CN116732008B

CN116732008B - Beta-glucosidase and application thereof

Info

Publication number: CN116732008B
Application number: CN202310873695.3A
Authority: CN
Inventors: 尹以瑞; 黄裕颖; 郑宏钊; 胡伟; 鲜文东; 朱丹; 吕志华; 朱倩; 万玲; 王妃
Original assignee: Dali University
Current assignee: Dali University
Priority date: 2023-07-17
Filing date: 2023-07-17
Publication date: 2024-08-20
Anticipated expiration: 2043-07-17
Also published as: CN116732008A

Abstract

The invention discloses beta-glucosidase and application thereof, and relates to the technical field of genetic engineering. The amino acid sequence of the beta-glucosidase is shown in SEQ ID NO: 1. The beta-glucosidase has excellent heat stability, pH tolerance and strong tolerance to glucose, can be suitable for high temperature, strong acid and strong alkali environments, and has potential value in the industries of food, health care, feed and medical treatment.

Description

Beta-glucosidase and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to beta-glucosidase and application thereof.

Background

Beta-glucosidase beta-glucosides belong to the class of cellulases, also called gentiobinase, cellobiase and amygdalase, can hydrolyze beta-D-glycosidic bonds and release the corresponding ligands simultaneously during the reduction process. Beta-glucosidase plays a very important role, mainly in biomass conversion of microorganisms; the decomposition of glycolipids and exogenous glucosides in animals, lignification, catabolism of cell wall oligosaccharides, and the like, has also been widely used in food processing, agricultural production, and medical diagnosis. Beta-glucosidase is mainly involved in hydrolyzing cellobiose and soluble cellooligosaccharides in the degradation process of cellulose, and has the function of converting cellulose into available glucose, which is a key rate-limiting enzyme. And cellulose and hemicellulose can be hydrolyzed into monosaccharide under the action of beta-glucosidase, and the monosaccharide is continuously fermented into ethanol, so that the bioethanol is a potential raw material of bioethanol, and the bioethanol is characterized by reproducibility, low toxicity and the like, can be developed into a liquid fuel, and provides a way for searching renewable resources.

Beta-glucosidase is classified according to different structures and is mainly distributed in 6 families of glycoside hydrolases GH1, GH3, GH5, GH9, GH30, GH116 and the like, wherein most beta-glucosidase is mainly distributed in two families of GH1 and GH 3. Bacterial-derived β -glucosidase is mainly distributed in the GH1 and GH3 families, and most fungal-derived β -glucosidase is distributed in the GH3 family. At present, the industrially used beta-glucosidase is mainly derived from fungi such as trichoderma, penicillium and the like, and most of the beta-glucosidase has the problems of low enzyme activity, low stability to temperature and pH and the like. The decomposition of cellulose is usually carried out at 50℃or above, where the heat resistance of the beta-glucosidase is particularly important, ensuring that the temperature does not affect its activity and thus reduces the hydrolysis of cellulose. Thermophilic beta-glucosidase not only improves the catalytic efficiency of the substrate, but also reduces the risk of pollution and energy consumption, and in industrial applications, finding a beta-glucosidase that is not inhibited by the end product glucose is also a focus of attention. Currently, the uncultured microorganisms account for about 99% of the total number of microorganisms in the environment, and the fact that most microorganisms cannot be cultivated can be dealt with by metagenomics, and accessibility of enzymes cannot be achieved by using conventional methods, so that the utilization of a metagenomic method in combination with modern biotechnology such as genetic engineering to dig novel beta-glucosidase has potential value.

Disclosure of Invention

In view of the above, the main purpose of the invention is to provide a beta-glucosidase gene, a recombinant vector, a host cell, beta-glucosidase and application thereof, which aim to solve the problems of poor thermal stability and glucose tolerance of the existing beta-glucosidase, and provide a beta-glucosidase with strong thermal stability, wide pH tolerance range and strong glucose tolerance and application thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a beta-glucosidase having an amino acid sequence as set forth in SEQ ID NO: 1.

