CN103146725B - Thermally-stabilized beta-glucosidase gene and its application - Google Patents
Thermally-stabilized beta-glucosidase gene and its application Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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Abstract
The invention discloses a thermally-stabilized beta-glucosidase gene S-bgl6 and its application. The nucleotide sequence of the gene is represented by SEQ ID NO:1, the amino acid sequence of beta-glucosidase S-bgl6 coded by the gene is represented by SEQ ID NO:2, and 75% of the enzymatic activity of the beta-glucosidase gene S-bgl6 coded by the gene is still reserved after the beta-glucosidase gene S-bgl6 is positioned in an environment having a temperature of 50DEG C for 72h. The beta-glucosidase gene S-bgl6 can be used in the decomposition of cellobiose.
Description
Technical field
The present invention relates to a thermally-stabilised beta-glucosidase gene and application thereof, the albumen of this genes encoding can be used for degradation of fibers disaccharides.
Background technology
Beta-glucosidase claims again β-D-Glucose glycosides glucose hydrolysis enzyme, and it can be hydrolyzed the β-D-Glucose glycosidic bond that is incorporated into end irreducibility, discharges D-Glucose and corresponding aglucon simultaneously.1837, Liebig and Wohler found this enzyme first in Semen Armeniacae Amarum.Research is afterwards found, beta-glucosidase is present in the mould and bacterial body of the many plants of nature, insect, yeast, aspergillus, wood widely, it participates in the carbohydrate metabolism of organism, to maintaining organism normal physiological function (the Ramakrishnan Anish that plays an important role, Mohammad Safikur Rahman, and Mala Rao, 2007, Application of cellulase from an alkalothermophilic Thermomonospora sp.in biopolishing of denims, Biotechnol.Bioeng., 96 (1): 48-56.).
Along with increasingly sharpening of fossil energy crisis, substituting fossil energy with recyclability biomass energy is effective way (Nie Heng, the Zhu Jing that solves current energy shortage situation; Duan Xu, etc., 2011; fiber substance is produced the progress of ethanol, 38(3): 28-34.).Lignocellulose is recyclability material (Tang Kaiyu, Zhang Quan, the Tong Mingyou of occurring in nature quantity maximum, 2009, the progress of beta-glucosidase fermentation technique, 17(3): 65-70.), the key of lignocellulose recovery energy is that Mierocrystalline cellulose can efficient degradation be fermentability monose.Enzymic degradation Mierocrystalline cellulose has the incomparable advantage of physico-chemical process--and mild condition, efficiency harmful chemical high, the follow-up fermentation of unrestraint produce (Sassner P., Martensson C.G., Galbe M., et al, 2008, Steam pretreatment of H
2sO
4-impregnated salix for the production of bioethanol, Bioresource Technology, 99 (1): 137-145.).In cellulosic enzymic degradation, need to be by comprising beta-glucan glycosides enzyme, prozyme synergy (the Ma Yinghui of 3 kinds of lytic enzyme compositions of cellobiohydrolase and beta-glucosidase, Wang Lianjie, 2009, the pretreated latest Progress of stalk, Cellulose Science and technology, 17(3): 71-78.), but in current used cellulose degradation prozyme, ubiquity beta-glucoside enzyme activity is low, the defect of poor heat stability, cause cellobiose accumulation, thereby suppress the activity of other 2 enzymes, greatly affect enzymolysis efficiency (Garcia-Kirchenr O., Seguar-Granados M., Rodriguez-Pascual P., 2005, Effect of media composition and growth conditions on production of β-glucosidase by Aspergillus niger C-6, Applied Biochemistry and Biotechnology, 121(1/3): 347-359.).Therefore the further investigation of beta-glucosidase being subject to people more and more pays close attention to.
