# Patent

## Title
Thermophilic ethanol-resistant β-glucosidase and encoding gene and application thereof

## Abstract

The present invention discloses thermophilic ethanol-resistant β-glucosidase and an encoding gene and application thereof. The amino acid sequence of the thermophilic ethanol-resistant β-glucosidase of the present invention is as shown in SEQ ID NO: 2, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO: 1. The nature of the thermophilic ethanol-resistant β-glucosidase of the present invention is conductive to the application of cellulose high-temperature simultaneous saccharification and fermentation, the enzyme has a relatively high enzyme activity and obvious active effect, cellobiose is kept at low concentration level during the whole fermentation period, the inhibition of terminal products can be effectively eliminated, the thermophilic ethanol-resistant β-glucosidase can be applied on the process of fuel ethanol production, and the important application value of the thermophilic ethanol-resistant β-glucosidase of the present invention in the aspect of bioenergy is further indicated.

## Background
FIELD OF THE INVENTION
The invention belongs to the field of enzyme gene engineering and enzyme engineering and specifically relates to thermophilic ethanol-resistant β-glucosidase and an encoding gene and application thereof.
BACKGROUND OF THE INVENTION
β-Glucosidase (EC3. 2. 1, 21) belongs to the class of cellulose hydrolases and is one kind of enzyme, which can used for hydrolysis or transferring β-1,4-glycosidic bonds. The β-glucosidase is an important constituent in cellulase system. No less than three kinds of enzymes, namely endoglucanase, cellobiohydrolase and β-glucosidase are needed for the cellulose conversion into glucose. Cellulose can be degraded into cellobiose by endoglucanase and cellobiohydrolase, and further decomposed into glucose by β-glucosidase. Releasing of glucose from cellobiose is the key rate-limiting step for the cellulose hydrolysis.
However, the content of β-glucosidase in cellulase system is very low, which is less than 1%, and the activity is generally relatively low, which is a main bottleneck for cellulose enzymatic conversion. For a long time, the poor enzymes properties, low yields of the enzymes and low specific activity of the enzymes are main bottlenecks on their actual application. Although β-glucosidase widely exists in nature, fungi, bacteria and other microbes have low enzyme production efficiency, are difficult to obtain quantities of β-glucosidase, and still have relatively poor thermostability. At present, the activity of β-glucosidase can not meet the needs of industrial production yet, and the cost is further relatively high. In the saccharification process of cellulose, the most appropriate action temperature of the cellulase is about 50° C. generally, while the most appropriate temperature for yeast fermentation is about 30° C. How to coordinate the temperature of the two processes is the key for efficiently producing ethanol by simultaneous saccharification and fermentation (SSF), and one of the methods for solving the contradiction is using a thermophilic yeast instead of traditional yeast. Thus, cloning and expression of β-glucosidase genes have been one of important links on cellulose studies. So far, β-glucosidase genes have been cloned from hundreds of microbes, and many β-glucosidase genes from microbes are heterologously expressed. In recent years, gene engineering technology was used to construct recombinant microorganisms to secret and express β-glucosidases with high activity and thermal stability, which is a research hotspot in the field of cellulose studies.
The known patents and literature reports of thermophilic β-glucosidase and the genes thereof are as follows: β-glucosidase gene fragment BCC2871 from Periconia sp. reported by Piyanun Harnpicharnchai, et al., [Protein Expres Purif, 67: 61-69, 2009]; β-glucosidase gene Cel3a from thermophilic fungal Talaromyces emersonii reported by Patrick Murraya, et al., [Protein Expres Purif, 38: 248-257, 2004]; β-glucosidase gene PtBglu3 from Paecilomyces thermophila reported by Qiaojuan Yan, et al., [Protein Expres Purif, 84: 64-72, 2012]; β-glucosidase gene BglC from Thermobifida Fusca reported by Xiao-Qiong Pei et al., [Bioresour. Technol., 102: 3337-3342, 2011]; and DSM 50691 thermophilic β-glucosidase gene Tt-bgl from Thermotoga thermarum reported by Linguo Zhao, et al., [J Mol Catal B-Enzym, 95: 62-69, 2013] and so on. There are a variety of types of β-glucosidase produced by microbes, and β-glucosidases from different, sources showed different characteristics. Due to different industrial applications, the β-glucosidase with new properties needs to be developed continuously for being better applicable to the industrial production needs. Wherein, thermophilic enzymes have peat advantages, including higher reaction rate, long-time reaction stability low pollution possibility and strengthen the tolerance to chemical reagents, and high enzyme activities under the required special conditions and so on. So the development of thermophilic β-glucosidase has become the research hotspot.
In our previous studies, we obtained two strains of strains producing β-glucosidase: Trichoderma viride W2 with patent number of ZL 201010577713.6 and Hypocrea sp. W63 with patent number of ZL 201110417104.9, both of them showed thermophilic and ethanol-resistant properties. Wherein the optimal pH value of the Trichoderma viride W2 is 4.8, the optimal temperature is 70° C. The activity of β-glucosidase can be largely improved by 1.6 times at 10% (v/v) of ethanol concentration, which is stable below 30% (v/v) of ethanol concentration; and the optimal pH value of Hypocrea sp. W63 is 4.8, the optimal temperature is 65° C., and the concentration of ethanol at 10% (v/v) also has the greatest effect of promoting on enzyme activity, which can improve the enzyme activity of β-glucosidase by nearly 1 time and the highest ethanol resistance concentration also is 30% (v/v).
Under normal circumstances, the cellulose produced by fungi mainly comprises endoglucanase and cellobiohydrolase, and the content of β-glucosidase in the cellulose system is the lowest, which is less than 1%. Many kinds of enzyme produced by gene engineering strain have the advantages of high genetic stability, rapid enzyme production, high enzyme yield and so on, and the produced recombinant enzymes are intended to meet the demands of future industrial applications. According to worldwide reports, the gene cloning of thermophilic β-glucosidases were mainly focused on cloned from thermophilic bacteria and fungi. However, the β-glucosidase produced by the thermophilic microbes do not have thermophilicity and thermal stability necessarily. As for β-glucosidases producing, microbes Trichoderma is one of the species researched most extensively before, but there are no report about β-glucosidase genes with thermophilic ethanol-resistant performance, which are cloned from Hypocrea sp.
SUMMARY OF THE INVENTION
The first purpose of the present invention is to provide thermophilic ethanol-resistant β-glucosidase and an encoding gene thereof.
The thermophilic ethanol-resistant β-glucosidase of the present invention, wherein the amino acid sequence is as shown in SEQ ID NO: 7.
A gene encoding the thermophilic ethanol-resistant β-glucosidase of the present invention, wherein the thermophilic ethanol-resistant β-glucosidase with the amino acid sequence as shown in SEQ ED NO: 2 is encoded.
Preferably, the nucleotide sequence of the gene encoding the thermophilic ethanol-resistant β-glucosidase is as shown in SEQ ID NO: 1.