A DNA molecule encoding a β -glucosidase, said DNA molecule being (a) or (b):

(a) The nucleotide sequence is shown as SEQ ID NO:2 is shown in the figure;

(b) SEQ ID NO:2, by substitution deleting or inserting one or several nucleotides to form a DNA molecule encoding said beta-glucosidase.

A recombinant vector comprising a DNA molecule encoding a β -glucosidase and regulatory sequences operably linked to the DNA molecule for expression.

A host cell comprising a DNA molecule encoding a β -glucosidase or the recombinant vector.

Use of beta-glucosidase in degrading cellulose.

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

In the technical scheme, the invention provides the beta-glucosidase gene y50bg4, the recombinant plasmid carrying the gene and the recombinant strain, wherein the beta-glucosidase gene y50bg4 can code a beta-glucosidase gene, the optimal pH of the beta-glucosidase is 6.0, and the activity of the beta-glucosidase after 24 hours in a pH4.0-10.0 buffer solution shows very remarkable activation and has very strong pH tolerance; the optimal temperature of the enzyme is 60 ℃, the activity of the enzyme can be kept for more than 2 hours at 50-60 ℃ without being reduced, and the beta-glucosidase provided by the invention has the excellent properties of strong pH tolerance and thermal stability, and can be applied to industries of fuel energy, food processing, papermaking, textile, medical care and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a phylogenetic relationship of recombinant β -glucosidase and related proteins;

FIG. 2 is a SDS-PAGE electrophoresis of recombinant beta-glucosidase;

FIG. 3 shows the relative activity of recombinant β -glucosidase at different temperatures;

FIG. 4 shows the relative activities of recombinant β -glucosidase at different pH values;

FIG. 5 is a thermal stability assay for recombinant β -glucosidase;

FIG. 6 is the thermostability of recombinant β -glucosidase and commercial cellulase at 60 ℃;

FIG. 7 is a recombinant β -glucosidase pH tolerance assay;

FIG. 8 is a graph showing the tolerance of recombinant β -glucosidase to glucose;

FIG. 9 is a synergistic effect of recombinant β -glucosidase and commercial cellulase in corn stover hydrolysis;

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Experimental materials and reagents

Gene: in this example, genomic DNA was extracted from the bottom sediment collected in hot sea (longitude 98.4440, latitude 24.94777) in Tengchong county, baoshan, yunnan, by using a soil DNA extraction kit (brand MOBIO, U.S. and product number: 12888-50), and then sent to a sequencing company for metagenomic sequencing and annotation, and the functional gene of beta-glucosidase (shown as SEQ ID NO: 2) was screened out by data analysis, and designated as y50bg4. And performing PCR amplification, identification, clone expression, purification of beta-glucosidase and enzymatic property on the functional gene, and cloning an expression vector pET-28a (+).

Enzymes and other biochemical reagents: restriction enzymes (EcoRI and HindIII) were purchased from Thermo Scientific, T4-DNA ligase was purchased from Beijing full gold Biotechnology Co., ltd, and other biochemical reagents were purchased from domestic common biochemical reagent company.

Culture medium:

LB basal medium (g/L): yeast extract 5, pancreatic protein 10, sodium chloride 10, ph7.4; the antibiotic Kanamycin (KANAMYCIN) was used at a final concentration of 50mg/L, 2% agar was added to the solid isolation medium, and the medium was sterilized at 121℃for 20min.

Example 1

Extraction of DNA from soil sample and Gene Synthesis

Genomic DNA was extracted using a soil DNA extraction kit (brand MOBIO, U.S. cat# 12888-50) as an experimental sample, with reference to the instructions provided by the manufacturer. And then sending the obtained product to a sequencing company for metagenome sequencing and annotation, and screening out the functional gene of the beta-glucosidase through data analysis to obtain the functional gene with the beta-glucosidase, which is named as y50bg4.