Except important role aspect degraded cellulose, beta-glucosidase is also widely used at other field, particularly in medicine, food, chemical industry, bio-transformation, there is important using value (Michael E.H., Mark F.R., Charles E., 1999, Cellulase for commodity products from cellulosic biomass.Curr.Opin.Biotechnol., 10 (4): 358 ~ 364.).In medical science, beta-glucosidase be applied in some cancer diagnosis and treatment in, and obtained many successes (Shao Jinhui, Han Jinxiang, etc., 2005, beta-glucosidase is cured the application in field workers and peasants. the chemistry of life, 5(01): 22-24.).In juice production, beta-glucosidase can be by hydrolysis the β-D-glycosidic link in fruit juice discharge terpenoid substance and strengthen the fragrance of fruit juice; Can be hydrolyzed color (the LETRAON-MASSON M.P. of the glycosyl removal fruit juice on anthocyanogen simultaneously, PELLERIN P., 1998, Purification and characterization of two β-D-glucosidases from an Aspergillusnigerenzyme preparation:affinity and specificity toward glucosylated compounds characteristic of the processing of fruits, Enzyme Microb.Tech., 22 (5): 374-382.).In wine brewing, beta-glucosidase is one of important enzyme material, not only can catalysis produce all kinds of terpenoid substances, and can be hydrolyzed the various aglycons with aromatic odour of various glucosides class materials generation, these materials have determined a kind of characteristic (Gonzalez-Pombo P. of wine jointly, Farinal L., Carrauf F., et al, 2011, A novel extracellular β-glucosidase from Issatchekia terricola:Isolation, immobilization and application for aroma enhancement of white Muscat wine, Process Biochem., 46 (1): 385-389.).Many flavor precursors and pharmacological component mainly exist with glucosides form, need this food flavor enzyme of beta-glucosidase to be discharged volatility aglycone, play flavouring effect (Jin Fengxie, village's fine jade, the red sudden strain of a muscle of fish, etc., 2009, special herbal medicine glycoside glycosides enzyme microorganism and fermentation and enzymatic property. biotechnology journal, 25 (12): 1863-1870.).In addition, beta-glucosidase also has the activity that shifts glucosyl group, can be used for production (the Yongling Qin of oligomeric dragon gallbladder sugar, Yunkai Zhang, Haiyan He, et al, 2011, Screening and identification of a fungal β-glucosidase and the enzymatic synthesis of gentiooligosaccharide.Appl Biochem Biotechnol.163 (8): 1012-9.), synthetic (the Wang Mengliang of enzyme process of rhodioside, Li Wanli .2009, immobilized β-glucosidase catalyzes and synthesizes the research of rhodioloside. biotechnology, 19 (1): 68-70.) and the synthetic (Zhu Yun of nonionogenic tenside alkyl glycoside, 2007, glucose enzyme process biosynthesizing alkyl glycoside technique. Zhejiang chemical industry, 38 (11): 3-6.).
The beta-glucosidase gene of at present existing up to a hundred microorganisms, plant and animal-origins is cloned and is sequenced, wherein many microbe-derived beta-glucosidase genes have obtained heterogenous expression (Korea Spro have laughed at, Chen Jienan, Wang Yiqiang, Deng, 2008, the cloning and expression progress of beta-glucosidase gene.Biotechnology circular, 3:8 ~ 13.).By contrast, the activity of beta-glucosidase of plant origin is low more than microbe-derived beta-glucosidase, therefore, research at present mainly concentrates on microbe-derived upper (Shi Cairui, Wang Yiqiang, Chen Jienan, Deng, 2011, produce beta-glucosidase Microbial Breeding progress, biotechnology communication, 3:59-65.).And in microorganism, study more be that yeast and filamentous fungus are as (Meng Xianwen such as the bacillus in the moulds such as Trichoderma (Trichoderma), Aspergillus (Aspergillus) and bacterium, Song little Hong, Chen Lijun, Deng, 2009, the progress of beta-glucosidase. diary processing, 10:42-44.).But the cellulase activity that has multiple mycotoxins in Trichoderma tunning and obtain is lower, and especially beta-glucoside enzyme activity is very low, cause cellobiose to accumulate in reaction system and affect enzymolysis efficiency, thereby its range of application is restricted.