Hypocrea sp. W63 (ZL201110417104.9) produces the thermophilic ethanol-resistant β-glucosidase, the enzyme has the specificity of hydrolyzing a 4-nitrophenyl β-D-glucopyranoside (pNPG) substrate, the optimal pH value is 4.8, the optimal temperature is 65° C. and the concentration of ethanol in the reaction is 10% (v/v), thereby having the greatest effect of promoting the enzyme activity, improving enzyme activity of the β-glucosidase by nearly 1 time and enabling the ethanol tolerance to be as high as 30% (v/v).
The present invention obtains thermophilic ethanol-resistant β-glucosidase gene epB-BGL from Hypocrea sp. W63, the nucleotide sequence with the length of 2202 bp as shown in SEQ ID NO: 1, and the thermophilic ethanol-resistant β-glucosidase gene encodes a protein consisting of 733 amino acids, and the amino acid sequence is as shown in SEQ ID NO: 2. The present invention obtains a recombinant plasmid which contains a gene fragment and takes pPIC9K as an the expression vector, and Pichia pastoris GS115 is used as an expression host, the resulting recombinant strain containing the plasmid above has the ability of secreting the thermophilic ethanol-resistant β-glucosidase gene. Thus, the present invention successfully cloned and heterogenous expressed the thermophilic ethanol-resistant β-glucosidase gene into other host through gene engineering or molecular biological methods, and the thermophilic ethanol-resistant β-glucosidase of the present invention is produced by other host.
Compared with the known β-glucosidase, the thermophilic ethanol-resistant β-glucosidase of the present invention is a novel β-glucosidase with new functions. By sequence analysis on BLAST program, the encoding amino acid sequence of the thermophilic ethanol-resistant β-glucosidase gene of the present invention with other reported amino acid sequences of the β-glucosidase the amino acid sequence as shown in SEQ ID NO: 2 has the highest homology with that of the β-glucosidase from Trichoderma reesei (Genbank index number AAA18473.1) with the similarity of the amino acid sequences is 79%, and the β-glucosidases with the similarity higher than 79% is from Trichoderma atroviride EMI 206040 (Kubicek, C. P., et al., Genome Biol. 12 (4), R40 (2011), without functional identification); and Trichoderma virens Gv29-8 (Kubicek, C. R, et al., Genome Biol. 12 (4), R40 (2011), without functional identification). Those proteins with similarity higher than were performed with sequence identification or protein structure research, it is found that in the strains with the similarity of more than 79%, sequence comparison or research of the protein structures is performed, but so far, no related literature reports about functional characterization of the β-glucosidase genes of the corresponding strains have been found. It can be seen that the thermophilic ethanol-resistant β-glucosidase gene epB-BGL of the present invention is a new gene. The thermophilic ethanol-resistant β-glucosidase of the present invention has submitted the nucleotide sequence into GenBank under the number of 10502670, the nucleotide sequence is as shown in SEQ ID NO: 1.
The second purpose of the invention is to provide a recombinant expression vector, wherein containing the thermophilic ethanol-resistant β-glucosidase gene of the present invention.
Preferably, the recombinant expression vector is a pPIC9K expression vector.
The third purpose of the present invention is to provide a host cell, wherein is a eukaryotic cell or prokaryotic cell contains the recombinant expression vector.
The prokaryotic cell can be various organisms, such as a yeast engineering strain, Escherichia coli or Bacillus subtilis. 
Preferably the yeast engineering strain is Pichia pastoris GS115 strain.
The fourth purpose of the present invention is to provide an application of the thermophilic ethanol-resistant β-glucosidase in the production of ethanol from cellulose with process of simultaneous saccharification and fermentation at high-temperature.
By similarity analysis of the amino acid sequences of the enzymes, the known β-glucosidases respectively belongs to glycoside hydrolases families 1 (GH1) and glycoside hydrolases families 3 (GH3). By sequence comparison and analysis of the thermophilic ethanol-resistant β-glucosidase of the present invention, the thermophilic ethanol-resistant β-glucosidase is one member in the GH3 family of the glycoside hydrolases.
In addition, the enzymatic properties of the thermophilic ethanol-resistant β-glucosidase of the present invention are different, from those of the known β-glucosidases; and according to the activity determination of the reaction enzyme activity, the reaction temperature is 40-90° C. and the optimal temperature is 70° C.; the reaction pH value is 4.0-6.5, and the optimal pH value is 5.0; and ethanol can promote the enzyme activity of β-glucosidase when the ethanol concentration is within 30% (v/v) in the reaction system. Wherein when the ethanol concentration is 10-20% (v/v), the β-glucosidase activity was most largely improved by 86.29%. All this properties mentioned above proving that the enzyme has the characteristic of keeping a relatively high enzyme activity in the presence of the ethanol at high temperature. The thermophilic ethanol-resistant β-glucosidase has the theoretical molecular weight of 76740 daltons and the pI of 6.01. The pure enzyme had the highest specific activity of 194.25 U/mg on pNPG. which showed the enzyme can specifically catalyze and hydrolyze β-glycosidic bonds to convert cellobiose into glucose and is a key enzyme in cellulase system. By applying the thermophilic ethanol-resistant β-glucosidase of the present invention was applied to the production of ethanol by simultaneous saccharification at high-temperature, biomass raw materials were firstly pre-hydrolyzed, and then an enzyme solution of the thermophilic ethanol-resistant β-glucosidase and thermostability yeast was added for high-temperature simultaneous saccharification and fermentation, so that the inhibition of cellobiose can be effectively eliminated and the yield of the ethanol was improved by 39%. Under the most appropriate conditions for enzymolysis of the cellulose enzyme and fermentation of the yeast in the process for producing the fuel ethanol from the raw material of cellulose by simultaneous saccharification and fermentation, the enzyme kept a relatively high enzyme activity and can be applied to produce fuel ethanol, so the thermophilic ethanol-resistant β-glucosidase of the present invention has an important application value in the aspect of bioenergy.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a construction pattern diagram of a recombinant plasmid pPIC9K;
FIG. 2 is gel electrophoresis analysis of thermophilic ethanol-resistant β-glucosidase cDNA of the present invention, wherein M lane is Marker, and lane 1 is cDNA of a target gene;
FIG. 3 is an EcoRI and AvrII double enzyme digestion electrophoresis analysis of a cloned plasmid pPIC9K-epB of thermophilic ethanol-resistant β-glucosidase of the present invention, wherein M lane is Marker, and lane 1 is EcoRI and AvrII double enzyme digestion of the plasmid pPIC9K-epB;
FIG. 4 is a SDS-PAGE analysis of purification of thermophilic ethanol-resistant β-glucosidase expressed by P. pastoris GS115, wherein M lane is Marker, and lane 1 is Micro-Prep DEAE column purification;
FIG. 5 is mass spectrometry analysis of thermophilic ethanol-resistant β-glucosidase protein of the present invention;
FIG. 6 shows optimal reaction temperature and pH value of thermophilic ethanol-resistant β-glucosidase of the present invention;
FIG. 7 shows the effects of addition of ethanol on enzyme activity of thermophilic ethanol-resistant β-glucosidase of the present invention;
FIG. 8 shows the effects of thermophilic ethanol-resistant β-glucosidase of the present invention on high-temperature simultaneous saccharification and fermentation.