Amplification of functional Gene and construction of recombinant expression vector

Taking the synthetic gene as a template, and carrying out primer:

y50bg4-F CATGAATTCAATAGATGGAGGGCTAAAATGT, as shown in SEQ ID NO:3 is shown in the figure;

y50bg4-R GTCAAGCTTTTCTTTATACTTTTTAATTATTTC, as shown in SEQ ID NO:4 is shown in the figure;

The functional gene is amplified by PCR using high-fidelity enzyme gold plate Mix (Beijing qingke biotechnology Co., ltd.) under the following amplification conditions: pre-denaturation at 94 ℃,4min, denaturation at 94 ℃,30s, annealing at 55 ℃,30s, extension at 72 ℃,90s,32 cycles, final extension at 72 ℃,10min. The PCR products were identified by 1.0% agarose gel electrophoresis, 100V,90min, and observed under UV light. The correct fragment was purified using the crude gel recovery purification kit.

Double digestion is carried out on the PCR gel recovery product and the vector pET28a (+) according to instructions by using restriction enzymes (EcoRI and HindIII), the digested product is recovered, the digested PCR product and the vector are connected through T4-DNA ligase, the ligation product was introduced into E.coli strain E.coli DH 5. Alpha. Competence by Ca2+ chemical transformation, positive clones were screened by colony PCR, finally, further verifying through sequencing of the Qinkeke biotechnology limited responsible company to obtain a recombinant vector pET28a-y50bg4, and submitting the obtained sequence to GenBank (http: i/www.ncbi.nlm.nih.gov).

The sequencing result is analyzed to obtain an open reading frame, and the beta-glucosidase gene is found to have 1341 bases and code 446 amino acids. The theoretical relative molecular mass is 52kDa, and the theoretical isoelectric point is 6.4. The amino acid sequences were aligned in the NCBI database and phylogenetic tree (FIG. 1) was constructed using the Maximum Likelihood (ML) method with a poisson correction model, with the highest similarity being the Caloramator mitchellensis-derived β -glucosidase gene, which was 80.65%. The beta-glucosidase was assigned to the glycosyl hydrolase GH1 family according to NCBI amino acid sequence conservation analysis.

Beta-glucosidase y50bg4 gene nucleotide sequence (1341 bp), as shown in SEQ ID NO:2 is shown as follows:

AATAGATGGAGGGCTAAAATGTTAGATAAAAATTTTATATTTGGAGTTGCTACATCATCCTATCAAATAGAGGGTGCACATGATAAAGATGGAAGAACACCCTCAATTTGGGATACATTTTCAAAAATAAAGGGAAAAACCTTTAATATGGATAATGGAGATGTTGCATGCGATCATTATCATAGGTATAAAGAAGACGTAGAATTAATGAAGGAATTAGGTGTTGATGCATATAGGCTTTCTATATCATGGCCACGCATATTTCCTAAGGAAGGTGAATATAACTCTAAGGGAATGGAGTTTTACAAAAACCTTTTAAAGGAGCTAAAAGAGAAAGGCATTAAGGTCGCAGTAACTTTATATCACTGGGACTTACCTCAATGGGTTCAAGACAAAAACCGATGGGAAGACAGAAACAATATCAATTATTTTTTAGAATATGCTAAAAAATGTTTTATTGAGCTCGATGAGTATGTAGATATGTGGATAACTCATAATGAACCTTGGTGTGCATCATTCTTGTCAAACGCATTAGGAGAGCATGCGCCGGGTAAAAGAAGTATAACATCAGCTGTTAAGGTTGCACATCATCTACTATTATCCCATGGTATGACTGTTAGGATGTATAGAGAGCTTGGATTTAAAAAGCCTATCGGGATTACTTTGAATTTAAGTCCTTATTATCCAGCAACTGATGAATTTAAGGATTTAATAGCTGCTAATAATGGGGATGGATTTTTAAACAGATGGTTCCTCGAGCCGATTTTTAAAGGTCATTATCCTTTTGATATGATTAATCTTTATTCTCAAAGAGTGGAGGATTTTAGCTTTATAAAAAAAGGTGATTTTAGTATTATAGGTGAAAAATGCGATTTTCTTGGTATAAATTTTTATAACAGAATAGTTGTTGAATATGATCCAACAAATGTTTTAATGTTAAGACCTGCTTTTACTGATTACAAAAAGACAAGCATGGGATGGGATGTATCGCCTAATGAATTTATAGATCTTATAAGGATGGTTAGAAATAAATATACTGATTTACCTATATATATTACAGAGAATGGTGCTGCCTTTGAAGATTATGTAGAGAATGGAAAAGTTCATGATATTGATAGAGTTATTTATATTGAGGACCATATTAAAGCTATAGAAAAAATGAACGAAGAAAAATTAAATGTTGCAGGTTATTTTTGCTGGTCGCTCCTTGATAATTTTGAATGGGCACACGGATATTCCAAACGTTTTGGAATTGTTTATGTTGATTTTAACACGCAAGAGAGAATAAAGAAGGATAGCTTTTATAAATATAAGGAAATAATTAAAAAGTATAAAGAATAG;

