C, Lee Y C.Enterprise of biological activity of dietary fibers in soy milk aglycone with beta-glucosidase-dietary promoter [ J.origin ],289, 2005,38(5):551-559.).

Xylan is the main component of plant hemicellulose, is the most abundant polysaccharide in nature except cellulose, exists in almost all parts of various terrestrial plants, and accounts for one third of the total amount of plant carbohydrates. Xylan is a hybrid polymer, the main chain is composed of a plurality of xylopyranosyl groups connected through beta-1, 4-glycosidic bonds, and the side chain is connected with a plurality of different substituents: o-ethylphthalyl, 4-O-methyl-D-glucuronic acid residue, L-arabinose residue and the like. Xylans are generally classified into two types, hardwood xylans and softwood xylans. The hardwood xylan is polymerized by xylose in the county of O-acetyl-4-O-methylglucal, contains more than 70 pyranose xylose residues, is connected by beta-1, 4-glucosidic bonds, has the polymerization degree of 150-200, and has 1 4-O-methylglucoyl group at the C2 position every 10 xylose residues. Beech, birch, and Oat xylans all belong to the class of hardwood xylans (arita Telemann, Maija Tenkanen, Anna Jacobs, et al. Characterication of O-acetyl- (4-O-methylglucurono) xylolan iso fatty from birch and beech [ J ]. Carbohydrate Research,2002,337:373-377.Kay hetrich, Steffen Fischer, Nils Schroder, et al. Derivatification and Characterication of xylolan from Oat Spelts [ mp ] Macromol Symp,2006,232:37-48. softwood.) xylans polymerized from arabino-4-O-methylglucuronyl Xylan residues with a degree of polymerization of 70-130, an average length shorter than that of Xylan, and a branching at position 2C. Wheat Arabic belongs to the family of softwood xylans (Kay Hetrich, Steffen Fischer, Nils Schroder, et al. Derivatification and charaterization of xylolan from Oat varieties [ J ]. Macromol Symp,2006,232: 37-48.).

The Wanghai et al studied the components and structures of corncob xylan and commercial birch xylan by thin layer chromatography, ion chromatography, infrared spectroscopy and dissolution rate. The research result shows that: monosaccharide components in the corncob xylan mainly comprise xylose, arabinose and a small amount of glucose, and mainly comprise straight-chain xylan with few branched chains; monosaccharide components in the alcohol-insoluble corncob xylan mainly comprise xylose, arabinose, a small amount of glucose and galactose, but the arabinose is slightly higher than that of the alcohol-insoluble corncob xylan, and the alcohol-insoluble corncob xylan has straight-chain and branched-chain xylans and more branched chains; birch xylan is mainly xylose, other monosaccharides are not detected, straight-chain and branched-chain xylan exists, and branched-chain xylan exists more (Wanghai, Liriter, Shibo. research on composition and structure of corncob xylan and birch xylan [ J ] food science, 2004,25: 36-42.).

In the pulp bleaching operation, xylan and lignin in unbleached wood pulp form a complex, so that the pulp is difficult to bleach, by adding xylanase and cellulase to act together, the xylan is degraded, so that the bleaching agent is directly contacted with lignin, thereby achieving the purpose of bleaching and reducing the use of environmental pollutants (KULK KARNI N, RAO M.application of xylylase from alkallitic therophilic Bacillus sp.NCIM 59in biobased of basic pulp [ J ]. J Biotechnol,1996,51(2):167-, can improve fermentation efficiency, increase ethanol yield, reduce viscosity of fermentation liquor, and improve beer taste (LI Y, LU J, GU G, et al. students on water-extractable antioxidant degradation and brewing [ J ]. Food Chem,2005,93(1): 33-38.).

