CN103602647A - Beta-xylosidase mutant and use thereof - Google Patents
Beta-xylosidase mutant and use thereof Download PDFInfo
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- CN103602647A CN103602647A CN201310641295.6A CN201310641295A CN103602647A CN 103602647 A CN103602647 A CN 103602647A CN 201310641295 A CN201310641295 A CN 201310641295A CN 103602647 A CN103602647 A CN 103602647A
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- xylobiase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)
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Abstract
The invention discloses a beta-glucosidase mutant and a use thereof. The beta-glucosidase mutant has an amino acid sequence as shown in SEQ ID NO:1. The mutant enzyme has high xylose and glucose tolerance compared with non-mutated beta-glucosidase, and has wide use in degradation of xylo-oligosaccharide.
Description
Technical field
The invention belongs to zymetology field, relate to a kind of xylobiase mutant, also relate to this mutant in the purposes in degraded wood oligose.
Background technology
Xylan is the chief component of hemicellulose, accounts for the 30-35% of hemicellulose gross dry weight, is distributed widely in the cell walls of higher plant, is the huge renewable resources of occurring in nature one class storage, is also a class hemicellulose that is easier to extraction, degraded and utilizes.Zytase is the prozyme system that a class can hydrolyzed xylan generates wood sugar, mainly by endo-xylanase (EC.3.2.1.8) and xylobiase (EC3.2.1.37), form (Beg Q.K., Kapoor M., Mahajan L., et al.2001, Microbial xylanase and their industrial applications:A review.Appl.Microbiol Biotechnol., 56:326-338.).
Xylobiase (β-D-xylosidase) is a kind of circumscribed lytic enzyme, it and endo-xylanase act synergistically, the degraded product xylo-oligosaccharide of degraded endo-xylanase, from non-reducing end, discharge wood sugar (Rasmussem L.E., Sorensen H.R., Vind J., et al.Mode of action and properties of the β-xylosidase from Talaromyces emersonii and Trichoderma reesei.2006, Biotechnology and Bioengineering, 94 (5): 869-876.).In xylan hydrolysis process, xylobiase can reduce the polymerization degree of wood sugar hydrolysate, remove largely the inhibition of product to zytase, rate-limiting enzyme (Jiang Zhengqiang in xylan hydrolysis process, Yang Shaoqing, Zhu Huifang etc., the character of thermophilic Paecilomyces varioti xylosidase and with the synergy of zytase, 2008, food and fermentation industries, 34 (7): 12-16.)。
The wide material sources of xylobiase, now separated obtaining from the microorganisms such as bacterium, actinomycetes and fungi and part higher plant.The research that xylobiase is produced in microorganism fermentation at present mainly concentrates on abroad, the xylobiase of reporting is generally intracellular enzyme (Polizeli MLTM, Rizzatti ACS, Monti R, et al.Xylanase from fungi:properties and industrial application.2005, Applied Microbiology and Biotechnology, 67 (5): 577-591.), minority fungi also can produce the outer xylobiase of born of the same parents, the bacterial strain of report has both at home and abroad: Aspergillus sydowii, Talaromyces thermophilus (Guerfali M., Gargouri A., Belghith H, Talaromyces thermophilus β-D-xylosidase:purification, characterization and xylobiose synthesis, 2008, Biotechnoloty and Applied Biochemistry, 150 (3): 267-279.), Paecilomyces thermophila (Wang Lan, Jiang Zhengqiang, Yang Shaoqing, thermophilic Paecilomyces varioti produces the optimization of the outer xylosidase fermentation condition of born of the same parents, 2007, 34 (3): 519-523.), aspergillus niger and Trichodermareesei (, in generation Yuan etc., the separation and purification of Trichodermareesei xylobiase, enzyme are character and Hydrolytic Mechanism research for Jiang little Hua, Zhu Junjun, 2013, biomass chemical engineering, 47 (1): 27-32.), but its yield of enzyme is low and have problems such as purge process complexity.Therefore the genetic engineering bacterium that, utilizes molecular biology method to obtain high yield xylobiase is a feasible important channel.With respect to external Er Yan, China about to the research of the recombinant bacterial strain of xylobiase or fewer.Xue Yemin etc. from Thermoanaerbacter ethanolicusJW200, clone and at expression in escherichia coli the xylobiase (Xue Yemin of a high heat stability.Lu Chen.Mao Zhonggui etc.The stability of the enzyme of the clone of arabinofuranosidase/xylosidase gene, expression and expression product.2003。