DETAILED DESCRIPTION OF THE EMBODIMENTS
The following, embodiments are used for further describing the invention rather than limiting the invention.
Materials used in the embodiments of the invention comprise: fungal total RNA fast extraction kit (purchased from Sangon Biotech Co., Ltd.); cDNA first strand synthesis kit (purchased from Thermo Company); Pichia pastoris GS115 and primer synthesis (purchased from Invitrogen Company); pPIC9K expression vector (purchased from Invitrogen Company); competent cell Trans-T1 and pEASY-Blunt Zero Cloning Kit (purchased from TransGen Biotech); PCR reagent, restriction endonuclease EcoRI, AvrII, SalI and T4 DNA ligase (purchased from Takara Company), cellulase compel (purchased from Genencor Company) and thermostability yeast NCYC587 (purchased from UK National Collection of Yeast Cultures); and Hypocrea sp. W63, collected in the China General Microbiological Culture Collection Center (CGMCC) on Sep. 1, 2011, with the collection number of CGMCC No. 5209 (the strain is published in Chinese Patent No. 201110417104.9).
The preparation of P. pastoris GS115 competent cell is known in the prior art.
Embodiment 1: Cloning of Thermophilic Ethanol-Resistant β-Glucosidase Gene of the Present Invention
1. Extraction of Total RNA of Hypocrea sp. W63
Early studies have proved that β-glucosidase produced by Hypocrea sp. W63 has thermophilic ethanol-resistant enzymatic performances, maintained high enzyme activity in the presence of ethanol at high temperature and is different from the known β-glucosidase. Thus, in the embodiment, a fungal total RNA fast extraction kit was used for extracting RNA of Hypocrea sp. W63 and the specific operation steps were as follows:
(1) Taking 450 μL of Buffer Rlysis-F and adding into a 1.5 mL RNase-free centrifugal tube for later use;
(2) Taking a fresh culture, centrifugally collecting thalli, fully grinding 0.1 g of mycelia in liquid nitrogen, transferring into a 1 mL, centrifugal tube, mentioned in (1) immediately shaking and uniformly mixing, and placing at room temperature for 5 min;

(3) Adding 0.2 mL of chloroform into the pyrolysis sample of (2) and uniformly mixing by a vortex oscillator; and centrifuging at 4° C. and 12000 g for 5 min, and carefully sucking out supernatant fluid;

(4) Transferring the supernatant fluid to a 1.5 mL RNase-free centrifugal tube, adding 1⁄2 volume of anhydrous ethanol and fully and uniformly mixing;

(5) Putting an adsorption column into a collection tube, completely adding the solution of (4) into the adsorption column with a pipette, standing for 1 min, centrifuging at 12000 g for 1 min, and pouring away waste liquid in the collection tube;

(6) Respectively adding 3 μL of 10×DNase I Buffer; 1.5 μL of Recombinant DNase I (5 U/μL); 1.5 μL of RNase Inhibitor (40 U/μL); and 24 μL of DEPC-treated water, reacting at the temperature of 37° C. for 20-30 min and removing DNA interference;

(7) Adding 500 μL of GT Solution into the adsorption column, standing for 1 min, centrifuging at 10000 g for 1 min and pouring away the waste liquid in the collection tube;

(8) Putting the adsorption column into the collection tube, adding 500 μL of NT Solution, standing for 1 min, centrifuging at 10000 g for 1 min and pouring away the waste liquid in the collection tube;

(9) Putting the adsorption column into the collection tube, centrifuging at 12000 g for 2 min, and opening a cover for several minutes (Volatilizing the residual ethanol); and

(10) Putting the adsorption column into a new 1.5 mL RNase-free centrifugal tube, adding 30-50 μL of DEPC-treated water, standing for 2 min, centrifuging at 12000 g for 2 min and putting the obtained RNA at −70° C. for cryopreservation.

