WO2015180362A1 - 一种嗜温耐乙醇β-葡萄糖苷酶及其编码基因和应用 - Google Patents

一种嗜温耐乙醇β-葡萄糖苷酶及其编码基因和应用 Download PDF

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WO2015180362A1
WO2015180362A1 PCT/CN2014/088168 CN2014088168W WO2015180362A1 WO 2015180362 A1 WO2015180362 A1 WO 2015180362A1 CN 2014088168 W CN2014088168 W CN 2014088168W WO 2015180362 A1 WO2015180362 A1 WO 2015180362A1
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glucosidase
ethanol
resistant
mesophilic
mesophile
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French (fr)
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袁振宏
梁翠谊
许敬亮
陈小燕
庄新姝
张宇
郭颖
周卫征
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中国科学院广州能源研究所
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    • 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
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • 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
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention belongs to the field of enzyme genetic engineering and enzyme engineering, and particularly relates to a mesophilic ethanol-resistant ⁇ -glucosidase and a gene encoding the same and application thereof.
  • ⁇ -glucosidase (EC 3.2.1.21) belongs to the class of fiber hydrolases and is a class of enzymes that catalyze the hydrolysis or transfer of ⁇ -1,4-glycosidic bonds. It is an important component of the cellulase system. In the process of cellulase hydrolysis of cellulose, the enzymatic action of cellulose into glucose requires at least three synergistic effects, glucan endonuclease, dextran. Exonuclease and ⁇ -glucosidase. Endoglucanase and exoglucanase degrade cellulose into cellobiose, which is then broken down into glucose by beta-glucosidase. Beta-glucosidase hydrolyzes cellobiose to release glucose, a key rate-limiting step in cellulose hydrolysis.
  • ⁇ -glucosidase has the lowest content in the cellulase system, less than 1%, and the activity is generally low, which is the bottleneck of cellulase hydrolysis. For a long time, the enzyme properties are not good enough, the enzyme yield is low, and the specific activity of the enzyme is low, which has been an important factor affecting the practical application of the enzyme.
  • ⁇ -glucosidase is widely present in microorganisms, microorganisms such as fungi and bacteria are not efficient in producing enzymes, and it is difficult to obtain a large amount of products, and thermal stability is poor. At present, the activity of ⁇ -glucosidase still cannot meet the needs of industrial production, and the cost is high.
  • the optimum temperature of cellulase is usually about 50 °C, and the optimum temperature for yeast fermentation is about 30 °C. How to make the temperature coordination of these two processes is the key to the efficient production of ethanol by simultaneous saccharification and fermentation (SSF).
  • SSF simultaneous saccharification and fermentation
  • One of the contradictions is to use high temperature resistant yeast. Therefore, the cloning and expression of the ⁇ -glucosidase gene has become one of the important links in the study of cellulase. So far, hundreds of microbial ⁇ -glucosidase genes have been cloned, and many microbial-derived ⁇ -glucosidase genes have also been heterologously expressed. In recent years, the construction of engineering bacteria by genetic recombination technology in the world, the secretion of highly active, thermostable ⁇ -glucosidase research is a hot spot in the field of cellulase.
  • thermophila ⁇ -glucosidase gene PtBglu3 [protein expression and purification (Protein Expres Purif) 84:64–72, 2012]; Thermo-Qiong Pei et al. reported thermophilic Thermobifida Fusca ⁇ -glucosidase gene BglC [Bioresour. Technol. 102:3337–3342, 2011] ; Thermotoga thermarum DSM 5069T high temperature ⁇ -glucosidase gene Tt-bgl reported by Linguo Zhao et al. [JMol Catal B-Enzym 95: 62–69, 2013].
  • the heat-resistant enzyme has great advantages, can improve the reaction speed, prolong the action time, reduce pollution, enhance the tolerance to chemical agents, and can carry out the reaction under the special conditions required, thus developing mesophilic ⁇ - Glucosidase has become a research hotspot.
  • Trichoderma viride W2 has an optimum pH of 4.8, an optimum reaction temperature of 70 ° C, and an ethanol concentration of 10% (v/v). It has the greatest promoting effect on enzyme activity, the ⁇ -glucosidase activity is increased by 1.6 times, the ethanol tolerance is up to 30% (v/v), and the optimum reaction pH of Hypocrea sp. W63 is 4.8.
  • the optimum reaction temperature is 65 ° C, and the ethanol concentration of 10% (v/v) has the greatest promotion effect on the enzyme activity, nearly double the ⁇ -glucosidase activity, and the ethanol tolerance is up to 30% (v/v). ).
  • the cellulase produced by the fungus is mainly endoglucanase and exoglucanase, and the ⁇ -glucosidase has the lowest content in the cellulase system, less than 1%.
  • Genetically engineered bacteria constructed by molecular biology techniques have the advantages of high genetic stability, rapid enzyme production, and large enzyme production.
  • the recombinant enzymes produced are intended to meet the needs of a large number of industrial applications in the future.
  • the thermophilic ⁇ -glucosidase gene cloning mainly comes from thermophilic bacteria and fungi.
  • thermophilic microorganisms does not necessarily have Thermophilic and thermal stability.
  • Trichoderma is one of the earliest and most widely studied species in the study of ⁇ -glucosidase-producing microorganisms, but the ⁇ -glucosidase gene with mesophilic and ethanol-resistant properties cloned from Hypocrea has not been reported.
  • a first object of the present invention is to provide a mesophilic ethanol-resistant ⁇ -glucosidase and a gene encoding the same.
  • the mesophilic ethanol-resistant ⁇ -glucosidase of the present invention is characterized in that the amino acid sequence thereof is shown in SEQ ID NO: 2.
  • the gene encoding mesophilic alcohol-resistant ⁇ -glucosidase of the present invention is characterized by encoding a mesophilic ethanol-resistant ⁇ -glucosidase having an amino acid sequence as shown in SEQ ID NO: 2.
  • the gene encoding mesophilic alcohol-resistant ⁇ -glucosidase preferably, has a nucleotide sequence as shown in SEQ ID NO: 1.
  • hypocrea sp. W63 (ZL201110417104.9) produces mesophilic ethanol-resistant ⁇ -glucosidase, which has a substrate specificity for hydrolysis of 4-nitrophenyl ⁇ -D-glucopyranoside (pNPG), and its optimum pH is suitable.
  • the value is 4.8
  • the optimum reaction temperature is 65 °C
  • the ethanol concentration in the reaction is 10% (v/v), which has the greatest promotion effect on the enzyme activity, and the ⁇ -glucosidase enzyme activity is nearly doubled, and the ethanol tolerance is as high as 30% (v/v).
  • the mesophilic ethanol-resistant ⁇ -glucosidase gene epB-BGL obtained from Hypocrea sp. W63 has a density of 2202 bp and a nucleotide sequence thereof as shown in SEQ ID NO: 1, which is thermophilic and ethanol resistant.
  • the ⁇ -glucosidase gene encodes a protein consisting of 733 amino acids, the amino acid sequence of which is shown in SEQ ID NO: 2.