Beta-glucosidase Y50Bg4 amino acid sequence (446 residues) as shown in SEQ ID NO:1 is shown in the figure NRWRAKMLDKNFIFGVATSSYQIEGAHDKDGRTPSIWDTFSKIKGKTFNMDNGDVACDHYHRYKEDVELMKELGVDAYRLSISWPRIFPKEGEYNSKGMEFYKNLLKELKEKGIKVAVTLYHWDLPQWVQDKNRWEDRNNINYFLEYAKKCFIELDEYVDMWITHNEPWCASFLSNALGEHAPGKRSITSAVKVAHHLLLSHGMTVRMYRELGFKKPIGITLNLSPYYPATDEFKDLIAANNGDGFLNRWFLEPIFKGHYPFDMINLYSQRVEDFSFIKKGDFSIIGEKCDFLGINFYNRIVVEYDPTNVLMLRPAFTDYKKTSMGWDVSPNEFIDLIRMVRNKYTDLPIYITENGAAFEDYVENGKVHDIDRVIYIEDHIKAIEKMNEEKLNVAGYFCWSLLDNFEWAHGYSKRFGIVYVDFNTQERIKKDSFYKYKEIIKKYKE-;

Inducible expression of recombinant proteins

And transforming the constructed recombinant vector pET28a-y50bg4 into E.coli BL21 (DE 3) to obtain recombinant strain E.coli BL21 (DE 3) -y50bg4. Single colony is picked from a flat plate and inoculated into LB liquid culture medium containing 50 mug/mL kanamycin resistance at 37 ℃,220rpm for overnight culture, 1% of inoculum size is inoculated into LB liquid culture medium containing 50 mug/mL kanamycin for culture, then the culture is carried out again to 25 ℃,220rpm for continuous culture for 8 hours; and then centrifuging at 4 ℃ and 7000r/min for 20min, harvesting thalli, and storing the thalli in a refrigerator at-20 ℃ for standby.

Separation and purification of recombinant proteins

The cells were resuspended in a centrifuge tube using a PBS solution (pH 7.6) containing 10mM imidazole, sonicated in an ice-water mixture for 30min, centrifuged using a high-speed refrigerated centrifuge at 4℃at 7000r/min for 20min, and the supernatant was collected. The Ni-NTA chromatographic column is used for separation and purification, and the steps are as follows: the Ni-NTA column was equilibrated with 5 column volumes of equilibration solution (10 mM imidazole in PBS, pH 7.6), the supernatant was loaded onto the Ni-NTA column, the column was repeated once, washed with 10 column volumes of equilibration solution after column passage, and finally the target protein was eluted with 3 column volumes of eluent (250 mM imidazole in PBS, pH 7.6), the protein solution was collected by 1.5mL centrifuge tubes, and 1.0mL each tube was collected. The protein concentration of the collected protein solution was measured by the Bradford method, and SDS-PAGE electrophoresis analysis was performed, and the target protein was successfully purified, and the purification result is shown in FIG. 2.