China Agricultural University's journal.8(5):9-13。)。Wu Ailian carries out rite-directed mutagenesis to the xylobiase from Thermoanaerobacterium sp.JW/SL YS485 and makes than the product enzyme level of wild bacterial strain, to have improved 55 times of (Wu Ailian containing the intestinal bacteria of mutant, the directional transformation of Glycosylase in thermophilc anaerobe, 2007, Nanjing: Nanjing Normal University).Beam is gorgeous wait people at E. coli the xylobiase gene of Bacillus halodurans C-125.(beam is gorgeous, Li Xingyu, Mao Zichao etc., expression and the enzyme CHARACTERISTICS IDENTIFICATION of Alkaliphilic bacillus C-125 xylosidase gene, 2009,25 (9): 1386-1393 to make its stability improve and increase tolerance to wood sugar.)。
At present, xylobiase is with a wide range of applications in many-sides such as the energy, papermaking, medicine.Aspect energy industry, utilize the synergy of zytase and xylobiase to generate wood sugar by efficient degradation xylan, the wood sugar producing can be continued to be converted into large industrial chemicals (as ethanol, lactic acid etc.) (Katahira S., Fujita Y., Mizuike H., et al, Construction of a xylan-fermentating yeast strain through codisplay of xylanolytic enzyme of the surface of xylose utilizing Saccharomyces cerevisiae cells.2004, Appl.Environ.Microbiol., 70 (9): 5407-5414.).In paper industry, xylobiase can be used as bio-bleaching agent, xylobiase carries out to Straw Pulp whiteness (the hair Lianshan Mountain that pre-treatment can obviously improve bleached pulp separately or with zytase synergy, You Jixue, Song Xiangyang etc., endo-xylanase and the impact of xylosidase pre-treatment on Wheat Straw Pulp Bleaching performance, 2003, chemistry of forest product and industry, 23 (2): 7-11.)。In medicine industry, xylobiase can be hydrolyzed removes taxol (Chen Tianjiao, the Zhu Ping that xylose residues manufacture has important physiological function, the catalysis characteristics of 7-wood sugar Taxan glycosyl hydrolase Lxyl-pl-1 and Lxyl-pl-2,2013, fungus research, 11 (2): 151.Wang Fen, Zhu Ping, 7-wood sugar Taxan glycosyl hydrolase Lxyl-pl-1 and the prediction of Lxyl-pl-2 active centre and preliminary functional study, 2013, fungus journal, 32 (5): 846-854.)。In field of food, xylobiase is applied to the aspects such as food bakes, prepared by xylo-oligosaccharide, drinks is brewageed and has obtained good result of use.Aspect environment protection; the application of xylobiase can more thoroughly be degraded many agriculture and industry wastes and some domestic refuses; the use of xylobiase has simultaneously greatly reduced the use of many harmful organic or inorganic chemical reagent; thereby (model is round, and Li Xiuting rises super to be conducive to the protection of environment; the progress of production by biological xylobiase; 2013, food research and development, 34 (2): 116-121.)。
Yet the end product wood sugar feedback inhibition of hemicellulose xylan hydrolysis reaction is the difficult point of application xylobiase.The accumulation of the wood sugar that xylobiase produces when hydrolysis wood oligose can suppress activity (Saha B.C.Purification and properties of an extracellular β-xylosidase from a newly isolated Fusarium proliferatum of xylobiase, 2003, Bioresour.Technol., 90:33-38.).Industrial, be used for eliminating the method that wood sugar suppresses and mainly contain: improve the working concentration of enzyme and with hyperfiltration process, shift the wood sugar of accumulation.These two kinds of methods inevitably can make production cost promote, hemicellulose and cellulosic hydrolysis simultaneously carried out simultaneously, Mierocrystalline cellulose is hydrolyzed and generates glucose under the effect of cellulase system, and glucose equally also can produce the effect suppressing to the activity of xylobiase.So find a kind of method that improves the xylose and glucose tolerance of xylobiase, it is one of focus of studying at present xylobiase application.