  • a recombinant plasmid containing pPIC9K as an expression vector containing the gene fragment is obtained by a molecular biological method, and Pichia pastoris GS115 is used as an expression host, and the recombinant strain is secreted to express mesophilic ethanol-resistant ⁇ -glucosidase. Therefore, the present invention successfully clones the mesophilic ethanol-resistant ⁇ -glucosidase gene into other recipient bacteria by genetic engineering or molecular biological means, and produces the mesophilic ethanol-resistant ⁇ -glucosidase of the present invention from other recipient bacteria. .
  • the mesophilic ethanol-resistant ⁇ -glucosidase of the present invention is a novel novel ⁇ -glucosidase, by the thermophilic ethanol-resistant ⁇ -glucose of the present invention.
  • Sequence Analysis of Gene Derivative Amino Acids of Glycosidases the amino acid sequence of SEQ ID NO: 2 is compared with the amino acid sequence of other reported ⁇ -glucosidases of the mesophilic ethanol-resistant ⁇ -glucosidase of the present invention.
  • the ⁇ -glucosidase (Genbank index number AAA18473.1) derived from Trichoderma reesei has the highest homology, and their amino acid sequence similarity is 79%, and the similarity is higher than 79% of ⁇ -glucosidase source.
  • IMI 206040 of Trichoderma atroviride Kubicek, CP et al, Genome Biol. 12 (4), R40 (2011), not functionally identified
  • Gv29-8 of Trichoderma virens, Trichoderma virens (Kubicek, C.P. et al., Genome Biol. 12(4), R40 (2011), no functional identification).
  • amylose-resistant ethanol ⁇ -glucosidase of the present invention is aligned with other reported ⁇ -glucosidase amino acid sequences, and it is found that among the strains having a similarity of more than 79%, sequence alignment or protein structure is studied. So far, no relevant literature reports have been found on the identification of the function of the ⁇ -glucosidase gene of the corresponding strain.
  • the mesophilic ethanol-resistant ⁇ -glucosidase gene epB-BGL of the present invention is a novel gene.
  • the mesophilic ethanol-resistant ⁇ -glucosidase of the present invention has been submitted to a nucleotide sequence on GenBank, and its nucleotide sequence is shown in SEQ ID NO: 1, and its sequence number is KJ502670.
  • a second object of the present invention is to provide a recombinant expression vector comprising the mesophilic ethanol-resistant ⁇ -glucosidase gene of the present invention.
  • the recombinant expression vector wherein the expression vector is preferably a pPIC9K expression vector.
  • a third object of the present invention is to provide a host cell characterized by comprising a eukaryotic cell or a prokaryotic cell of the above recombinant expression vector.
  • the prokaryotic cells may be various bacteria such as yeast engineering bacteria, Escherichia coli or Bacillus subtilis.
  • the yeast engineered bacteria is preferably a Pichia pastoris GS115 strain.
  • a fourth object of the present invention is to provide a use of mesophilic and ethanol-resistant ⁇ -glucosidase in the high-temperature simultaneous saccharification of ethanol for fermentation of ethanol.
  • the currently known ⁇ -glucosidase belongs to the first and third families of glycoside hydrolase, respectively.
  • the temperature- and ethanol-resistant ⁇ -glucosidase sequence of the present invention it is considered to be a member of the third family of glycoside hydrolase.
  • the enzymatic properties of the mesophilic ethanol-resistant ⁇ -glucosidase of the present invention are different from the known ⁇ -glucosidase, and the reaction enzyme activity is determined by activity, and the reaction temperature is 40-90 ° C, and the optimum reaction is carried out.
  • the temperature is 70 ° C; the reaction pH is 4.0-6.5, the optimum pH value is 5.0; the ethanol concentration can promote the ⁇ -glucosidase activity within 30% (v/v), wherein the reaction system
  • concentration of ethanol was 10-20% (v/v)
  • the effect of ⁇ -glucosidase activity was the strongest, and the activity of ⁇ -glucosidase was increased by 86.29%, which proved that the enzyme has high enzyme retention in the presence of high temperature and ethanol.
  • the mesophilic ethanol-resistant ⁇ -glucosidase has a molecular weight of 76,740 daltons and a pI of 6.01.
  • the optimal specific activity of the enzyme is 194.25 U/mg, and its specific substrate is pNPG.
  • This enzyme is an enzyme that specifically catalyzes the hydrolysis of ⁇ -glucosidic bonds to degrade cellobiose into glucose, which is a cellulase system.
  • a key enzyme of the present invention, the mesophilic and ethanol-resistant ⁇ -glucosidase of the present invention is applied to high-temperature simultaneous saccharification to produce ethanol, firstly prehydrolyzing the biomass raw material, and then adding mesophilic resistance
  • the high-temperature simultaneous saccharification and fermentation of ethanol ⁇ -glucosidase enzyme solution and high temperature resistant yeast can effectively eliminate cellobiose inhibition and increase ethanol production by 39%.
  • the enzyme Under the optimum conditions of cellulase enzymatic hydrolysis and yeast fermentation in the simultaneous saccharification and fermentation process of producing fuel ethanol from cellulose, the enzyme has high enzyme activity, and the process can be applied to produce fuel ethanol.
  • the mesophilic and ethanol-resistant ⁇ -glucosidase of the invention has important application value in energy.
  • Figure 1 is a schematic diagram showing the construction of recombinant plasmid pPIC9K
  • Figure 2 is a gel electrophoresis pattern of the mesophilic ethanol-resistant ⁇ -glucosidase cDNA of the present invention, wherein the M lane is Marker, and the lane 1 is the cDNA of the target gene;
  • FIG. 3 is a diagram showing the EcoRI and AvrII double-cut electrophoresis of the thermophilic ethanol-resistant ⁇ -glucosidase cloning plasmid pPIC9K-epB of the present invention, wherein the M lane is Marker, the lane 1 is the EcoRI of the plasmid pPIC9K-epB, and the AvrII double digestion ;
  • Figure 4 is an electrophoresis diagram of P. pastoris GS115 expressing mesophilic ethanol-resistant ⁇ -glucosidase purification, wherein the M lane is Marker and the lane 1 is Micro-Prep DEAE column purification;
  • Figure 5 is a secondary peak view of the mesophilic ethanol-resistant ⁇ -glucosidase protein profile of the present invention.
  • Figure 6 is the optimal reaction temperature and pH value of the mesophilic ethanol-resistant ⁇ -glucosidase of the present invention.
  • Figure 7 is a graph showing the effect of ethanol addition on the thermophilic ethanol-resistant ⁇ -glucosidase enzyme activity of the present invention.
  • Figure 8 is a graph showing the effect of adding the thermophilic ethanol-resistant ⁇ -glucosidase of the present invention to high-temperature simultaneous saccharification and fermentation.
  • the materials used in the examples of the present invention include: a fungal total RNA rapid extraction kit (purchased from Bioengineering Co., Ltd.); a cDNA first strand synthesis kit (purchased from Thermo); Pichia pastoris (Pichia pastoris) GS115, primer synthesis (purchased from Invitrogen); pPIC9K expression vector (purchased from Invitrogen); competent cells Trans-T1, pEASY-Blunt Zero Cloning Kit (purchased from TransGen Biotech); PCR reagent, restriction Endonucleases EcoRI, AvrII and SalI, T4 DNA ligase (purchased from Takara), cellulase (purchased from Genencor), high temperature yeast NCYC587 (purchased from the National Yeast Collection, UK); Hypocrea sp .) W63 was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on September 1, 2011. (CGMCC), the deposit number is: CGMCC No.