2. cDNA Cloning of Thermophilic Ethanol-Resistant β-Glucosidase Gene of the Present Invention

Seven amino acid sequences with relatively high similarity of β-glucosidase were searched in GenBank database, then Multiple Sequence Alignment in DNAman software was used for homologous comparison of these sequences, and the design of a pair of primers P1 and P3 was performed according to two highly conserved sequences and the known sequences of the homologous β-glucosidase genes. A cDNA first strand synthesis kit was adopted for cDNA synthesis. PCR amplification was performed to obtain cDNA sequence, using 1 μL of the above total RNA solution as a template for reverse transcription of cDNA connection and P1 and P3 primer set as follow.









P1: 



5-WSN ATH TGG GAY ACN TT-3



 



P3: 



5-CC NAR YTG YTT NCK CAT-3






Touchdown PCR

Reaction conditions were as follows: pre-denaturation at 94° C. for 3 min; then 10 cycles as follows: denaturating at 94° C. for 30 s; annealing at 60-45° C. for 30 s; extending at 72° C. for 30 s; then another 25 cycles: denaturating at 94° C. for 30 s; annealing at 45° C. for 30 s; extending at 72° C. for 30 s, and performing 25 cycles; and finally extending at 72° C. for 6 min. 1% agarose gel electrophoresis was performed to detect whether a target gene with an appropriate size exists or not. Then, target gene with the size of about 1.8 kb was obtained and preserved at −20° C. for further study.
The specific primers were designed according to the comparison of the known sequences, and the PCR reaction was performed. The reaction procedure was as follows: pre-denaturation at 94° C. for 5 min; then cycling as follows: denaturating at 94° C. for 30 s; annealing at 52° C. for 30 s; extending at 72° C. for 30 s and performing 30 cycles; and finally extending at 72° C. for 10 min. 1% agarose gel electrophoresis analysis was performed for the PCR product analysis. Connection, transformation, identification and sequencing of the target gene fragment were performed. The fall-length cDNA with the size of about 2.2 kb of the target gene (as shown in FIG. 2) was obtained.
The obtained sequence was analyzed by using BLAST on NCBI, the encoding gene of the thermophilic ethanol-resistant β-glucosidase was confirmed, which was named as epB-BGL. The correct open-reading frame was constituted of 2202 bp nucleotides; and the thermophilic ethanol-resistant β-glucosidase gene epB-BGL encoded the protein containing 733 amino acids, and DNAstar software was used for predicting to get the results that the theoretical molecular weight of the protein was 76550.45 daltons and the isoelectric point was 6.01. The β-glucosidase from Trichoderma reesei (Genbank index number A.A.A18473.1) had the highest homology with a catalytic functional domain of the epB-BGL gene, and the similarity between the amino acid sequences was 79%. The nucleotide sequence of the thermophilic ethanol-resistant β-glucosidase gene epB-BGL of the present invention has been submitted the nucleotide sequence on GenBank and the sequence number ID is KJ502670.
Embodiment 2: Expression of β-Glucosidase of the Present Invention in Pichia pastoris GS115 and Purification
1. Construction of an epB-BGL Gene Expression Plasmid
According to the obtained full-length cDNA sequence of the thermophilic ethanol-resistant β-glucosidase, a pair of expression primers ep3 and ep5 were designed, the predict signal peptide sequence and 3′ and 5′ non-encoding region sequences were removed, and EcoRI and AvrII enzyme digestion sites were introduced into the two ends of upstream and downstream primers respectively to ensure directional insertion of the epB-BGL gene into vector pPIC9K. The spacing interval between the two primers was about 2.5 kb, and amplification products contained mature protein encoding sequences of the thermophilic ethanol-resistant β-glucosidase.









ep3: 



5-′CGGAATTCATGCTTTACACAGCCGTAGCG-3'



 



ep5: 



5′-CCCCTAGGCTATGAGACCGTGAAGCTTCC-3'






The amplification full-length cDNA PCR product of the thermophilic ethanol-resistant β-glucosidase was taken as the template, and the ep5 and the ep3 were taken as the upstream and downstream primers for performing PCR thereby obtaining the gene expression sequence of the mature peptide of the thermophilic ethanol-resistant β-glucosidase. After the target gene was recovered, a pEASY-Blunt Zero Cloning Kit was used for connecting the target fragment with the pPIC9K expression vector, and transformed into Escherichia coli Trans-T1 competent cell for propagation, Positive clones were screened by colony PCR and enzyme digestion identification of the plasmid DNA, sequencing was performed, and the plasmid which was sequenced and identified correctly was named as pPIC9K-epB.
EcoRI and AvrII were used for double enzyme digestion of the plasmid pPIC9K-epB which was sequenced and verified to contain a correct reading frame of the thermophilic ethanol-resistant β-glucosidase gene, the inserted fragment was recovered, the connection with the yeast expression plasmid pPIC9K which was also subjected to double enzyme digestion was performed by T4 DNA ligase, the Escherichia coli competent cell Trans-T1 was transformed, the transformed Trans-T1 was subjected to ampicillin resistance screening, the colony was subjected to culture at 37° C. overnight with shaking, then the plasmid was extracted, the recombinant plasmid was subjected to enzyme digestion identification and sequencing, the enzyme digestion identification result was as shown in FIG. 3, the recombinant plasmid was named as pPIC9K-epB and the construction pattern diagram was as shown in FIG. 1.
2. Expression of the Recombinant Plasmid pPIC9K-epB in Pichia pastoris GS115 and Purification
Sal I restriction enzyme (positioned in a His4 region) was used for performing lining of the recombinant plasmid pPIC9K-epB, the same performance was carried out on empty pPIC9K vector plasmid as positive control.
Yeast Transformation and Screening:
(1) Preparing P. pastoris GS115 competent cell;
(2) Transforming P. pastoris GS115 competent cell by electric shock with the recombinant expression plasmid pPIC9K-epB, wherein the specific steps of the electrical control method were as follows:
a) Uniformly mixing 10 μL of linear DNA and 80 μL of thalli obtained in step (1) and transferring into a 0.2 cm pre-cooled electric transformation cup;
b) Putting electric transformation cup on ice for ice-bathing for 5 min;
c) Performing electric shock for 4-5 msec according to a yeast transformation program built in the electric transformation cup under the conditions that the voltage was 1.5-1.8 kV, the capacitance was 25 μF and the resistance was 200Ω; and
d) Immediately adding 1 mL of 1M sorbitol solution into the electric, transformation cup after the completion of the electric shock, uniformly mixing the thalli, transferring into a new EP tube, uniformly coating the thalli on an MD flat plate, culturing at 30° C. till the appearance of the single colony, observing the size of the colony, counting and screening the transformants with plump shape on the MD flat plate.