Taking beta-glucosidase as keyword lookup State Intellectual Property Office patent retrieval database, there are altogether 163 patents of invention.Wherein have gene and application thereof that 30 patents of invention are description encoding beta-glucosidases, and other 133 is relevant with the application of beta-glucosidase on various fields.There are 8 from the grand genomic library of various not culturing micro-organisms at these 30 to the gene of encoding beta-glucosidase and the beta-glucosidase gene of applying in relevant patent of invention thereof, other 22 is different biological from 20 kinds, there is thermophilic deamination genus bacillus, Trichodermareesei, Agrobacterium tumefaciems, Saksenaea vasiformis XH8, Pyrococcus furiosus, thermophile bacteria, aspergillus niger, Duan Shi wood is mould, aspergillus niger, blackwing subterranean, Clostridium sp.WGC702, Aspergillus strain, thermophilic Paecilomyces varioti, Dictyoglomus thermophilum DSM3960, chaetomium thermophilum, yeast saccharomyces cerevisiae, Extreme basophilic bacteria, thermophilic Paecilomyces varioti, Ke Shi endospore bacillus and new sphingomonas bacteria, 1 from trans-resveratrol.The beta-glucosidase that the present invention obtains is from the streptomycete GX6(Streptomyces sp.GX6 screening voluntarily), lower with the amino acid whose homology of disclosed beta-glucosidase in gene database, be a new beta-glucosidase.
Summary of the invention
The genome of the streptomycete GX6 that the present invention screens from Nanning, be cloned into a thermally-stabilised beta-glucosidase gene, in e. coli host cell, express this genes produce beta-glucosidase, can degrade taking cellobiose as raw material and generate glucose, and S-bgl6 also remains with 75% enzyme activity for 72 hours 50 DEG C of insulations.
Thermally-stabilised beta-glucosidase gene S-bgl6 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:1.To clone and obtain the genome of streptomycete GX6 building from laboratory.The sequence of this beta-glucosidase gene S-bgl6 is by 2319 based compositions, open reading frame (the Open Reading Frame that contains complete beta-glucosidase gene S-bgl6, ORF), the initiator codon of S-bgl6 gene is ATG, and terminator codon is TGA.
The protein of SEQ ID NO:2 is the beta-glucosidase product S-bgl6 of gene S-bgl6 coding, formed by 772 amino acid, with S-bgl6 catalysis territory homology the highest be Streptomyces ambofaciens ATCC23877(Streptomyces ambofaciens ATCC23877) putative beta-glucosidase(supposition beta-glucosidase) both consistence=538/750 (72%), similarity=599/750 (79%).
Gene S-bgl6 can decompose cellobiose at the recombinant products S-bgl6 of expression in escherichia coli.
The invention still further relates to the expression vector that contains gene of the present invention, and for transforming the host of gene of the present invention.
The coded beta-glucosidase of thermally-stabilised beta-glucosidase gene S-bgl6 of the present invention also retains 75% enzyme activity in 72 hours 50 DEG C of insulations, and has the purposes of decomposing cellobiose.
Brief description of the drawings
Fig. 1 is that the Vitamin C2 that screening contains beta-glucosidase gene S-bgl6 recombinant bacterial strain is selected lithograph.
Fig. 2 is the SDS-PAGE figure of beta-glucosidase S-bgl6 purified.
Fig. 3 is the HPLC figure of beta-glucosidase S-bgl6 hydrolysis fiber disaccharides.
Recognize from Fig. 1, the recombinant bacterial strain that contains beta-glucosidase gene S-bgl6 can screen and on flat board, form black circle at Vitamin C2.
Recognize from Fig. 2, the molecular weight of beta-glucosidase S-bgl6 purified is 82.5kDa.
A in Fig. 3 and B are respectively the standard specimens of glucose and cellobiose.
Recognize from the C of Fig. 3, beta-glucosidase S-bgl6 can be hydrolyzed into glucose by cellobiose.
Embodiment
Following implementation method is in order better to explain the present invention, and the object should not be construed as limiting the invention.
Material used comprises in an embodiment of the present invention: intestinal bacteria (Escherichia coli) strain XL1-blue is purchased from Dalian TaKaRa company; Expression vector pSE380 is purchased from Stratagene company, and the reagent such as restriction enzyme, modifying enzyme are purchased from TaKaRa, MBI.
Below will by embodiment, the present invention is described in detail:
1) extraction of streptomycete Streptomyces sp.GX6 genomic dna
The genomic dna of streptomycete Streptomyces sp.GX6 is the bacterial genomes DNA extraction test kit Biospin Bacteria Genomic DNA Extraction Kit(catalog number (Cat.No.) BSC12S1 according to BioFlux company) operation instruction extract.
Send Hua Da genome company to measure genome sequence the genomic dna of the streptomycete Streptomyces sp.GX6 having extracted.