  • Example 1 Thermophilic and ethanol-resistant ⁇ -glucosidase gene cloning of the present invention
  • RNA of Hypocrea sp. W63 is extracted by the fungal total RNA rapid extraction kit, and the specific steps are as follows:
  • the adsorption column is placed in a collection tube, centrifuged at 12,000 g for 2 min, and the lid is opened for several minutes (volatile residual ethanol);
  • cDNA first-strand synthesis kit 1 ⁇ L of the above total RNA solution was used as a template for reverse transcription cDNA ligation, and then seven amino acid sequences with higher ⁇ -glucosidase similarity were searched from the GenBank database, using DNAman software. The Multiple Sequence Alignment in these sequences performs a homology comparison of these sequences, and a pair of primers P 1 and P 3 are designed based on two highly conserved sequences of the homologous ⁇ -glucosidase gene and known sequences. The reverse transcribed cDNA was subjected to PCR amplification.
  • Reaction conditions pre-denaturation at 94 ° C for 3 min; then the following cycles: denaturation at 94 ° C for 30 s; annealing at 60-45 ° C for 30 s; extension at 72 ° C for 30 s, 10 cycles; denaturation at 94 ° C for 30 s; annealing at 45 ° C for 30 s; extension at 72 ° C for 30 s , for 25 cycles; the last 72 ° C extension for 6 min.
  • a 1% agarose gel electrophoresis was performed to detect the presence or absence of a target band of an appropriate size.
  • the PCR product was then stored at -20 °C.
  • the target band was obtained and the size was about 1.8 kb.
  • a PCR reaction is carried out by designing specific primers based on known sequence alignments.
  • the reaction procedure was as follows: pre-denaturation at 94 ° C for 5 min, followed by the following cycles: denaturation at 94 ° C for 30 s, annealing at 52 ° C for 30 s, extension at 72 ° C for 30 s, for a total of 30 cycles, and finally at 72 ° C for 10 min.
  • the PCR product was analyzed by 1% agarose gel electrophoresis. Linkage, transformation, identification and sequencing of the target gene fragment.
  • the full-length cDNA of the gene of interest was obtained, which was about 2.2 kb in size (as shown in Figure 2).
  • epB-BGL mesophilic alcohol-resistant ⁇ -glucosidase
  • the lyase gene epB-BGL encodes a protein of 733 amino acids.
  • the theoretical molecular weight of the protein is predicted to be 76550.45 Daltons by DNAstar software, and the isoelectric point pI is 6.01.
  • the most homologous to the epB-BGL gene catalytic domain is the ⁇ -glucosidase (Genbank index number AAA18473.1) derived from Trichoderma reesei, which has an amino acid sequence similarity of 79%.
  • the mesophilic ethanol-resistant ⁇ -glucosidase gene epB-BGL of the present invention has been submitted to the nucleotide sequence on GenBank to obtain the sequence number KJ502670.
  • Example 2 Expression and purification of the ⁇ -glucosidase of the present invention in yeast engineering bacteria
  • a pair of expression primers ep3 and ep5 were designed, and the signal peptide sequence and the 3' and 5' non-coding region sequences were removed to ensure the targeted insertion of the epB-BGL gene.
  • the vector pPIC9K was introduced with EcoRI and AvrII restriction sites at both ends of the upstream and downstream primers. The two primers are spaced apart by about 2.5 kb, and the amplified product contains a mesophilic alcohol-resistant ⁇ -glucosidase mature protein coding sequence.
  • the PCR product of the full-length cDNA of mesophilic and ethanol-resistant ⁇ -glucosidase was used as a template, and ep5 and ep3 were used as primers for PCR to obtain the gene expression sequence of mesophilic ethanol-resistant ⁇ -glucosidase mature peptide.
  • the target gene fragment was recovered, the target fragment was ligated with pPIC9K expression vector using pEASY-Blunt Zero Cloning Kit, and transformed into E. coli competent cell Trans-T1, and the positive clone was screened by colony PCR and restriction endonuclease analysis of plasmid DNA. After sequencing, the correct plasmid identified by sequencing was named pPIC9K-epB.
  • the plasmid pPIC9K-epB containing the correct reading frame of the mesophilic ethanol-resistant ⁇ -glucosidase gene was sequenced and digested with EcoRI and AvrII to recover the insert, and the same double-digested yeast expression plasmid pPIC9K was used for T4 DNA.
  • the ligase was ligated and transformed into E. coli competent cell Trans-T1.
  • the transformed Trans-T1 was screened by Amp resistance.
  • the colony was cultured at 37 ° C overnight, and the plasmid was extracted and identified by restriction endonuclease digestion and sequencing.
  • the recombinant plasmid was named pPIC9K-epB, and its construction pattern is shown in Fig. 1.
  • the constructed recombinant plasmid pPIC9K-epB was linearized with SalI restriction endonuclease (located in the His4 region), and the empty pPIC9K vector plasmid and the linearized enzyme of the recombinant plasmid pPIC9K-epB containing the epB-BGL gene were simultaneously digested.
  • the yeast transformation was simultaneously performed as a positive control.
  • the electric shock is: voltage 1.5-1.8kV, capacitance 25 ⁇ F; resistance 200 ⁇ . Electric shock time is 4-5msec
  • the yeast transformants to be tested after the electric shock transformation are inoculated on the MD plate in a certain direction with a sterile disposable toothpick, and cultured inversion at 30 ° C for 2-4 d, and the glycerol species are stored.
  • yeast cells were recovered by centrifugation at 3000 g for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in an appropriate volume of BMMY medium to an OD600 value of 1.0-2.0 (about 100-200 mL).
  • cover 8 layers of sterile gauze put it in a shaker, continue to culture at 28-30 °C and start to induce expression (note that the induction temperature should be strictly controlled, not more than 30 °C);
  • the extracted crude enzyme solution was precipitated with ammonium sulfate of 90% saturation for 4 hours, centrifuged at 14,000 rpm for 20 min at 4 ° C, and the precipitate was collected and dissolved in an appropriate amount of buffer A (20 mmol/L Tris-HCl buffer, pH 7.0).
  • buffer A 20 mmol/L Tris-HCl buffer, pH 7.0.
  • the supernatant was applied to a Desalting desalting column pre-equilibrated by buffer A, 2 mL of the peak at A280 nm was collected, and the enzyme solution collected after desalting was applied to a Micro-Prep DEAE column pre-equilibrated with buffer A.