(3) Correspondingly inoculating the yeast transformants onto a fresh MD flat plate with sterile disposable toothpick, performing inverted culture at 30° C. for 2-4 d, and preserved in glycerine.

Culture of the Yeast Engineering Strain and Induction of Secretion and Expression of the Thermophilic Ethanol-Resistant β-Glucosidase:
(1) Selecting the positive colony, inoculating into a 250 mL shaking flask containing 25 mL of BMGY culture medium and performing shaking culture at 28-30° C. (250-300 rpm) till the logarithmic growth phase (OD600 value of 2-6, about 16-18 h), thereby taking the GS115 strain harboring pPIC9K empty vector as the control;

(2) Centrifuging at 3000 g at room temperature for 5 min to recover yeast cells, abandoning supernatant, re-suspending the cells in an appropriate volume of BMMY culture medium, culturing till the OD600 value is 1.0-2.0 (about 100-200 mL), then putting a culture solution into a 500 mL shaking flask, covering eight layers of sterile gauze, putting into a shaking table, continuously culturing at 28-30° C. and starting to induce the expression (note: the induction temperature should be strictly controlled and should not exceed 30° C.);

(3) After the starting of the induction of the expression, replenishing 100% of methanol every 24 h till the final concentration was 1% to maintain the induction; and

(4) Inducing the expression for 5 d, taking the fermentation supernatant, centrifuging at 4° C. and 12000 rpm and performing detection and analysis on the enzyme activity of an expression product.

Purification of the Expression Product of the Thermophilic Ethanol-Resistant β-Glucosidase Engineering Strain:

precipitating an extracted crude enzyme solution with ammonium sulfate with the saturation degree of 90% for 4 h, then centrifuging at 4 and 14000 rpm for 20 min, collecting a precipitate, re-dissolving with an appropriate amount of buffer solution A (20 mmol/L of Tris-HCl buffer solution, pH 7.0), applying the solution to a desalting column pre-equilibrated by buffer solution A, collecting 2 mL of a peak appeared at A280 nm, applying an enzyme solution collected after desalting to a Micro-Prep DEAE column pre-equilibrated by the buffer solution A, firstly eluting an enzyme protein with 3 times the column volume of the buffer solution A till the A280 was constant, and further performing gradient elution with 5 times of the buffer solution A (20 mmol/L of Tris-HCl buffer solution, pH 7.0) and a buffer solution B (1 mol/L of Tris-HCl buffer solution, pH 7.0), wherein the flow rate was 1 mL/min and 1 mL was collected for each tube. The determination of enzyme activity and the determination of protein concentration were performed on each tube the SDS-PAGE electrophoresis purity detection was finally performed (the result was as shown in FIG. 4), and the highest specific activity of the enzyme after purification was 194.25 U/mg. After SDS-PAGE electrophoresis, gel cutting recovery was performed, protein spectrum identification and analysis were performed on the target protein, the tandem MS (MS/MS) determined molecular weight of the enzyme was 76740 daltons, which was similar to the theoretically calculated molecular weight (76550 daltons), the MS results was as shown in FIG. 5.