2) analysis of encoding beta-glucosidase gene order in the genome sequence of streptomycete Streptomyces sp.GX6
By NCBI(National Center for Biotechnology Information for the genome sequence of the streptomycete Streptomyces sp.GX6 obtaining, http://www.ncbi.nlm.nih.gov) on software ORF finder (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi) and Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analyze.Result obtains altogether 8 and has the open reading frame (Open Reading Frame, ORF) of higher homology with beta-glucosidase, and the present invention only relates to one of them ORF.
3) nucleotide sequence analysis of beta-glucosidase gene S-bgl6
With NCBI(National Center for Biotechnology
Information, http://www.ncbi.nlm.nih.gov) on software ORF finder
(http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi) and
Blast (
http:// blast.ncbi.nlm.nih.gov/Blast.cgi)dNA sequence dna is analyzed.The open reading frame (Open Reading Frame, ORF) of gene S-bgl6 is made up of 2319 Nucleotide, and sequence is as shown in SEQID NO:1.Wherein, the initiator codon of S-bgl6 gene is ATG, and terminator codon is TGA.
4) amino acid sequence analysis of the product S-bgl6 of beta-glucosidase gene S-bgl6 coding
One of beta-glucosidase gene S-bgl6 coding is containing 772 amino acid whose protein, with the theoretical molecular size of this protein of DNAStar software prediction be 82494.18 dalton.
With simple assemblies structural research instrument (Simple Modular Architecture Research Tool, SMART, http://smart.embl-heidelberg.de) analyze the unit construction of beta-glucosidase S-bgl6, result is to be family's 3 glycosyl hydrolases (glycosyl hydrolase) functional domains from 30-252 position and the 321-539 amino acids of N end.
5) clone of beta-glucosidase gene S-bgl6 and expression
Use upstream primer 5 '-CACTCATGATGCACCACCACCACCACCACACCGCGACCGAAGAAGACGCCG-3 ' and downstream primer 5 '-CATAAGCTTTCAGCTCACCTGCCGACCGCTC-3 ', by polymerase chain reaction (PCR) amplification beta-glucosidase gene S-bgl6, cut after beta-glucosidase gene S-bgl6 with restriction enzyme Pag I and Hind III enzyme, be connected with the expression vector pSE380 cutting through Nco I and Hind III enzyme.Again connection product is transformed in E.colistrain XL1 blue by CaCl2 method, is applied to containing on the LA flat board of 100 μ g/mL penbritins.Single bacterium colony point plate that conversion obtains sieves substratum again to the LA that contains 100 μ g/mL penbritins, Vitamin C2, ferric ammonium citrate, and (every 100mL sieves again substratum and contains: Vitamin C2: 0.2g, ferric ammonium citrate: 0.5g, peptone: 1g, yeast powder: 0.5g, NaCl:0.5g, agar powder: 1.5g) on flat board, flat board is placed in to 37 DEG C of thermostat containers to be cultivated 5 hours, each turning point bacterium colony is dripped to 1 μ L LB substratum one by one and be diluted to 1% IPTG abduction delivering, then, flat board being placed in to 37 DEG C of thermostat containers continues to cultivate 6 hours.After time arrives, carry out the stifling broken born of the same parents of chloroform, then flat board is placed in to 37 DEG C of thermostat containers the S-bgl6 enzyme of expressing is reacted 4 hours with Vitamin C2 and ferric ammonium citrate.Observe screening dull and stereotyped.
Then be further extracted in the dull and stereotyped plasmid DNA that can form the clone of black circle of screening, and by its called after pSE-S-bgl6, with after restriction enzyme NcoI and HindIII complete degestion pSE-S-bgl6, carry out 0.8% agarose gel electrophoresis analysis, outside the DNA fragmentation of result pSE-S-bgl6 apart from an about 4.5kb, also have a size to be about the DNA fragmentation of 1.9kb.