Wheat arabinoxylan is mainly present in the cell wall of wheat, is the main polymer of the wheat cell wall, and the structure of which has been identified. It is mainly composed of arabinose and xylose, the main molecular chain is composed of beta-1, 4-D-xylopyranose residue, some substituents are connected with the main molecular chain through O-2 and O-3 atoms, and the main substituent is alpha-D-arabinofuranose. In grains, arabinoxylan is not simply physically embedded in cell walls, but is fixed in the cell walls through ester-like cross-linking, so that most grains are not easy to be water-soluble. The molar ratio of arabinoxylans (A/X) in various plants varies widely, and is mainly influenced by factors such as plant species, species and ecological environment, and due to the variation of A/X, various arabinoxylans exhibit distinct anti-nutritional properties in the gastrointestinal tract of animals. When wheat is used as a feed for poultry, arabinoxylan in the wheat increases the viscosity of digesta and affects the absorption of nutrients, and this adverse effect can be eliminated by adding xylanase to the feed to degrade arabinoxylan (research on anti-nutritional factor-arabinoxylan in wheat such as zukiocai, lintongkang [ J ] feed research, 2006,8:53-55.)

The patent retrieval database of the national intellectual property office is searched by taking the beta-glucosidase as the name of the invention, 192 invention patents appear in total, and 61 repeated contents exist in the patent retrieval database. In the 131 patent inventions, there are 48 patents on beta-glucosidase genes and applications, 11 patents on beta-glucosidase producing strains and applications, 32 patents on beta-glucosidase preparation screening methods and immobilization, 23 patents on beta-glucosidase mutants and applications, 16 patents on beta-glucosidase in terms of salidroside synthesis, preparation of steviol, resveratrol, arctiin, gentiooligosaccharide and the like, and 1 patent on polypeptides for enhancing beta-glucosidase activity at low temperature. In these patents, the application of β -glucosidase is related to many aspects including hydrolysis of cellobiose, degradation of cellulose to produce bioethanol, synthesis of salidroside, etc. Wherein, the beta-glucosidase in the patent application No. CN201010557211.7, a beta-glucosidase and a coding gene and application thereof can hydrolyze cellopolysaccharide, barley glucan, lichenin and laminarin. The fibrous polysaccharide is macromolecular polysaccharide formed by glucose with beta-1, 4-glycosidic bonds; beta-glucan in barley takes beta-glucopyranose as a basic unit, is connected into a plurality of cellotriose and cellotetraose groups through beta-1, 4-glycosidic bonds, and is further connected into a polysaccharide polymer through beta-1, 3-glycosidic bonds, and forms a specific helical structure. Wherein the beta-1, 3-linkage and the beta-1, 4-linkage account for about 30% and 70%, respectively (bacterial A., Stone B.A. isolation and architecture of aldehyde cells from floor and barrel [ J ]. Australian Journal of Plant Physiology,1981,8, 453-); lichenin is mainly D-glucan with a straight-chain structure connected by cellotriose molecules through beta-1, 3 bonds; laminarin is a neutral glucan composed of beta-1, 3-glucan and some beta-1, 6-glycosidic linkages. They all contain only glucosidic bonds and no glucosidic and arabinoside bonds.

However, there is no patent of β -glucosidase which can act on xylan polysaccharides such as beech xylan, birch xylan, oat xylan, corncob xylan and wheat arabinoxylan alone. Therefore, the beta-glucosidase SPBGL5 is novel in property and has application value.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

The invention clones a beta-glucosidase gene SPBGL5 from sphingomonas ATCC 31461, connects the gene with a cloning expression vector pSE380, then transforms the gene into escherichia coli XL1-Blue for induced expression, carries out nickel affinity chromatography purification on target protein, and obtains SPBGL5 protein which can act alone to degrade beech xylan, birch xylan, oat xylan, corncob xylan and wheat araboxylan.

The technical scheme provided by the invention is as follows:

the invention provides application of beta-glucosidase SPBGL5 in decomposition of beech xylan polysaccharide substances.

The invention also provides application of the beta-glucosidase SPBGL5 in decomposing birchwood xylan polysaccharide substances.

The invention also provides application of the beta-glucosidase SPBGL5 in decomposing oat xylan polysaccharide substances.

The invention also provides application of the beta-glucosidase SPBGL5 in decomposing corn cob xylan polysaccharide substances.