Embodiment 3: Enzymatic Properties of Thermophilic Ethanol-Resistant β-Glucosidase of the Present Invention
The enzyme activity of the thermophilic ethanol-resistant β-glucosidase of the present invention was determined as follows: using 4-nitrophenyl β-D-glucopyranoside (pNPG) as substrate, total reaction whim is 2 mL, firstly uniformly mixing 1 mL of pNPG (5 mmol/L) and 0.9 mL of Na2HPO4-citric acid buffer solution with the pH of 5.0, further adding 0.1 mL of appropriately diluted thermophilic ethanol-resistant β-glucosidase preparation obtained in the previous step, incubating at 50° C. for 10 min, immediately adding 3 mL of Na2CO3 solution of 1 mol/L to terminate the reaction, placing at room temperature for 5 min and determining the absorbance at 400 nm (OD).
The definition of the enzyme activity was as follows: the amount for catalyzing the production of 1 μmol/L of p-nitrophenol within 1 min was defined as one enzyme unit.
The determination of the enzymatic properties of the thermophilic ethanol-resistant β-glucosidase was as follows: performing enzyme activity determination reaction on the β-glucosidase preparation according to a pNPG determination method by regulating the buffer solution with different pH values, different temperatures and different ethanol concentrations under the situation that the other conditions are constant, wherein the conditions under which the highest enzyme activity was determined to be 100% were the relative activity for determining the thermophilic ethanol-resistant β-glucosidase.
The thermophilic ethanol-resistant β-glucosidase of present invention kept the high enzyme activity at high temperature in the presence of ethanol, the reaction temperature was 40-90° C. and the optimal temperature was 70° C. (as shown in FIG. 6A); the reaction pH value was 4.0-6.5, and the optimal pH value was 5.0-5.5 (as shown in FIG. 6B); and ethanol could promote the enzyme activity of the thermophilic ethanol-resistant β-glucosidase when the concentration of ethanol was within 30% (v/v), wherein when the ethanol concentration in the reaction system was 10-20% (v/v), the effect of promoting the activity of the thermophilic ethanol-resistant β-glucosidase was the strongest, and the activity of the thermophilic ethanol-resistant β-glucosidase was improved by 86.29%.
Embodiment 4: Application of the Thermophilic Ethanol-Resistant β-Glucosidase of the Present Invention to High-Temperature Simultaneous Saccharification and Fermentation
(1) Crushing the substrate, namely bagasse to 60 meshes, pretreatment with 2% of NaOH at 80° C. for 2 h, washing with running water till neutral pH and drying at 65° C. till constant weight;
(2) Culturing the thermostability yeast NCYC587 with a YM liquid culture medium at 42° C. for 24 h for activation;
(3) Adding 30 FPU/g substrate of cellulase into the reaction system and performing pre-hydrolysis at 50° C. for 24 h;
(4) Further addling 15 FPU/g substrate of the thermophilic ethanol-resistant β-glucosidase preparation into the reaction system, inoculating the yeast into the reaction system according to the inoculation amount of 10%, fermenting at 45° C., sampling at 0 h, 24 h, 48 h, 96 h and 120 h by taking the inoculation of the yeast as 0 h, and performing HPLC detection of the content of ethanol and the content of reducing sugar.
The reaction system was as follows: loading 200 mL of reaction solution into a 500 mL shaking flask, wherein the reaction solution contained 30 g of bagasse after alkali treatment, inorganic salt components, namely 0.5 g/L of (NH4)2HPO4, 0.025 g/L of MgSO4.7H2O, 1.0 g/L of yeast extract and the balance of Na2HPO4-citric acid buffer solution with the pH of 5.0.
The preparation which was not added with the thermophilic ethanol-resistant β-glucosidase of the invention was taken as the control for researching the effects of thermophilic ethanol-resistant β-glucosidase when being applied to high-temperature simultaneous saccharification and fermentation.
The results were as shown in FIG. 8, when the thermophilic ethanol-resistant β-glucosidase was applied to high-temperature simultaneous saccharification and fermentation, the highest yield of the ethanol could be obtained by fermenting for 24 h, and the content of the produced ethanol was as high as 28.2 g/L, which was improved by 39% in comparison with the yield of the ethanol in the control. Thus, it was clear that the nature of the thermophilic ethanol-resistant β-glucosidase of the invention was conductive to the application of cellulose high-temperature simultaneous saccharification and fermentation was kept at low concentration level during the whole fermentation period, the inhibition of terminal products was effectively eliminated, and under the most appropriate conditions for hydrolysis of the cellulose and fermentation for producing fuel ethanol from the raw material by simultaneous saccharification and fermentation, the enzyme had a relatively high enzyme activity and obvious active effect and could be applied on the process of producing fuel ethanol, indicating that the thermophilic ethanol-resistant β-glucosidase of the invention had an important application value in the aspect of bioenergy.
The above detailed description is only used for specifically describing the feasible embodiments of the invention, the embodiments are not used for limiting the scope of patent of the invention, and equivalent implementations or variations which do not deviate from the invention should be within the scope of the patent of the invention.










Sequence Table
















<110>
Guangzhou Institute of Energy Conversion, Chinese Academy of



Sciences


 


<120>
Thermophilic ethanol-resistant β-glucosidase and encoding gene and



application thereof


 


<160>
   2


 


<210>
   1


 


<211>
2202


 


<212>
DNA


 


<213>
Hypocrea sp. W63


 


<400>
   1


 








ATGCTTTACA CAGCCGTAGC GGCGTTGGCC ATTGCCACAA CGCCCTTTGT AAGGGCAGGG
  60


 


AGTGCAGTCA CTCCTCCAGC AGGCTCTCCT TGGGCCACCG CATATTCCAA AGCTCAGACG
 120


 


GCATTGGCCA AGCTCTCGCT CCAGGATAAA GTCGGCATCG TGACTGGCGT TGGCTGGAAC
 180


 


AAGGGGCCCT GCGTTGGAAA CACATCTCCA GCGTCAAGTA TCAGCTATCC TCAGTTGTGT
 240


 


CTTCAAGATT CACCACTGGG CATCCGGTTC TCAACGGGCA ACACTGCTTT CACGCCAGGC
 300


 


GTCCAAGCCG CCTCAACATG GGATCTAGAT TTAATCGGCC AGCGCGGTCA ATTCATCGGC
 360


 


CAGGAGAATA AAGCCGCAGG CGTTCACGTC ACTTTGGGGC CGGTGGCTGG GCCGCTAGGA
 420


 


AAGACGCCAC AGGGCGGCCG CAACTGGGAG GGCTTCAGTC CCGATCCATA TCTCACTGGG
 480


 


TTGGCAATGG CTGCAACCAT TAACGGCATT CAGGGCGCTG GAGTGCAAGC AACTGCCAAG
 540


 


CACTACATCC TCAACGAGCA GGAGCTGAAC CGGGAGAGCA TGTCCAGCAA TGCTGACGAC
 600


 


CGCACCCTTC ATGAGCTTTA TGCGTGGCCC TTTGCCGACG CCGTAAACGC CAATGTGGCT
 660


 


GCCGTCATGT GCTCGTACAA CCGAGTCAAT AGCACGTATG CGTGTGAGGA CACGTATACG
 720


 