Enzymatic Property study of beta-glucosidase

(1) The method for measuring the enzyme activity of the beta-glucosidase comprises the following steps: the glucose oxidase-peroxidase detection kit detection method is used. Beta-glucosidase activity was determined using cellobiose as substrate. mu.L of 10-fold diluted enzyme solution was added to 90. Mu.L of a buffer containing 1% (w/v) cellobiose, reacted at an optimum temperature for 30min, and frozen at-80℃for 2-3min to terminate the reaction. 10. Mu.L of the reaction mixture was added to a 96-well plate, 200. Mu.L of a glucose oxidase-peroxidase detection kit buffer was added, and after incubation at 37℃for 10 minutes, the absorbance was measured at OD492nm using an enzyme-labeled instrument. One unit (U) is defined as the amount of enzyme required to release 2. Mu. Mol glucose from cellobiose per minute.

(2) Determination of the optimum temperature and the temperature stability of beta-glucosidase

The enzyme activity of the purified beta-glucosidase was measured at pH6.0 at different temperatures (30-85 ℃) with a gradient of every 5 ℃, and the analytical results (FIG. 3) showed that: the optimal temperature is 60 ℃, the enzyme activity is gradually increased to 60 ℃ to reach the highest activity at 30-60 ℃, and the enzyme activity is 30% at 65 ℃.

The enzyme solutions were placed at three temperatures: the stability of the temperature was measured at 50℃and 55℃and 60℃for various times (20-120 min), the residual activity was measured every 20min, and the measurement results are shown in FIG. 5, which shows that: the residual activity of the enzyme was still 100% after incubation at 50, 55, 60 ℃ for 2 h. The enzyme and the commercial cellulase from fungi Trichoderma reesei are incubated at 60 ℃ for 80min simultaneously, and residual activities of the two enzymes are respectively measured, and the result is shown in figure 6, the activity of the commercial cellulase is lost by more than 80% after the commercial cellulase is incubated for 60min, and the residual activity of Y50Bg4 is still kept at 100%, so that the result shows that the recombinant enzyme has better thermal stability.

(3) Determination of optimal pH and pH stability of beta-glucosidase

The purified expressed beta-glucosidase was subjected to enzymatic reactions at different pH to determine its optimum pH. The buffers used were disodium hydrogen citrate monobasic buffer of pH2.6-8.0, glycine-hydrochloric acid buffer of pH 8.0-10, and the pH adaptation results of purified beta-glucosidase measured at 60℃in buffer systems of different pH (FIG. 4) showed that: the optimum pH of the beta-glucosidase is 6.0, and the enzyme activity of the beta-glucosidase is kept at 60% in the pH range of 5.0-10.0.

The residual activity was measured with a buffer having a pH of (1.0-10.0) at a gradient of every 1pH, and the pure enzyme solutions were allowed to stand at 4℃for 12h and 24h, respectively, as shown in FIG. 7, which shows that: in the pH range of 3.0-10.0, the activity of the positive control enzyme is taken as 100% after 12h treatment, the activity of the enzyme has a certain activation effect, the activity of the enzyme can be improved by 65% at the highest, the activation effect is more obvious after 24h treatment, the activity of the enzyme is improved by 132% at the highest, the pH tolerance range of the recombinase is very wide, and the activation effect of the enzyme activity is higher with the increase of the treatment time.