  • the enzyme protein was first eluted with 3 column volumes of buffer A to A280, followed by 5 times buffer A (20 mmol/L Tris-HCl buffer, pH 7.0 and buffer B (1 mol/L Tris-). Gradient elution of the same proportion of buffer solution in HCl buffer, pH 7.0), flow rate of 1 mL/min, per tube Collect 1 mL. Each tube was tested for enzyme activity and protein concentration, and finally purified by SDS-PAGE (the results are shown in Figure 4). The optimal specific activity of the enzyme after purification was 194.25 U/mg. After SDS-PAGE electrophoresis, the gel was recovered and the protein of the target protein was identified and analyzed. The molecular weight of the enzyme was 76,740 Daltons, which is similar to the theoretical molecular weight (76550 Daltons). The figure is shown in Figure 5.
  • Example 3 Enzymatic properties of mesophilic and ethanol-resistant ⁇ -glucosidase of the present invention
  • the enzyme activity of the mesophilic ethanol-resistant ⁇ -glucosidase of the present invention is determined by using 4-nitrophenyl ⁇ -D-glucopyranoside (pNPG) as a substrate, and the reaction system is 2 mL, first 1 mL of pNPG (5 mmol/L) and 0.9 mL of pH 5 .0Na 2 HPO 4 - citrate buffer mixed, then add 0.1mL of the appropriate dilution of the mesophilic ethanol-resistant ⁇ -glucosidase preparation obtained in the previous step, react at 50 ° C for 10 min, immediately add 3mLlmol / L Na 2 CO 3 The solution was terminated, and allowed to stand at room temperature for 5 min, and the light absorption value (OD) was measured at 400 nm.
  • pNPG 4-nitrophenyl ⁇ -D-glucopyranoside
  • Enzyme activity definition The amount of 1 ⁇ mol/L p-nitrophenol catalyzed by 1 min was defined as one enzyme unit.
  • ⁇ -glucosidase preparation according to the pNPG determination method, other conditions are unchanged, adjust different pH buffer, different temperature, different ethanol concentration for enzyme activity The reaction was measured to determine the highest enzyme activity of 100%, which was the optimum reaction condition for measuring mesophilic and ethanol-resistant ⁇ -glucosidase.
  • the mesophilic and ethanol-resistant ⁇ -glucosidase of the invention has the characteristics of maintaining high enzyme activity in the presence of high temperature and ethanol, and the reaction temperature is 40-90 ° C, and the optimum reaction temperature is 70 ° C (as shown in FIG. 6A ).
  • the reaction pH is 4.0-6.5, the optimum pH value is 5.0-5.5 (as shown in Figure 6B); the ethanol concentration is 30% (v/v), and the mesophilic ethanol-resistant ⁇ -glucosidase
  • the activity has a promoting effect, in which the ethanol concentration in the reaction system is 10-20% (v/v), the thermophilic and ethanol-resistant ⁇ -glucosidase activity is most enhanced, and the mesophilic and ethanol-resistant ⁇ -glucosidase activity is increased 86.29. %.
  • Example 4 The mesophilic and ethanol-resistant ⁇ -glucosidase of the present invention is applied to high-temperature simultaneous saccharification and fermentation
  • Substrate The bagasse is pulverized to 60 mesh, 2% NaOH, pretreated at 80 ° C for 2 h, washed with tap water to neutral, and dried to constant weight at 65 ° C;
  • the high temperature yeast NCYC587 was activated by culturing the YM liquid medium at 42 ° C for 24 h;