CTGCAGACAC TGCTGAAGAA CCAGCTGGGA TTCCCTGGAT ACGTCATGAC CGATTGGAAC
 780


 


GCGCAGCACT CGACTGTGCA A4GTGCGCTG GCAGGTCTCG ACATGTCGAT GCCTGGCACT
 840


 


GACTTCAACG GTGGCAGCCT GTACTGGGGA TCGGCTCTGA CCAATGCTGT GAACAGCAAC
 900


 


CAGGTCCCTC TGAGCAGGCT CAATGACATG GTGACGCGTA TCCTCGCTGC GTGGTACTTG
 960


 


ACGGGCC&IG ATTCGGGCTT TCCATCAGTC AGCTTCAGCA GGAATGTCCA GGGAAGTCAC
1020


 


AACACCAACG TACGATCCAT CGCCAGAGAC GGCATTGTAC TTCTCAAGAA CACCGGCAAT
1080


 


ATTCTGCCGC TGAAAACGCC GTCGAGCATC GCCCTCATTG GATCAGCCAC CATTGTCGGT
1140


 


GCCCACGCAA ATAACTCGCC CTCATGCTCT GACCATCGCT GCAACCTTGG TGCCCTCGGA
1200


 


ATGGGATGGG GATCTGGTAC CGCCAACTAC CCATACTTTG TGGCGCCTTA CGACGCCATC
1260


 


AACACAAAGG CTTCTTCGAT TGCCGCCAAA CTGACTCTGA GCAGTACTGA TAACACCTCT
1320


 


GCTGGCGCGT CTGCGGCAAG CGGCAAGGAC GTTGCCATTG TCGTCATCAC TGCAGACTCA
1380


 


GGCGAAGGCT ACATCACTGT TGAGGGCAAC GCAGGCGATC GTAATGACCT GAACGCATGG
1440


 


CACAGCGGCA CTGCTCTGGT ACAGGCCGTG GCAGCAGCCA ACAGCAACGT CATCGTCGTT
1500


 


GTCCACAGTG TCGGCGCCAT TAATCTAGAG CAGATTGTCG CTCTCTCCCA GGTCAAGGCG
1560


 


ATTGTTTGGG CGGGTCTCCC CTCTCAGGAG AACGGCAATG CGCTGGTCGA TATCCTATGG
1620


 


GGAGCCATCA GCCCGTCTGG CAAGCTGGTG TATACAATTG CCAAGAGCCC AAGCGACTAT
1680


 


AACACGCGCA TTTCTTCGGG CGACGACAAC TACAGCGAGG GGCTGTTTAT CGATTACAAG
1740


 


CACTTTGACG ACGCCGGCAT CACGCCGCGA TACGAGTTCG GCTTTGGACT GGCCTACACA
1800


 


AACTTTACCT ACTCTGGCCT TTCCATCACC TCAAACGCCA AGTCTGGACC AGCCACCGGC
1860


 


GCTGTGGTTC CTGGAGGCCC CAGCGATCTG TTCCAGGATG TCGCCACCGT GACTGTGAGC
1920


 


ATCAAAAACA CTGGAGCCGT GACTGGCGCC GAGGTTGCCC AGCTGTACAT CACCTACCCG
1980


 


TCCTCTGCAC CCAGAACCCC CGTGAGGCAG CTTCGAGGCT TCGACAAGCT CAGCTTGACG
2040


 


GCTGCCCAGA GCGGAACCGC GACGTTCAAC ATTCGCAAGC GAGATCTGAC CTACTGGAAC
2100


 


GTGGCGTCGC AGCAGTGGGT GGTGCCGTCG GGGACTTTTG GCGTGAGCGT TGGAGCGAGC
2160


 


AGCAGAGATT TGCGTTTGAC GGGAAGCTTC ACGGTCTCAT AG
2202


 








<210>
  2


 


<711>
733


 


<212>
pRT


 


<213>
Hypocrea sp. W63


 


<400>
  2


 







Met Leu Tyr Thr Ala Val Ala Ala Leu Ala Ile Ala Thr Thr Pro


1               5                   10                  15


 


Phe Val Arg Ala Gly Ser Ala Val Thr Pro Pro Ala Gly Ser Pro


                20                  25                  30


 


Trp Ala Thr Ala Tyr Ser Lys Ala Gln Thr Ala Leu Ala Lys Leu


                35                  40                  45


 


Ser Leu Gln Asp Lys Val Gly Ile Val Thr Gly Val Gly Trp Asn


                50                  55                  60


 


Lys Gly Pro Cys Val Gly Asn Thr Ser Pro Ala Ser Ser Ile Ser


                65                  70                  75


 


Tyr Pro Gln Leu Cys Leu Gln Asp Ser Pro Leu Gly Ile Arg Phe


                80                  85                  90


 


Ser Thr Gly Asn Thr Ala Phe Thr Pro Gly Val Gln Ala Ala Ser


                95                  100                 105


 


Thr Trp Asp Leu Asp Leu Ile Gly Gln Arg Gly Gln Phe Ile Gly


                110                 115                 120


 


Gln Glu Asn Lys Ala Ala Gly Val His Val Thr Leu Gly Pro Val


                125                 130                 135


 


Ala Gly Pro Leu Gly Lys Thr Pro Gln Gly Gly Arg Asn Trp Glu


                140                 145                 150


 


Gly Phe Ser Pro Asp Pro Tyr Leu Thr Gly Leu Ala Met Ala Ala


                155                 160                 165


 


Thr Ile Asn Gly Ile Gln Gly Ala Gly Val Gln Ala Thr Ala Lys


                170                 175                 180


 


His Tyr Ile Leu Asn Glu Gln Glu Leu Asn Arg Glu Ser Met Ser


                185                 190                 195


 