Zytase and the xylobiase end product that hydrolytic activity can be hydrolyzed conventionally in the process of hydrolyzed hemicellulose xylan is mainly that wood sugar suppresses; the glucose that the activity of xylan also can be stored in suppresses (M.L.T.M.Polizeli; A.C.S.Rizzatti; R.Monti; et al; Xylanase from fungi:properties and industrial applications.2005, Appl.Microbiol.Biotechnol., 67:577-591.).The method that improves the xylose and glucose tolerance of xylobiase is exactly to find the beta-glucosidase of high xylose and glucose tolerance.Q.J.Yan etc. obtain the xylobiase of a resistance to wood sugar from Paecilomyces thermophila, it is 139mM (Q.L.Yan that the wood sugar of this xylobiase suppresses constant K i, L.Wang, Z.Q.Jiang, et al.A xylose-tolerant β-xylosidase from Paecilomyces thermophila:Characterization and its co-action with the endogenous xylanase, 2008, Bioresource Technology, 99:5402-5410.).The people such as Zanoelo obtain a highest xylobiase of current wood sugar tolerance from Scytalidium thermophilum, its the highest wood sugar tolerance concentration is 200mM (Zanoelo F.F., Polizeli M.L., Jorge J.A., Purification and biochemica properties of a thermostable xylose-tolerance β-xylosidase from Scytalidium thermophilum, 2004, J.Ind.Microbiol.Biotechnol., 31:170-176.).The people such as Vivian Machado Benassi by the xylobiase from Aspergillus nigerUSP-67 is immobilized in Polyethyleneimine – sepharose polyethylene imine based-sepharose on, thereby the tolerance of the xylose and glucose of this xylobiase is all increased, beta-glucosidase after immobilization is at 100mM wood sugar and not suppressed (the Vivian Machado Benassi of 200mM glucose Water Under solution activity, Tony Marcio da Silva, Benevides Costa Pessela, et al, Immobilization and biochemical properties of a β-xylosidase activated by glucose/xylose from Aspergillus niger USP-67with transxylosylation activity.2013, Journal of Molecular Catalysis B:Enzymatic, 89:93-101.).
Take xylobiase as name lookup State Intellectual Property Office patent retrieval database, occur altogether 6 patents of invention.About the gene of xylobiase and the patent of application thereof, have 5, these 5 xylobiase genes are respectively from aspergillus niger, thermophilic Paecilomyces varioti J18, paper shape grape ear mould, acid heat acidocaldarius and these 5 kinds of bacterial strains of thermophilic Paecilomyces varioti; Patent about xylobiase application has 1.
Take xylobiase as keyword lookup State Intellectual Property Office patent retrieval database, there are altogether 15 patents of invention, wherein 4 is about xylobiase gene and application thereof, have 3 relevant with the preparation of cellulolytic enzyme, other 8 patents are all relevant to the application of xylobiase.
The approach of finding and cloning the beta-glucosidase of high xylose and glucose tolerance has 2: the one, and the microorganism of screening novel enzyme; The 2nd, enzyme is carried out to suitable protein engineering transformation.Microbial strains seed selection is the most frequently used in industrial production and academic research and one of the simplest means, but workload is large, randomness is strong, therefore is often difficult to screen desirable strain.Protein engineering transformation is that method emerging, that utilize molecular biology and information biology is carried out orthomutation and rationality transformation to albumen, thereby obtain the method for ideal protein or enzyme, its advantage is that workload is relatively little and the probability of success is larger, but the zymologic property of most muteins does not change or becomes poorer.Therefore, the beta-glucoside enzyme mutant of acquisition xylose and glucose tolerance better effects if is the study hotspot of this area always.
Summary of the invention
The object of the invention is to for above-mentioned technical problem, by beta-glucosidase is carried out to protein engineering transformation, thereby obtain a kind of beta-glucoside enzyme mutant of high xylose and glucose tolerance.
For achieving the above object, the technical solution adopted in the present invention is:
A kind of beta-glucoside enzyme mutant provided by the invention, its aminoacid sequence is SEQ ID NO:1, this mutant is to obtain by the tryptophane of 138 of xylobiase gene Exyl is mutated into halfcystine.
The present invention also provides the host cell that contains beta-glucoside enzyme mutant of the present invention, and described host cell can be prokaryotic cell prokaryocyte or eukaryotic cell.
Beta-glucoside enzyme mutant of the present invention tool in degraded wood oligose has been widely used.
The preparation method of beta-glucoside enzyme mutant of the present invention comprises the following steps:
1) clone obtains beta-glucosidase gene;
2) genetic modification step 1) being obtained obtains beta-glucosidase mutant gene;
3) by step 2) the beta-glucosidase mutant gene that obtains expresses and purifying, obtains the purified product of beta-glucoside enzyme mutant.