The recombination bacillus coli XL1-blue inoculation that contains plasmid pSE-S-bgl6 is contained in the LB substratum of 100 μ g/mL penbritins to 600mL, and 37 DEG C of shaking culture, treat OD
600be 0.4 o'clock, adding IPTG to make its final concentration is 0.5mmol/L, and 30 DEG C, 220 turn induction 10 hours.The centrifugal 3min of 11000rpm, collects thalline, with the resuspended thalline of phosphoric acid buffer of 4mL pH7.0100mmol/L, and the broken born of the same parents of ultrasonic wave 9 minutes.The centrifugal 20min of 12000rpm, gets supernatant and carries out protein purification below.Add the nickel affinity chromatography colloid of 1mL50% by every 4mL supernatant liquor, turn and shake 60 minutes with 200 at 4 DEG C, mixture is filled into pillar, collect effluent.Add 1mL dcq buffer liquid (50mmol/L NaH
2pO
4, 300mmol/L NaCl, 20mmol/L imidazoles, pH8.0) in pillar, slowly stir, collect effluent.Repeat rinse step 4 times.Add elution buffer (50mmol/LNaH
2pO
4, 300mmol/LNaCl, 250mmol/L imidazoles, pH8.0) and elute protein.Collect the protein soln of wash-out, with polyacrylamide gel electrophoresis (SDS-PAGE) checking of sex change, find that there is the protein band of object size.
6) mensuration of beta-glucosidase S-bgl6 hydrolysis fiber disaccharides
Beta-glucosidase S-bgl6 taking cellobiose as substrate, act on 30 minutes at the Sodium phosphate dibasic phosphate sodium dihydrogen buffer solution of pH6.5 and 50 DEG C.After reaction times arrives, in 10 minutes termination reactions of 100 DEG C of heating.The high performance liquid chromatography for product (HPLC) of S-bgl6 hydrolysis fiber disaccharides detects.
The result that HPLC detects shows: S-bgl6 can be hydrolyzed into glucose by cellobiose.
HPLC condition: instrument: Agilent1100 chromatographic instrument; Chromatographic column: nh 2 column; Moving phase: acetonitrile: water (70:30); Flow velocity: 1.0mL/min; Detector: RID(refraction detector).
7) mensuration of beta-glucosidase S-bgl6 thermostability
It is to be incubated 72 hours in the water-bath of 50 DEG C that the S-bgl6 of purifying is placed on to temperature.The S-bgl6 enzyme liquid that sampled, took out respectively every 3 hours for latter 24 hours 10 μ L for 48 hours every 6 hours starting.Then under pH6.5 and 50 DEG C of conditions, measure the enzyme of S-bgl6 taking p-nitrophenyl-β-D-glucopyranoside (pNPG) as substrate and live, wherein live in contrast to be kept at the enzyme that the S-bgl6 of 4 DEG C measures under the same conditions.
Found that beta-glucosidase S-bgl6 still remains with 75% enzyme activity 50 DEG C of insulations after 72 hours.
Claims (4)
1. a thermally-stabilised beta-glucosidase gene
s-bgl6, it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:1.
2. according to the protein of the coded by said gene of claim 1, its aminoacid sequence is as shown in SEQ ID NO:2.
3. an expression vector, is characterized in that, it contains gene claimed in claim 1.
4. a host cell, is characterized in that, it is prokaryotic cell prokaryocyte or the eukaryotic cell that expression vector transforms described in claim 3.
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| CN102149819A (en) * | 2008-09-12 | 2011-08-10 | Ifp新能源公司 | Beta-glucosidase variants having improved activity, and uses thereof |
| WO2012044915A2 (en) * | 2010-10-01 | 2012-04-05 | Novozymes, Inc. | Beta-glucosidase variants and polynucleotides encoding same |
| CN102719458A (en) * | 2012-03-15 | 2012-10-10 | 广西大学 | Gene encoding alkaline beta-glucosidase and application thereof |
| CN102791853A (en) * | 2009-09-23 | 2012-11-21 | 丹尼斯科美国公司 | Novel glycosyl hydrolase enzymes and uses thereof |
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| CN102149819A (en) * | 2008-09-12 | 2011-08-10 | Ifp新能源公司 | Beta-glucosidase variants having improved activity, and uses thereof |
| CN102791853A (en) * | 2009-09-23 | 2012-11-21 | 丹尼斯科美国公司 | Novel glycosyl hydrolase enzymes and uses thereof |
| WO2012044915A2 (en) * | 2010-10-01 | 2012-04-05 | Novozymes, Inc. | Beta-glucosidase variants and polynucleotides encoding same |
| CN102719458A (en) * | 2012-03-15 | 2012-10-10 | 广西大学 | Gene encoding alkaline beta-glucosidase and application thereof |
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