  • the reaction system is: 500 mL of a 500 mL shake flask reaction solution, and the reaction solution contains 30 g of alkali-treated bagasse, and an inorganic salt component: (NH 4 ) 2 HPO 4 0.5 g/L, MgSO 4 ⁇ 7H 2 O0.025 g/ L, yeast extract 1.0 g / L, the balance is pH 5.0Na 2 HPO 4 - citrate buffer.
  • the effect of the mesophilic ethanol-resistant ⁇ -glucosidase on high-temperature simultaneous saccharification fermentation was studied by using the mesophilic ethanol-resistant ⁇ -glucosidase preparation of the present invention as a control.
  • thermophilic ethanol-resistant ⁇ -glucosidase was applied to high-temperature synchronous saccharification and fermentation, and the highest yield of ethanol was obtained after fermentation for 24 hours.
  • the ethanol content was as high as 28.2 g/L, compared with the control. Ethanol production increased by 39%.
  • the mesophilic and ethanol-resistant ⁇ -glucosidase property of the invention is beneficial to the application of the high-temperature synchronous saccharification fermentation technology of cellulose, and the cellobiose remains at the bottom concentration level during the whole fermentation, effectively eliminating the inhibition of the terminal product, in the cellulose
  • the enzyme Under the optimum conditions of cellulase enzymatic hydrolysis and yeast fermentation in the high-temperature simultaneous saccharification and fermentation process for producing fuel ethanol, the enzyme has high enzymatic activity and obvious effect, and can be applied to produce fuel ethanol. It is indicated that the mesophilic and ethanol-resistant ⁇ -glucosidase of the present invention has important application value in energy.

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Abstract

提供一种嗜温耐乙醇β-葡萄糖苷酶及其编码基因和应用。嗜温耐乙醇β-葡萄糖苷酶的氨基酸序列如SEQIDNO：2所示，其编码基因的核苷酸序列如SEQIDNO：1所示。嗜温耐乙醇β-葡萄糖苷酶的性质有利于其在纤维素高温同步糖化发酵技术中的应用，该酶具有较高的酶活和明显的作用效果，纤维二糖在整个发酵期间保持在低浓度水平，能有效消除终端产物抑制，可以用于生产燃料乙醇。

Description

一种嗜温耐乙醇β-葡萄糖苷酶及其编码基因和应用 技术领域：
本发明属于酶基因工程和酶工程领域，具体涉及一种嗜温耐乙醇β-葡萄糖苷酶及其编码基因和应用。
背景技术：
β-葡萄糖苷酶(EC3.2.1.21)属于纤维水解酶类，是一类催化水解或转移β-1,4-糖苷键的酶。是纤维素酶系的重要组成部分,在纤维素酶水解纤维素的过程中，纤维素类物质经酶促作用成葡萄糖至少需要三种酶的协同作用，葡聚糖内切酶，葡聚糖外切酶和β-葡萄糖苷酶。葡聚糖内切酶和葡聚糖外切酶把纤维素降解成纤维二糖，它再被β-葡萄糖苷酶分解成葡萄糖。β-葡萄糖苷酶水解纤维二糖释放葡萄糖是纤维素水解的一个关键限速步骤。
但是，β-葡萄糖苷酶在纤维素酶系中含量最低，不足1％，且活性普遍偏低，是纤维素酶解转化的瓶颈。长期以来，酶性质不够优良、酶的产量低及酶的比活力低下一直是影响酶实际应用的重要因素。虽然β-葡萄糖苷酶在微生物中广泛存在，但是真菌、细菌等微生物的产酶效率不高，难以获得大量产品，且热稳定性较差。目前β-葡萄糖苷酶的活力仍不能满足工业生产的需要，而且成本较高。在纤维素糖化过程中，纤维素酶的最适作用温度通常为50℃左右，而酵母发酵的最适温度为30℃左右。如何使这两个过程的温度协调，是采用同步糖化发酵(SSF)高效生产乙醇的关键，解决这种矛盾办法之一就是采用耐高温酵母。因此，对β-葡萄糖苷酶基因的克隆与表达，已成为研究纤维素酶重要环节之一。到目前为止，已有上百个微生物的β-葡萄糖苷酶基因得以克隆，许多微生物来源的β-葡萄糖苷酶基因也已获得异源表达。近年来，国际上通过基因重组技术构建工程菌，分泌表达高活性、热稳定的β-葡萄糖苷酶研究是纤维素酶学领域研究的热点。
关于嗜温β-葡萄糖苷酶及其基因已有的专利和文献报道：Piyanun Harnpicharnchai等报道的Periconia sp.β-葡萄糖苷酶基因片段BCC2871[蛋白质表达与纯化(Protein Expres Purif)67：61–69，2009]；Patrick Murraya等报道嗜 热真菌Talaromyces emersonii的β-葡萄糖苷酶基因Cel3a[蛋白质表达与纯化(Protein Expres Purif)38：248–257，2004]；Qiaojuan Yan等报道的Paecilomyces thermophila β-葡萄糖苷酶基因PtBglu3[蛋白质表达与纯化(Protein Expres Purif)84：64–72，2012]；Xiao-Qiong Pei等报道的嗜热Thermobifida Fusca β-葡萄糖苷酶基因BglC[生物资源技术(Bioresour.Technol.)102：3337–3342，2011]；Linguo Zhao等报道的Thermotoga thermarum DSM 5069T高温β－葡萄糖苷酶基因Tt-bgl[分子催化杂志，B辑:酶催化(JMol Catal B-Enzym)95：62–69，2013]等。微生物产β-葡萄糖苷酶的种类众多，不同来源的β-葡萄糖苷酶具有不同的性质。由于工业上应用的不同，因此需要不断开发出具有新特性的β-葡萄糖苷酶以更好的适应工业生产的需要。其中耐热酶具有很大优越性，可以提高反应速度、延长作用时间，减少污染、增强对化学试剂的耐受性等，以及可以在所需要的特殊条件下进行反应，因此开发嗜温β-葡萄糖苷酶已成为研究热点。