The present invention uses the method for protein engineering transformation to carry out directional transformation to beta-glucosidase, obtained the beta-glucoside enzyme mutant of high xylose and glucose tolerance, this mutant is compared with protoenzyme, its tolerance to wood sugar is 2.37 times before sudden change, and the tolerance of glucose is 2.31 times before sudden change.Beta-glucoside enzyme mutant of the present invention can be used for efficient degradation wood oligose, at aspects such as the energy, papermaking, medicine, is with a wide range of applications.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE figure of xylobiase mutant purified of the present invention;
Fig. 2 is the HPLC figure that xylobiase mutant of the present invention is hydrolyzed various wood oligoses;
Fig. 3 is that the wood sugar of xylobiase mutant of the present invention and xylobiase suppresses constant K i value analysis chart;
Fig. 4 is that the glucose of xylobiase mutant of the present invention and xylobiase suppresses constant K i value analysis chart.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Do not deviating from the present invention spirit and essential in the situation that, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
The preparation of embodiment 1 xylobiase mutant
Material: intestinal bacteria (Escherichia coli) strain XL10-gold, intestinal bacteria M15 are purchased from Stratagene company; Ni-NTA histidine protein purification media is purchased from Intrivogen company; Restriction enzyme, modifying enzyme are purchased from TaKaRa ,MBI company; Wood oligose and wood sugar are purchased Sigma company; All the other reagent unless otherwise noted, are the conventional reagent of buying from the market.
1) clone of xylobiase gene Exyl
From GenBank gene database, find the gene order of a coding supposition glycosyl hydrolase of enterobacter cloacae Enterobacter cloacae subsp.cloacae ATCC13047 (YP_003612882.1).Design upstream primer Exyl-1:5 '-CTGAGATCTACTGCAATCTATAAGGACGCGGGAC-3 ' (comprising a BglII restriction enzyme site) and downstream primer Exyl-25 '-ACTAAGCTTCTACGCGTGCTGAACCTGACAGGTG-3 ' (comprising a HindIII restriction enzyme site), by polymerase chain reaction PCR β-xyloside gene Exyl that increases, with restriction enzyme BglII and HindIII enzyme, cut after xylobiase gene Exyl, be inserted into the expression vector pQE30 cutting with HindIII enzyme through BamHI and be connected.The recombinant plasmid called after pQE-Exyl obtaining.
2) acquisition of xylobiase mutant gene Exyl-W138C
Exyl-W138C gene is the tryptophane of 138 of gene Exyl to be mutated into halfcystine transform.Take 1) in the pQE-Exyl that builds be template, use upstream primer W138C1:5 '-TCAACC
tGCgATCCGGCGCTGGTACAGCGGGC-3 ' and downstream primer W138C2:5 '-CGGATC
gCAgGTTGAACCGTAGTTGAGCGACG-3 ' carries out inverse PCR reaction.PCR response procedures: 95 ℃ of the first steps 2 minutes; Second step carries out 30 circulations, circulation be 98 ℃ 10 seconds, 58 ℃ 15 seconds, 72 ℃ 7 minutes; The 3rd 72 ℃ of steps 10 minutes.PCR product is used 1 μ L Dpn I at 37 ℃ of effects one hour, then CaCl
2chemical method Transformed E .coli XL10-gold competent cell.Converted product clone delivers Shanghai biotechnology company limited and carries out the correct transformant of DNA sequencing Analysis deterrmination.
3) expression of xylobiase mutant gene Exyl-W138C and the partial purification of expression product
The recombination bacillus coli M15 inoculation that contains plasmid Exyl-W138C is contained in the LB substratum of 100 μ g/mL penbritins to 20mL, and 37 ℃ of shaking culture, treat OD
600be 0.4 o'clock, adding final concentration is the IPTG of 0.5mmol/L, induces 20 hours for 20 ℃.11000 leave heart 3min, collect thalline, with 4mL lysis buffer (50mmol/L NaH
2pO
4, 300mmol/L NaCl, 10mmol/L imidazoles, pH8.0) resuspended thalline, ultrasonic wave is broken born of the same parents 9min.12000 leave heart 10min, get supernatant and carry out protein purification below.The nickel affinity chromatography colloid that adds 1mL50% by every 4ml supernatant liquor, turns and shakes 60 minutes with 200 at 4 ℃, and mixture is filled into pillar, collects effluent.Add 1ml dcq buffer liquid (50mmol/L NaH
2pO
4, 300mmol/L NaCl, 20mmol/L imidazoles, pH8.0) in pillar, slowly stirs, and collects effluent.Repeat rinse step 4 times.Add elution buffer (50mmol/L NaH
2pO
4, 300mmol/L NaCl, 250mmol/L imidazoles, pH8.0) elute protein.Collect the protein soln of wash-out, with polyacrylamide gel electrophoresis (SDS-PAGE) checking of sex change, the SDS-PAGE figure of gained as shown in Figure 1, finds that there is an obvious target protein matter band.