Ser Asn Ala Asp Asp Arg Thr Leu His Gln Leu Tyr Ala Trp Pro


                200                 205                 210


 


Phe Ala Asp Ala Val Asn Ala Asn Val Ala Ala Val Met Cys Ser


                215                 220                 225


 


Tyr Asn Arg Val Asn Ser Thr Tyr Ala Cys Glu Asp Thr Tyr Thr


                230                 235                 240


 


Len Gln Thr Leu Leu Lys Asn Gln Leu Gly Phe Pro Gly Tyr Val


                245                 250                 255


 


Met Thr Asp Trp Asn Ala Gln His Ser Thr Val Gln Ser Ala Leu


                260                 265                 270


 


Ala Gly Leu Asp Met Ser Met Pro Gly Thr Asp Phe Asn Gly Gly


                275                 280                 285


 


Ser Leu Tyr Trp Gly Ser Ala Leu Thr Asn Ala Val Asn Ser Asn


                290                 295                 300


 


Gln Val Pro Leu Ser Arg Leu Asn Asp Met Val Thr Arg Ile Leu


                305                 310                 315


 


Ala Ala Trp Tyr Leu Thr Gly Gln Asp Ser Gly Phe Pro Ser Val


                320                 325                 330


 


Ser Phe Ser Arg Asn Val Gln Gly Ser His Asn Thr Asn Val Arg


                335                 340                 345


 


Ser Ile Ala Arg Asp Gly Ile Val Leu Leu Lys Asn Thr Gly Asn


                350                 355                 360


 


Ile Leu Pro Leu Lys Thr Pro Ser Ser Ile Ala Leu Ile Gly Ser


                365                 370                 375


 


Ala Thr Ile Val Gly Ala His Ala Asn Asn Ser Ala Ser Cys Ser


                380                 385                 390


 


Asp His Gly Cys Asn Leu Gly Ala Leu Gly Met Gly Trp Gly Ser


                395                 400                 405


 


Gly Thr Ala Asn Tyr Pro Tyr Phe Val Ala Pro Tyr Asp Ala Ile


                410                 415                 420


 


Asn Thr Lys Ala Ser Ser Ile Gly Ala Lys Leu Thr Leu Ser Ser


                425                 430                 435


 


Thr Asp Asn Thr Ser Ala Gly Ala Ser Ala Ala Ser Gly Lys Asp


                440                 445                 450


 


Val Ala Ile Val Val Ile Thr Ala Asp Ser Gly Gln Gly Tyr Ile


                455                 460                 465


 


Thr Val Gln Gly Asn Ala Gly Asp Arg Asn Asp Leu Asn Ala Trp


                470                 475                 480


 


His Ser Gly Thr Ala Leu Val Gln Ala Val Ala Ala Ala Asn Ser


                485                 490                 495


 


Asn Val Ile Val Val Val His Ser Val Gly Ala Ile Ala Leu Glu


                500                 505                 510


 


Gln Ile Val Ala Leu Ser Gln Val Lys Ala Ile Val Trp Ala Gly


                515                 520                 525


 


Leu Pro Ser Gln Gln Asn Gly Ala Ala Leu Val Asp Ile Leu Trp


                530                 535                 540


 


Gly Ala Ile Ser Pro Ser Gly Lys Leu Val Tyr Thr Ile Ala Lys


                545                 550                 555


 


Ser Pro Ser Asp Tyr Asn Thr Arg Ile Ser Ser Gly Asp Asp Asn


                560                 565                 570


 


Tyr Ser Gln Gly Leu Phe Ile Asp Tyr Lys His Phe Asp Asp Ala


                575                 580                 585


 


Gly Ile Thr Pro Arg Tyr Gln Phe Gly Phe Gly Leu Ala Tyr Thr


                590                 595                 600


 


Asn Phe Thr Tyr Ser Gly Leu Ser Ile Thr Ser Asn Ala Lys Ser


                605                 610                 615


 


Gly Pro Ala Thr Gly Ala Val Val Pro Gly Gly Pro Ser Asp Leu


                620                 625                 630


 


Phe Gln Asp Val Ala Thr Val Thr Val Ser Ile Lys Asn Thr Gly


                635                 640                 645


 


Ala Val Thr Gly Ala Gln Val Ala Gln Leu Tyr Ile Thr Tyr Pro


                650                 655                 660


 


Ser Ser Ala Pro Arg Thr Pro Val Arg Gln Leu Arg Gly Phe Asp


                665                 670                 675


 


Lys Leu Ser Leu Thr Ala Gly Gln Ser Gly Thr Ala Thr Phe Asn


                680                 685                 690


 


Ile Arg Lys Arg Asp Leu Thr Tyr Trp Asn Val Ala Ser Gln Gln


                695                 700                 705


 


Trp Val Val Pro Ser Gly Thr Phe Gly Val Ser Val Gly Ala Ser


                710                 715                 720


 


Ser Arg Asp Leu Arg Leu Thr Gly Ser Phe Thr Val Ser ***


                725                 730

## Claims

1. A recombinant expression vector, comprising a gene comprising the nucleotide sequence of SEQ ID NO: 1.

2. The recombinant expression vector according to claim 1, wherein the expression vector is pPIC9K.

3. A host cell, comprising:
the recombinant expression vector according to claim 1,
wherein the host cell is a eukaryotic cell or prokaryotic cell.

4. A host cell, comprising:
the recombinant expression vector according to claim 2,
wherein the host cell is a eukaryotic cell or prokaryotic cell.

5. The host cell according to claim 3, wherein the host cell is a yeast engineering strain, Escherichia coli or Bacillus subtilis.

6. The host cell according to claim 4, wherein the host cell is a yeast engineering strain, Escherichia coli or Bacillus subtilis.

7. The host cell according to claim 3, wherein the host cell is Pichia pastoris GS115.

8. The host cell according to claim 4, wherein the host cell is Pichia pastoris GS115.