我们之前研究中，获得了2株β-葡萄糖苷酶的产生菌：绿色木霉(Trichoderma viride)W2，专利号ZL 2010 1 0577713.6和肉座菌(Hypocrea sp.)W63，专利号ZL 2011 1 0417104.9，这2株菌均有嗜温、耐乙醇的特性，其中绿色木霉(Trichoderma viride)W2最适反应pH值为4.8，最适反应温度为70℃，乙醇浓度为10％(v/v)对酶活有最大促进作用，对β-葡萄糖苷酶酶活提高1.6倍，乙醇耐受能力高达30％(v/v)；肉座菌(Hypocrea sp.)W63最适反应pH值为4.8，最适反应温度为65℃，乙醇浓度为10％(v/v)对酶活有最大促进作用，对β-葡萄糖苷酶酶活提高将近1倍，乙醇耐受能力高达30％(v/v)。
通常情况下，真菌所产的纤维素酶系主要是内切葡聚糖酶和外切葡聚糖酶，β-葡萄糖苷酶在纤维素酶系中含量最低，不足1％。利用分子生物学技术构建的基因工程菌具有遗传稳定性高、产酶迅速、产酶量大等优点，生产的重组酶拟满足未来大量工业应用需求。据国内外已有的报道来看，嗜热型β-葡萄糖苷酶基因克隆主要集中来自于嗜热性细菌、真菌，然而这些嗜热型微生物所产的β-葡萄糖苷酶并不一定就具有嗜热及热稳定性能。在产β-葡萄糖苷酶微生物研究中，木霉是研究最早和最广泛的种属之一，但是从肉座菌属克隆的具有嗜温耐乙醇性能的β-葡萄糖苷酶基因尚未见报道。
发明内容：
本发明的第一个目的是提供一种嗜温耐乙醇β-葡萄糖苷酶及其编码基因。
本发明的嗜温耐乙醇β-葡萄糖苷酶，其特征在于，其氨基酸序列如SEQ ID NO：2所示。
本发明的编码嗜温耐乙醇β-葡萄糖苷酶的基因，其特征在于，编码氨基酸序列如SEQ ID NO：2所示的嗜温耐乙醇β-葡萄糖苷酶。
所述的编码嗜温耐乙醇β-葡萄糖苷酶的基因，优选，其核苷酸序列如SEQ ID NO：1所示。
肉座菌(Hypocrea sp.)W63(ZL201110417104.9)生产嗜温耐乙醇β-葡萄糖苷酶，该酶具有水解4-nitrophenyl β-D-glucopyranoside(pNPG)底物特异性，其最适反应pH值为4.8，最适反应温度为65℃，反应中乙醇浓度为10％(v/v)对酶活有最大促进作用，对β-葡萄糖苷酶酶活提高将近1倍，乙醇耐受能力高达30％(v/v)。
本发明从肉座菌(Hypocrea sp.)W63得到的嗜温耐乙醇β-葡萄糖苷酶基因epB-BGL，具有2202bp，其核苷酸序列如SEQ ID NO：1所示，该嗜温耐乙醇β-葡萄糖苷酶基因编码由733个氨基酸组成的蛋白质，其氨基酸序列如SEQ ID NO：2所示。本发明通过分子生物学方法获得了含有该基因片段的以pPIC9K为表达载体的重组质粒，以毕赤酵母GS115为表达宿主，使重组菌株胞外分泌表达嗜温耐乙醇β-葡萄糖苷酶。因此，本发明成功通过基因工程或分子生物学手段，将嗜温耐乙醇β-葡萄糖苷酶基因克隆到其他受体菌，由其他受体菌产生本发明的嗜温耐乙醇β-葡萄糖苷酶。
与已知的β-葡萄糖苷酶相比，本发明的嗜温耐乙醇β-葡萄糖苷酶是一种新型的具有新功能的β-葡萄糖苷酶，通过对本发明的嗜温耐乙醇β-葡萄糖苷酶的基因推导氨基酸的序列比较分析，本发明的嗜温耐乙醇β-葡萄糖苷酶与其他已报道的β-葡萄糖苷酶的氨基酸序列相比，SEQ ID NO：2所示的氨基酸序列与来源于里氏木霉Trichoderma reesei的β-葡萄糖苷酶(Genbank索引号AAA18473.1)同源性最高，它们的氨基酸序列相似性为79％，相似性高于79％的β-葡萄糖苷酶来源于深绿木霉Trichoderma atroviride的IMI 206040(Kubicek,C.P.等，Genome Biol.12(4),R40(2011)，未做功能鉴定)；绿木霉Trichoderma virens的Gv29-8， (Kubicek,C.P.等，Genome Biol.12(4),R40(2011)，未做功能鉴定)。本发明的嗜温耐乙醇β-葡萄糖苷酶与其它已报道的β-葡萄糖苷酶氨基酸序列比对以后发现，相似性大于79％的菌株中，都是进行序列比对或蛋白结构的研究，目前为止并没有发现对相应菌株的β-葡萄糖苷酶基因的功能进行鉴定的相关文献报道。由此可见，本发明的嗜温耐乙醇β-葡萄糖苷酶基因epB-BGL是一个新的基因。本发明的嗜温耐乙醇β-葡萄糖苷酶已在GenBank上提交核苷酸序列，其核苷酸序列如SEQ ID NO：1所示，其序列号为KJ502670。
本发明的第二个目的是提供一种重组表达载体，其特征在于，含有本发明的嗜温耐乙醇β-葡萄糖苷酶基因。
所述的重组表达载体，其表达载体优选为pPIC9K表达载体。
本发明的第三个目的是提供一种宿主细胞，其特征在于，含有上述重组表达载体的真核细胞或原核细胞。
所述的原核细胞，可以为各种细菌，如酵母工程菌、大肠杆菌或枯草杆菌。
所述的酵母工程菌，优选为毕赤酵母GS115菌株。
本发明的第四个目的是提供嗜温耐乙醇β-葡萄糖苷酶在纤维素高温同步糖化发酵乙醇中的应用。
通过对酶的氨基酸序列相似性分析，目前已知的β-葡萄糖苷酶分别有属于糖苷水解酶第1和第3家族。通过对本发明的嗜温耐乙醇β-葡萄糖苷酶序列比对分析，认为其属于糖苷水解酶第3家族的一员。
此外，本发明的嗜温耐乙醇β-葡萄糖苷酶的酶学特性不同于已知的β-葡萄糖苷酶，其反应酶活经活性测定，其反应温度为40-90℃，其最适反应温度为70℃；反应pH值为4.0-6.5，最适反应pH值为5.0；乙醇浓度在30％(v/v)以内均能对β-葡萄糖苷酶酶活有促进作用，其中反应体系中乙醇浓度为10-20％(v/v)时对β-葡萄糖苷酶活力促进效果最强，β-葡萄糖苷酶活力提高86.29％，证明该酶具有在高温、乙醇存在条件下保持较高酶活的特点。该嗜温耐乙醇β-葡萄糖苷酶的分子量为76740道尔顿，pI为6.01。纯化后该酶的最佳比活力为194.25U/mg，其特异底物为pNPG，该酶是一种特异催化水解β-葡萄糖苷键将纤维二糖降解成葡萄糖的酶，是纤维素酶系中一类关键的酶，将本发明的嗜温耐乙醇β-葡萄糖苷酶应用在高温同步糖化上生产乙醇，首先对生物质原料预水解，然后加入嗜温耐 乙醇β-葡萄糖苷酶酶液和耐高温酵母进行高温同步糖化发酵，能有效消除纤维二糖抑制，乙醇产量提高39％。在以纤维素为原料生产燃料乙醇的同步糖化发酵工艺中的纤维素酶酶解和酵母菌发酵的最适条件下，该酶具有较高酶活，可以应用该工艺中以生产燃料乙醇，因此，本发明的嗜温耐乙醇β-葡萄糖苷酶在能源方面具有重要的应用价值。
附图说明：
图1是重组质粒pPIC9K的构建模式图；
图2是本发明的嗜温耐乙醇β-葡萄糖苷酶cDNA的凝胶电泳图，其中，M泳道是Marker，泳道1为目的基因的cDNA；
图3是本发明的嗜温耐乙醇β-葡萄糖苷酶克隆质粒pPIC9K-epB的EcoRI,AvrII双酶切电泳图，其中M泳道为Marker，泳道1为质粒pPIC9K-epB的EcoRI,AvrII双酶切；
图4是P.pastoris GS115表达嗜温耐乙醇β-葡萄糖苷酶纯化的电泳图，其中M泳道为Marker，泳道1为Micro-Prep DEAE柱纯化；
图5是本发明的嗜温耐乙醇β-葡萄糖苷酶蛋白质谱的二级峰图；
图6是本发明的嗜温耐乙醇β-葡萄糖苷酶最佳反应温度及pH值；
图7是乙醇添加对本发明的嗜温耐乙醇β-葡萄糖苷酶酶活的影响；
图8是添加本发明的嗜温耐乙醇β-葡萄糖苷酶对高温同步糖化发酵效果。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