4) xylobiase mutant Exyl-W138C is hydrolyzed the HPLC analysis of various wood oligoses
To get 5 μ L xylobiase mutant Exyl-W138C purifieds be 1% (w/v) with concentration respectively, and xylotriose, Xylotetrose, wooden pentasaccharides react 30 minutes under the condition of 45 ℃ of optimum temperutures and optimal pH 5.5, then reactant is placed on to 10 minutes termination reactions in boiling water, after cooling, carry out HPLC, detect its reaction product, gained HPLC schemes as shown in Figure 2.
HPLC working conditions: Agilent1100Series detector, Alltech2000ES type light scattering detector, Alltima Amino5 μ chromatographic column (250 * 4.6mm), XWK-III oil-free air pump; Moving phase: acetonitrile: distilled water=79:21; Flow velocity 1.0mL/min; Sample size 20 μ L; 28 ℃ of column temperatures.
5) wood sugar of xylobiase mutant Exyl-W138C suppresses the mensuration of constant K i value
Under the condition of 45 ℃ of optimum temperutures and optimal pH 5.5, in the system that is 0mM-0.8mM at substrate p-nitrophenyl-β-D-xyloside pNPX final concentration, adding respectively final concentration is 0mM, and the wood sugar of 300mM and 600mM is measured the enzyme activity of Exyl-W138C.In the reaction system of these three kinds of different concns wood sugars, the ratio vigor under different pNPX concentration is done linear regression analysis to corresponding pNPX concentration, and the wood sugar that uses the mapping of GraphPad Prism5.0 software simultaneously to calculate mutant Exyl-W138C suppresses constant K i value.
6) glucose of xylobiase mutant Exyl-W138C suppresses the mensuration of constant K i value
Under the condition of 45 ℃ of optimum temperutures and optimal pH 5.5, in the system that is 0mM-0.8mM at substrate p-nitrophenyl-β-D-xyloside pNPX final concentration, adding respectively final concentration is 0mM, the enzyme activity of the glucose assays Exyl-W138C of 200mM and 400mM.In the reaction system of these three kinds of different glucoses, ratio vigor under different pNPX concentration is done linear regression analysis to corresponding pNPX concentration, and the glucose that uses the mapping of GraphPad Prism5.0 software simultaneously to calculate mutant Exyl-W138C suppresses constant K i value.
The xylose and glucose of reference examples xylobiase Exyl suppresses the mensuration of constant K i value
1) wood sugar of xylobiase Exyl suppresses the mensuration of constant K i value
Xylobiase Exyl is under the condition of 45 ℃ of optimum temperutures and optimal pH 5.5, and in the system that is 0mM-0.8mM at substrate p-nitrophenyl-β-D-xyloside pNPX final concentration, adding respectively final concentration is 0mM, and the wood sugar of 200mM and 400mM is measured enzyme activity.In the reaction system of these three kinds of different concns wood sugars, the ratio vigor under different pNPX concentration is done linear regression analysis to corresponding pNPX concentration, and the wood sugar that uses the mapping of GraphPad Prism5.0 software simultaneously to calculate Exyl suppresses constant K i value; The wood sugar of xylobiase mutant Exyl-W138C and xylobiase Exyl suppresses constant K i value analysis chart as shown in Figure 3.
2) glucose of xylobiase Exyl suppresses the mensuration of constant K i value
Xylobiase Exyl before sudden change is also under the condition of 45 ℃ of optimum temperutures and optimal pH 5.5, in the system that is 0mM-0.8mM at substrate p-nitrophenyl-β-D-xyloside pNPX final concentration, adding respectively final concentration is 0mM, the glucose assays enzyme activity of 100mM and 200mM.In the reaction system of these three kinds of different glucoses, the ratio vigor under different pNPX concentration is done linear regression analysis to corresponding pNPX concentration, and the glucose that uses the mapping of GraphPad Prism5.0 software simultaneously to calculate Exyl suppresses constant K i value; The glucose of xylobiase mutant Exyl-W138C and xylobiase Exyl suppresses constant K i value analysis chart as shown in Figure 4.