The invention also provides application of the beta-glucosidase SPBGL5 in decomposing wheat arabinoxylan polysaccharide substances.

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

(1) the invention constructs a recombinant escherichia coli strain containing a beta-glucosidase gene SPBGL5 from sphingomonas ATCC 31461, and the gene is subjected to induction expression and nickel affinity chromatography purification in escherichia coli to obtain an expression product, namely beta-glucosidase SPBGL 5.

(2) The SPBGL5 provided by the invention has beta-glucosidase activity, beta-xylosidase activity and beta-arabinosidase activity, can be used for independently hydrolyzing beech xylan, birch xylan, oat xylan, corncob xylan, wheat arabinoxylan and lichen polysaccharide, and has great application potential in degrading various xylan polysaccharide substances.

Drawings

FIG. 1 is an SDS-PAGE pattern of a purified product of recombinant β -glucosidase SPBGL 5;

FIG. 2 is a graph of an HPAE-PAD analysis of the hydrolysis of beech xylan by recombinant β -glucosidase SPBGL 5; wherein, a-xylose standard sample; b-control group; c-Experimental group;

FIG. 3 is a graph of an HPAE-PAD analysis of the hydrolysis of birchwood xylan by recombinant β -glucosidase SPBGL 5; wherein, a-xylose standard sample; b-control group; c-Experimental group;

FIG. 4 is a graph of an HPAE-PAD analysis of the hydrolysis of oat xylan by recombinant β -glucosidase SPBGL 5; wherein, a-xylose standard sample; b-control group; c-Experimental group;

FIG. 5 is a graph of an HPAE-PAD analysis of recombinant β -glucosidase SPBGL5 hydrolysis of Corn xylan; wherein, a-xylose standard sample; b-control group; c-Experimental group;

FIG. 6 is a HPAE-PAD analysis of the hydrolysis of wheat arabinoxylan by recombinant β -glucosidase SPBGL 5; wherein, a-L-arabinose standard sample; b-xylose standard sample; c-control group; d-Experimental group.

Detailed Description

The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The materials and reagents used in the following examples are commercially available, unless otherwise specified.

Example 1：

1. Cloning of beta-glucosidase Gene spbgl5

Sphingomonas strain No. ATCC 31461 was purchased from American type culture Collection, and total DNA of the strain was extracted. The nucleotide sequence of the gene encoding β -glucosidase, having the sequence number NZ _ AGFU01000034, located 124633 … 127032 of the genomic sequence of the sphingomonas strain was obtained from the NCBI gene database. The gene is subjected to enzyme cutting site and ORF analysis by using Vector NTI and ORF finder, and the protein coded by the gene is subjected to structural domain analysis and signal peptide prediction by using SMART and SignalP 4.1.

According to the analysis result, signal peptide is removed, selected restriction enzyme cutting sites are adopted, 6 His labels (underlined parts) are added to the C end, 1 pair of PCR primers are designed to amplify target fragments, and the primer sequences are specifically as follows:

5-F：5′-TCCTCATGAAACAGCCTGCCGCAGCGCAGACCGCC-3′；

5-R：5′-CTCAAGCTTTCAATGATGGTGGTGGTGGTGCCTTACAGTCAGCGTAGCACTCTT-3′；

the beta-glucosidase gene spbgl5 was amplified by Polymerase Chain Reaction (PCR), cleaved with restriction enzymes Pag I and Hind III, and after cleaving the beta-glucosidase gene spbgl5, ligated with the Nco I and Hind III cleaved expression vector pSE 380. The ligation products were transformed into E.coli XL1-Blue, spread on LB plates (containing 1.5% (w/v) agar powder and 100. mu.g/mL ampicillin), and cultured by inversion at 37 ℃ for 14 to 16 hours. Single colonies on the transformation plates were spotted in parallel onto an esculin selection plate containing 100. mu.g/mL ampicillin (obtained by adding 0.5% ferric ammonium citrate, 0.2% (w/v) esculin and 1.5% (w/v) agar powder on the basis of LB medium) and an ordinary LB backup plate containing 1.5% (w/v) agar powder and 100. mu.g/mL ampicillin, and cultured for 12 to 14 hours at 37 ℃ in an inverted manner. After the single colonies grow out, 1 mu L of IPTG solution diluted by LB to 1% (w/v) is dripped into each single colony on the selection plate, inversion culture is continued, induction is carried out for 6-8 hours at 37 ℃, cells are fumigated by chloroform for 15min, color development is carried out for 1-2 hours at 37 ℃, and the phenomenon on the selection plate is observed.