在本发明的实施例中所用到的材料包括：真菌总RNA快速抽提试剂盒(购自生工生物工程有限公司)；cDNA第一链合成试剂盒(购自Thermo公司)；巴氏毕赤酵母(Pichia pastoris)GS115、引物合成(购自Invitrogen公司)；pPIC9K表达载体(购自Invitrogen公司)；感受态细胞Trans-T1、pEASY-Blunt Zero Cloning Kit(购自TransGen Biotech)；PCR试剂、限制性内切酶EcoRI、AvrⅡ和SalⅠ，T4 DNA连接酶(购自Takara公司)，纤维素酶(购自Genencor公司)，高温酵母菌NCYC587(购于英国国立酵母保藏中心)；肉座菌(Hypocrea sp.)W63于2011年9月1日保藏于中国微生物菌种保藏管理委员会普通微生物中心 (CGMCC)，其保藏编号为：CGMCCNo.5209(该菌株公布于中国专利201110417104.9)。
P.pastoris GS115感受态细胞的制备为现有技术。
实施例1：本发明的嗜温耐乙醇β-葡萄糖苷酶基因克隆
1、肉座菌(Hypocrea sp.)W63总RNA的提取
前期研究证明，由肉座菌(Hypocrea sp.)W63所产的β-葡萄糖苷酶具有嗜温耐乙醇的酶学性能，在高温和乙醇存在的情况下保持酶的高活性，不同于已知的β-葡萄糖苷酶。因此，在本实施例中，利用真菌总RNA快速抽提试剂盒提取肉座菌(Hypocrea sp.)W63的RNA，具体操作步骤如下：
(1)、取450μL Buffer Rlysis-F加入1.5mL RNase-free的离心管中备用；
(2)、取其新鲜培养物，离心收集菌体，0.1g菌丝在液氮中充分研磨，移入上述1mL离心管中，立即震荡混匀，室温放置5min；
(3)、向裂解样品中加入0.2mL氯仿，用旋涡震荡器混匀。于4℃下12,000g离心5min，小心吸取上清夜；
(4)、上清液转移至1.5mL RNase-free的离心管中，加入1/2体积无水乙醇，充分混匀；
(5)、将吸附柱放入收集管中，用移液器将全部溶液加至吸附柱中，静置1min，12,000g离心1min，倒掉收集管废液；
(6)、在吸附柱中分别加入10×DNase I Buffer 3μL；Recombinant DNase I(5U/μL)1.5μL；RNase Inhibitor(40U/μL)1.5μL；DEPC-treated water 24μL，37℃反应20-30min，除去DNA干扰；
(7)、在吸附柱中加入500μL GT Solution，静置1min，10,000g离心1min，倒掉收集管废液；
(8)、将吸附柱放入收集管中，加入500μL NT Solution，静置1min，10,000g离心1min，倒掉收集管废液；
(9)、将吸附柱放入收集管中，12,000g离心2min，打开盖子数分钟(挥发残留乙醇)；
(10)、将吸附柱放入1.5mL RNase-free的离心管中，加入30-50μLDEPC-treated water，静置2min，12,000g离心2min，将所得的RNA置于-70℃ 冻存。
2、本发明的嗜温耐乙醇β-葡萄糖苷酶基因cDNA克隆
采用cDNA第一链合成试剂盒，取上述的总RNA溶液1μL作为模板进行反转录cDNA连接，然后从GenBank数据库中搜索到7种β-葡萄糖苷酶相似性较高的氨基酸序列，利用DNAman软件中的Multiple Sequence Alignment对这些序列进行同源性比较，根据同源β-葡萄糖苷酶基因的两段高度保守序列和已知序列设计一对引物P1和P3。对反转录的cDNA进行PCR扩增。
P1：5-WSNATHTGGGAYACNTT-3
P3：5-CCNARYTGYTTNCKCAT-3
TOUCHDOWNPCR(降落PCR)：
反应条件：94℃预变性3min；然后进行以下循环：94℃变性30s；60-45℃退火30s；72℃延伸30s，进行10个循环；94℃变性30s；45℃退火30s；72℃延伸30s，进行25个循环；最后72℃延伸6min。进行1％的琼脂糖凝胶电泳检测有无适当大小的目的条带。然后于-20℃保存PCR产物。获得目的条带，大小约1.8kb。
根据已知序列比对设计特异引物，进行PCR反应。反应程序如下：94℃预变性5min，然后进行以下循环：94℃变性30s，52℃退火30s，72℃延伸30s，共进行30个循环，最后72℃延伸10min。PCR产物经1％的琼脂糖凝胶电泳分析。目的基因片段的连接、转化、鉴定和测序。获得目的基因全长cDNA，大小约2.2kb(如图2所示)。
将获得的序列用NCBI上的软件BLAST对序列进行分析，得到嗜温耐乙醇β-葡萄糖苷酶的编码基因，命名为epB-BGL，由2202个核苷酸组成，嗜温耐乙醇β-葡萄糖苷酶基因epB-BGL编码一个含733个氨基酸的蛋白质，用DNAstar软件预测该蛋白质理论分子量大小为76550.45道尔顿，等电点pI为6.01。和epB-BGL基因催化功能域同源性最高的是来源于里氏木霉Trichoderma reesei的β-葡萄糖苷酶(Genbank索引号AAA18473.1)，它们的氨基酸序列相似性为79％。本发明的嗜温耐乙醇β-葡萄糖苷酶基因epB-BGL已在GenBank上提交核苷酸序列，获得序列号为KJ502670。
实施例2：本发明的β-葡萄糖苷酶在酵母工程菌中表达和纯化
1、epB-BGL基因表达质粒的构建
根据已获得的嗜温耐乙醇β-葡萄糖苷酶全长cDNA序列，设计一对表达引物ep3、ep5，去除信号肽序列和3’、5’非编码区序列，为保证epB-BGL基因定向插入载体pPIC9K，在上下游引物两端分别引入EcoRI和AvrⅡ酶切位点。两引物间距约2.5kb，扩增产物包含嗜温耐乙醇β-葡萄糖苷酶成熟蛋白编码序列，
ep3：5'-CGGAATTCATGCTTTACACAGCCGTAGCG-3'
ep5：5'-CCCCTAGGCTATGAGACCGTGAAGCTTCC-3'
以扩增嗜温耐乙醇β-葡萄糖苷酶全长cDNA的PCR产物为模板，以ep5、ep3为上下游引物进行PCR，获得嗜温耐乙醇β-葡萄糖苷酶成熟肽的基因表达序列。将目的基因片段回收后，利用pEASY-Blunt Zero Cloning Kit将目的片段与pPIC9K表达载体进行连接，并转化大肠杆菌感受态细胞Trans-T1，通过菌落PCR、质粒DNA的酶切鉴定筛选出阳性克隆并测序，将测序鉴定正确的质粒命名为pPIC9K-epB。
将经测序验证含嗜温耐乙醇β-葡萄糖苷酶基因正确读码框的质粒pPIC9K-epB用EcoRI和AvrⅡ双酶切，回收插入片断，并与同样双酶切的酵母表达质粒pPIC9K用T4 DNA连接酶进行连接，转化大肠杆菌感受态细胞Trans-T1，转化的Trans-T1经Amp抗性筛选，菌落经37℃摇培过夜后抽提质粒，重组质粒进行酶切鉴定并测序，酶切鉴定结果如图3所示，将该重组质粒命名为pPIC9K-epB，其构建模式图如图1所示。
2、重组质粒pPIC9K-epB在巴氏毕赤酵母GS115中的表达、纯化
用SalⅠ限制性内切酶(位于His4区域内)将构建好的重组质粒pPIC9K-epB进行线性化酶切，同时将空pPIC9K载体质粒与含epB-BGL基因的重组质粒pPIC9K-epB的线性化酶切及酵母转化同步进行，作阳性对照。
酵母转化和筛选：
(1)、P.pastoris GS115感受态细胞的制备；
(2)、重组表达质粒pPIC9K-epB电击转化P.pastoris GS115感受态细胞，电击法的具体步骤如下：
a、将10μL线性化DNA与80μL的上述步骤(1)所得的菌体混匀，转至0.2cm预冷的电转化杯中；
b、将电转化杯置冰上冰浴5min
c、根据电转化仪的内置酵母转化程序进行电击：电压1.5-1.8kV，电容25μF；电阻200Ω。电击时间为4-5msec
d、电击完毕后，立即向电转杯加入1mL1M山梨醇溶液，将菌体混匀，转至新的EP管中，将菌体均匀涂布于MD平板上，30℃培养至单菌落出现，观察菌落的大小并计数，筛选在MD平板上菌落形态生长饱满的转化子。
(3)、将电击转化后的待检酵母转化子用无菌的一次性牙签按一定方向对应接种于MD平板上，30℃倒置培养2-4d，甘油种保存。
酵母工程菌的培养和嗜温耐乙醇β-葡萄糖苷酶的诱导分泌表达：