Result:
From Fig. 1, recognize, the molecular weight of the purified of xylobiase mutant Exyl-W138C is 85.6kDa.
A in Fig. 2 and B are respectively the standard specimens of wood sugar and xylo-bioses, xylotriose, Xylotetrose, wooden pentasaccharides; From C, D, the E of Fig. 2, recognize, xylobiase mutant Exyl-W138C can become wood sugar and xylo-bioses by xylotriose, Xylotetrose, wooden pentasaccharides complete hydrolysis.
From Fig. 3, recognize, it is 51.95mM that the wood sugar of xylobiase Exyl suppresses constant K i value, it is 123.8mM that the wood sugar of mutant Exyl-W138C suppresses constant K i value, as can be seen from the above data, the Ki of xylobiase mutant is 2.37 times before sudden change, and wood sugar tolerance has improved 1.37 times.
From Fig. 4, recognize, it is 164mM that the glucose of xylobiase Exyl suppresses constant K i value, it is 378.7mM that the glucose of mutant Exyl-W138C suppresses constant K i value, as can be seen from the above data, the Ki of xylobiase mutant is 2.31 times before sudden change, and glucose tolerance has improved 1.31 times.
In sum, xylobiase mutant of the present invention can fully be degraded the wood oligoses such as xylotriose, Xylotetrose, wooden pentasaccharides, with respect to its wood sugar tolerance of xylobiase of not sudden change, improve 1.37 times, glucose tolerance and improved 1.31 times, can be used for efficient degradation wood oligose, at aspects such as the energy, papermaking, medicine, be with a wide range of applications.
Claims (3)
1. a beta-glucoside enzyme mutant, is characterized in that: the aminoacid sequence of described beta-glucoside enzyme mutant is as shown in SEQ ID NO:1.
2. a host cell, is characterized in that: described host cell is prokaryotic cell prokaryocyte or the eukaryotic cell that contains beta-glucoside enzyme mutant claimed in claim 1.
According to a kind of beta-glucoside enzyme mutant claimed in claim 1 in degraded the purposes in wood oligose.
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CN105524902A (en) * | 2016-01-28 | 2016-04-27 | 中国农业科学院饲料研究所 | Beta-glucosaccharase mutant M36E with high catalytic efficiency as well as coding gene and applications thereof |
CN105524903A (en) * | 2016-01-28 | 2016-04-27 | 中国农业科学院饲料研究所 | Beta-glucosaccharase improved mutant E168Q as well as coding gene and applications thereof |
CN105754973A (en) * | 2016-05-05 | 2016-07-13 | 广西大学 | Mutant W233D of beta-glucosidase and application of mutant W233D |
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US7041814B1 (en) * | 1998-02-18 | 2006-05-09 | Genome Therapeutics Corporation | Nucleic acid and amino acid sequences relating to Enterobacter cloacae for diagnostics and therapeutics |
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CN105524902A (en) * | 2016-01-28 | 2016-04-27 | 中国农业科学院饲料研究所 | Beta-glucosaccharase mutant M36E with high catalytic efficiency as well as coding gene and applications thereof |
CN105524903A (en) * | 2016-01-28 | 2016-04-27 | 中国农业科学院饲料研究所 | Beta-glucosaccharase improved mutant E168Q as well as coding gene and applications thereof |
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CN105524902B (en) * | 2016-01-28 | 2019-03-12 | 中国农业科学院饲料研究所 | A kind of high catalytic efficiency β-glucosidase mutants M36E and its encoding gene and application |
CN105754973A (en) * | 2016-05-05 | 2016-07-13 | 广西大学 | Mutant W233D of beta-glucosidase and application of mutant W233D |
CN105754973B (en) * | 2016-05-05 | 2019-01-29 | 广西大学 | A kind of mutant W233D of beta-glucosidase and its application |
CN110904078A (en) * | 2019-12-11 | 2020-03-24 | 云南师范大学 | Sodium sulfate and ammonium sulfate resistant xylosidase mutant V322R and application thereof |
CN110904078B (en) * | 2019-12-11 | 2020-09-04 | 云南师范大学 | Sodium sulfate and ammonium sulfate resistant xylosidase mutant V322R and application thereof |
CN114981405A (en) * | 2020-01-28 | 2022-08-30 | 东丽株式会社 | Mutant strain of Trichoderma filamentous fungus |
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