Plasmid DNA of a clone producing a black circle on the selection plate was extracted and designated pSE-spbgl5, and the plasmid DNA was double digested with Pst I and Hind III, and two bands were detected by gel electrophoresis, a linear vector band of about 4.3kb and a target gene band of 2.2 kb.

2. Induced expression, purification and SDS-PAGE analysis of recombinant beta-glucosidase SPBGL5

(1) Induced expression of the protein SPBGL 5: streaking and activating strains, picking single strains from a plate, placing the single strains in a 5mL LB medium (containing 100 mug/mL ampicillin) finger-shaped bottle, and culturing at 37 ℃ and 200rpm for 12-14 hours, wherein the single strains are first-grade seeds; the first-stage seeds were measured at 1% inoculum size and inoculated into a100 mL Erlenmeyer flask containing 30mL LB medium (containing 100. mu.g/mL ampicillin), and cultured overnight at 37 ℃ and 200rpm, which were the second-stage seeds; the secondary seeds were inoculated into 5 flasks of 200mL LB medium (containing 100. mu.g/mL ampicillin) in 500mL Erlenmeyer flasks at 2% inoculum size, and cultured at 37 ℃ and 200rpm to OD₆₀₀When the concentration reached 0.4 to 0.6, IPTG was added to a final concentration of 0.5mmol/L and ampicillin was added to a final concentration of 100. mu.g/mL, and the mixture was induced at 30 ℃ and 200rpm for 8 hours.

(2) Purification of the protein SPBGL 5: the cells were collected by centrifugation and washed with 7mL lysine buffer (50mmol/L NaH)₂PO₄300mmol/L NaCl, 10mmol/L imidazole, pH8.0), adding 1mL of 10mg/mL lysozyme, and standing on ice for 15 minutes. Then, the cells were disrupted by ultrasonic waves for 18 minutes, and after completion, the cells were centrifuged at 12000rpm for 25 minutes at 4 ℃ to obtain a supernatant as a crude enzyme solution.

The crude enzyme solution was mixed with the filler resuspended in 1mL lysine buffer and placed in a finger bottle and allowed to act on a destaining shaker for 1 hour in ice to allow the His-tagged target protein to bind well to the Ni-NTA filler. With 1mL of precooled Wash buffer (50mmol/L NaH)₂PO₄300mmol/L NaCl, 20mmol/L imidazole, pH8.0) is resuspended and washed 5-7 times to remove heteroproteins, and 500. mu.L of precooled Elution buffer (50mmol/L NaH) is added₂PO₄300mmol/L NaCl, 250mmol/L imidazole, pH8.0) were washed 8 times and the eluted protein was collected using a pre-cooled EP tube.

Protein desalting: the desalting column was washed 3 to 5 times with 3mL of sterile deionized water, followed by 3 times with 3mL of a displacement buffer (pH 7.0McIlvaine buffer), 500. mu.L of the protein eluted with the Elution buffer was slowly eluted, 1mL of the displacement buffer (pH 7.0McIlvaine buffer) was then used to elute the target protein, and the desalted target protein was collected using a precooled EP tube. In this study, the purified protein was desalted using PD MiniTrap G-25column purchased from GE.

(3) SDS-PAGE analysis: and (3) taking the desalted protein to perform polyacrylamide gel electrophoresis analysis, and finding a protein band with a target size.

As can be seen from FIG. 1, the molecular weight of the purified product of the recombinant β -glucosidase SPBGL5 is about 87 kDa.