(1)、挑取上述阳性菌落，接种于含25mLBMGY培养基的250mL摇瓶中，28-30℃振荡培养(250-300rpm)至对数生长期(OD600达2-6，约16-18小时)，以转化pPIC9K空载体的GS115菌株作为对照；
(2)、室温下以3000g离心5min回收酵母细胞，弃去上清，将细胞重悬于适当体积的BMMY培养基中，至OD600值为1.0-2.0(约100-200mL)将培养液置于500mL摇瓶中，覆盖8层无菌纱布，置摇床中，28-30℃继续培养并开始诱导表达(注意诱导温度应严格控制，不要超过30℃)；
(3)、诱导表达起始后，每隔24h补加100％甲醇至终浓度为1％以维持诱导；
(4)、诱导表达5d，取发酵上清液，4℃，12000rpm离心，对表达产物的酶活性进行检测分析。
嗜温耐乙醇β-葡萄糖苷酶工程菌表达产物的纯化：
将提取的粗酶液用90％饱和度的硫酸铵沉淀4h后，4℃，14000rpm离心20min，收集沉淀，用适量的缓冲液A(20mmol/L Tris-HCl缓冲液，pH7.0)回溶，将上清液施于经缓冲液A预先平衡好的Desalting脱盐柱，收集A280nm处出峰的2mL，将脱盐后收集的酶液施于经缓冲液A预先平衡好的Micro-Prep DEAE柱，酶蛋白先用3倍柱体积的缓冲液A洗脱至A280不变后，再用5倍缓冲液A(20mmol/L Tris-HCl缓冲液，pH7.0和缓冲液B(1mol/L Tris-HCl缓冲液，pH7.0)的相同比例缓冲溶液进行梯度洗脱，流速为1mL/min，每管 收集1mL。每管进行酶活性测定和蛋白浓度测定，最后进行SDS-PAGE电泳检测纯度(结果如图4所示)，纯化后酶的最佳比活力为194.25U/mg。经SDS-PAGE电泳后切胶回收，对目的蛋白进行蛋白质谱鉴定分析，测得酶的分子量为76740道尔顿，与理论推算的分子量(76550道尔顿)相似，其蛋白质谱结果二级峰图如图5所示。
实施例3：本发明的嗜温耐乙醇β-葡萄糖苷酶酶学特性
本发明的嗜温耐乙醇β-葡萄糖苷酶的酶活力测定：用4-nitrophenyl β-D-glucopyranoside(pNPG)作为底物，反应体系为2mL，先将1mLpNPG(5mmol/L)和0.9mL pH5.0Na2HPO4-柠檬酸缓冲液混匀，再加入0.1mL适当稀释的上一步骤得到的嗜温耐乙醇β-葡萄糖苷酶制剂，50℃反应10min，立即加入3mLlmol/L的Na2CO3溶液终止反应，室温放置5min，于400nm处测光吸收值(OD)。
酶活定义：在1min内催化产生1μmol/L对硝基酚的量定义为一个酶单位。
嗜温耐乙醇β-葡萄糖苷酶的酶学性质的测定：β-葡萄糖苷酶制剂，按pNPG测定方法，其他条件不变情况下，调节不同pH缓冲液、不同温度、不同乙醇浓度进行酶活测定反应，以测得最高酶活为100％，该条件为测得嗜温耐乙醇β-葡萄糖苷酶最适反应条件。
本发明的嗜温耐乙醇β-葡萄糖苷酶具有高温和乙醇存在条件下保持高酶活的特性，其反应温度为40-90℃，其最适反应温度为70℃(如图6A所示)；反应pH值为4.0-6.5，最适反应pH值为5.0-5.5(如图6B所示)；乙醇浓度在30％(v/v)以内均能对嗜温耐乙醇β-葡萄糖苷酶酶活有促进作用，其中反应体系中乙醇浓度为10-20％(v/v)时对嗜温耐乙醇β-葡萄糖苷酶活力促进效果最强，嗜温耐乙醇β-葡萄糖苷酶活力提高86.29％。
实施例4：本发明的嗜温耐乙醇β-葡萄糖苷酶应用于高温同步糖化发酵
(1)、底物：甘蔗渣粉碎至60目，2％NaOH，80℃预处理2h，自来水冲洗至中性，65℃烘干至恒重；
(2)、高温酵母菌NCYC587用YM液体培养基42℃培养24h活化；
(3)、向反应体系加入纤维素酶30FPU/g底物酶量，50℃预水解24h；
(4)、再加入嗜温耐乙醇β-葡萄糖苷酶制剂15FPU/g底物进反应体系，按 10％的接种量接种酵母至反应体系中，45℃发酵，以接种酵母时算为0h，于0，24，48，96，120h取样，HPLC检测乙醇、还原糖含量。
所述的反应体系为：500mL摇瓶装反应液200mL，该反应液中含有碱处理甘蔗渣30g、无机盐成分：(NH4)2HPO40.5g/L，MgSO4·7H2O0.025g/L，酵母膏1.0g/L，余量为pH5.0Na2HPO4-柠檬酸缓冲液。
以不加本发明的嗜温耐乙醇β-葡萄糖苷酶制剂作为对照，研究嗜温耐乙醇β-葡萄糖苷酶应用于高温同步糖化发酵的效果。
结果如图8所示，所得嗜温耐乙醇β-葡萄糖苷酶应用于高温同步糖化发酵中，发酵至24h即可得乙醇最高产量，所产乙醇含量高达28.2g/L，与对照相比，乙醇产量提高39％。由此可见本发明的嗜温耐乙醇β-葡萄糖苷酶性质有利于纤维素高温同步糖化发酵技术的应用，整个发酵期间纤维二糖保持在底浓度水平，有效消除终端产物抑制，在以纤维素为原料生产燃料乙醇的高温同步糖化发酵工艺中的纤维素酶酶解和酵母菌发酵的最适条件下，该酶具有较高酶活和明显作用效果，可以应用该工艺中以生产燃料乙醇，表明本发明的嗜温耐乙醇β-葡萄糖苷酶在能源方面具有重要的应用价值。
上述详细说明是针对本发明的可行实施例的具体说明，该实施例并非用以限制本发明的专利范围，凡未脱离本发明的等效实施或变更，均应包含于本发明的专利范围中。
Figure PCTCN2014088168-appb-000001
Figure PCTCN2014088168-appb-000002
Figure PCTCN2014088168-appb-000003
Figure PCTCN2014088168-appb-000004
Figure PCTCN2014088168-appb-000005
Figure PCTCN2014088168-appb-000006

Claims (9)

  1. 一种嗜温耐乙醇β-葡萄糖苷酶，其特征在于，其氨基酸序列如SEQIDNO：2所示。
  2. 一种编码权利要求1所述的嗜温耐乙醇β-葡萄糖苷酶的基因。
  3. 根据权利要求2所述的编码嗜温耐乙醇β-葡萄糖苷酶的基因，其特征在于，其核苷酸序列如SEQIDNO：1所示。
  4. 一种重组表达载体，其特征在于，含有权利要求2或3所述的编码嗜温耐乙醇β-葡萄糖苷酶的基因。
  5. 根据权利要求4所述的重组表达载体，其特征在于，所述的表达载体为pPIC9K。
  6. 一种宿主细胞，其特征在于，含有权利要求4或5所述的重组表达载体的真核细胞或原核细胞。
  7. 根据权利要求6所述的宿主细胞，其特征在于，所述的原核细胞为酵母工程菌、大肠杆菌或枯草杆菌。
  8. 根据权利要求7所述的宿主细胞，其特征在于，所述的酵母工程菌为巴氏毕赤酵母GS115菌株。
  9. 权利要求1所述的嗜温耐乙醇β-葡萄糖苷酶在纤维素高温同步糖化发酵乙醇中的应用。
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CN111876399B (zh) * 2020-07-13 2022-03-01 中国水产科学研究院黄海水产研究所 北极来源的β-葡萄糖苷酶基因、及其编码的蛋白和应用
CN112538437B (zh) * 2020-12-09 2022-02-01 江南大学 一种通过代谢工程改造提高蒎烯生物合成的方法
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CN115094052B (zh) * 2022-06-08 2023-05-19 大理大学 一种嗜热、耐受重金属离子的β-葡萄糖苷酶及其应用
CN117384799B (zh) * 2023-11-29 2024-06-11 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) 一株鲁戈斯芽孢杆菌A78.1、菌剂、产β-葡萄糖苷酶的方法和应用

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