3. Determination of optimum temperature and optimum pH of recombinant beta-glucosidase SPBGL5

(1) Determination of optimum pH: the influence of different pH (pH 4.5-8.0McIlvaine buffer solution) on the enzyme activity is measured by taking 2 mmol/L4-nitrophenyl-beta-D glucopyranoside (pNPG) as a substrate at 37 ℃. Calculating the relative activity of the enzyme under each pH value by taking the highest activity as 100 percent, wherein the corresponding pH value with the highest activity is the optimal reaction pH value of the enzyme.

The optimum reaction pH of SPBGL5 was found to be 5.5 when pNPG was used as substrate.

(2) Measurement of optimum temperature: the influence of different temperatures (25-55 ℃) on the enzyme activity is measured under the condition of the optimum reaction pH by taking 2mmol/L pNPG as a substrate. Calculating the relative activity of the enzyme at each temperature by taking the highest activity as 100 percent, wherein the corresponding temperature with the highest activity is the optimal reaction temperature of the enzyme.

The optimum reaction temperature of SPBGL5 was determined to be 40 ℃ when pNPG was used as a substrate.

4. Determination of artificial substrate activity of recombinant beta-glucosidase SPBGL5

Taking 2mmol/L of 4-nitrophenyl-beta-D-xyloside (pNP-Xyl) and 4-nitrophenyl-beta-D-arabinoside (pNPA). The reaction is carried out for 20 minutes under the optimal reaction condition, and the relative activity of SPBGL5 in hydrolyzing pNP-Xyl and pNPA is calculated by the product yield and the activity of hydrolyzing pNPG is 100%.

The relative activities of SPBGL5 to hydrolyze pNPG, pNP-Xyl and pNPA were found to be 100%, 218.35% and 498.97%, respectively, as calculated.

5. Determination of hydrolysis of beech xylan, birch xylan, oat xylan, corncob xylan and wheat arabinoxylan by recombinant beta-glucosidase SPBGL5

Beech xylan, birch xylan, oat xylan, corncob xylan and wheat arabinoxylan at a final concentration of 0.5% (w/v) were taken to react with purified recombinant enzyme solution SPBGL5 diluted appropriately at ph5.5 and 40 ℃ for 6 hours, boiling was performed to terminate the enzyme reaction, and the reaction product was detected by High-Performance Anion-Exchange chromatography (HPAE-PAD).

HPAE-PAD operates specifically as follows:

the instrument comprises the following steps: dionex AS-AP, Dionex ICS-5000EG5, Dionex ICS-5000DC5,

Dionex ICS-5000SP5

A chromatographic column: CarboPac PA100Analytical Column (4X 250mm)

CarboPac PA100Guard Column(4×50mm)

Detection conditions are as follows: the column temperature was room temperature and the flow rate was 1 mL/min.

Mobile phase A: 18.2 omega of ultrapure water is added,

mobile phase B: 0.5mol/L of NaOH is added,

mobile phase C: 0.5mol/L NaOAC and 0.08mol/L NaOH.

Gradient elution procedures are as follows (Mandellia F, Brenellia L B, Almeida R F, et al. Simultaneous production of xylooligosaccharides and antioxidant compositions from sugar organic basic virus hydrolytics [ J ]. Industrial hoops and Products,2014,52(1): 770-775.):

as shown in fig. 2-6, the β -glucosidase SPBGL5 can act on four xylan macromolecules, beech xylan, birch xylan, oat xylan and corncob xylan, alone to hydrolyze to form xylose; while acting on wheat arabinoxylan, xylose and arabinose are produced.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims

1. Application of beta-glucosidase SPBGL5 in decomposing beech xylan, birch xylan, oat xylan, corncob xylan, and wheat araboxylan polysaccharide substances; the beta-glucosidase SPBGL5 obtains the nucleotide sequence of the gene coding the beta-glucosidase from the NCBI gene database, the gene sequence number is NZ _ AGFU01000034, and the gene sequence number is 124633 … 127032 of the genome sequence of the Sphingomonas strain.