(4) Influence of glucose on the enzymatic Activity of beta-glucosidase

Using p-nitrobenzene-. Beta. -D-glucopyranoside (pNPG) as substrate, 10. Mu.g of protein was added to 200. Mu.l of the reaction mixture containing 2.5mM pNPG (Sigma, st.Louis, MO, USA). After incubation for 5 minutes at the optimal temperature, the reaction was stopped by adding 450. Mu.l of 1MNA2CO 3. Release of p-nitrophenol the release of p-nitrophenol was measured by monitoring the absorbance at 405 nm, with reference to p-nitrophenol (Sigma, usa). To investigate the effect of D-glucose on the catalytic activity of LQ-BG8, different concentrations of D-glucose (0-3M) were added to the reaction system to determine the relative enzyme activity, and a control group was used without glucose. As shown in FIG. 8, the enzyme activity of the recombinant β -glucosidase decreased with increasing glucose concentration, and 83% of the enzyme activity was still maintained in the presence of 3000mM glucose. Thus, it was demonstrated that recombinant β -glucosidase was very tolerant to the product glucose. This property is of great significance for the application of recombinant beta-glucosidase in cellulose complex enzyme systems.

(5) Determination of hydrolysis of corn straw by beta-glucosidase

10 G of maize straw was ground, sieved with a 80 mesh sieve, boiled in 100ml of hot water for 30 minutes, filtered with filter paper and dried at 80 ℃. 0.2 g of corn stalk pretreated with hot water was added to 1 ml of buffer (pH 5.6), 0.2mg of cellulase (Sangon Biotechnology Co., china) and 0.03mg of LQ-BG8 were added to the reaction system. The mixture was incubated at 50 ℃. Samples were taken at 0, 1,2,3, 4, 5, 6, 7, 8, 18 and 24h after the start of the reaction. Glucose concentration in the reaction solution the glucose concentration in the reaction solution was measured using a glucose oxidase detection kit. The control group was a reaction solution without enzyme. The measurement result shows that after the Y50Bg4 enzyme solution is added into commercial cellulase for 24 hours as shown in fig. 9, the degradation rate is obviously increased compared with the degradation rate (100%) of the commercial cellulase only, and the degradation rate of the Y50Bg4 added into the corn stalk hydrolysis process is obviously increased, while the Y50Bg4 has no separate hydrolysis activity on the corn stalk, but can show good synergistic effect with the commercial cellulase, which also shows that the Y50Bg4 has a certain application value in improving the lignocellulose degradation.

(6) Determination of the influence of different Metal ions and organic reagents on the Activity of beta-glucosidase

To determine the effect of metal ions and organic reagents on beta-glucosidase, residual enzyme activity was determined by adding different metal ions (K⁺、Mg²⁺、Fe³⁺、Ca²⁺、Co²⁺、Ni²⁺、Mn²⁺、Pb²⁺、Cu²⁺、Zn²⁺ and Ag ⁺ to final concentrations of 1mM and 10mM, respectively, by enzyme solution at 60℃with different organic reagents [ disodium ethylenediamine tetraacetate (EDTA), tween-80 (Tween-80), ethanol (Ethanol), sodium Dodecyl Sulfate (SDS), phenylmethylsulfonyl fluoride (PMSF) and Dithiothreitol (DTT) ] to final concentrations of 0.1% and 1%, after 30min of treatment. The reaction mixture without additive was used as a control (100%) under standard conditions.

The measurement results are shown in Table 1:

TABLE 1 influence of different metal ions and chemical substances on the activity of the Y50Bg4 enzyme

The results show that: besides the metal ions K ⁺、Mg²⁺、Ca²⁺ and ethanol which do not have the inhibition effect, other ions and reagents all have the obvious inhibition effect, co ²⁺ and Ag ⁺ almost completely lose the activity of the recombinant beta-glucosidase, other ions lose more than 80% of the activity of the enzyme, and the surfactant Tween 80 and SDS have the strong inhibition effect on the recombinant beta-glucosidase; the addition of the metal chelating agent EDTA in the reaction system enables the enzyme activity of the recombinant beta-glucosidase to be only 11.86%, which indicates that the recombinant beta-glucosidase belongs to metal binding protein. In conclusion, the beta-glucosidase has excellent heat stability, pH tolerance and strong tolerance to glucose, can be suitable for high temperature, strong acid and strong alkali environments, and has potential value in the industries of food, health care, feed and medical treatment.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A beta-glucosidase, characterized in that the amino acid sequence of the beta-glucosidase is as set forth in SEQ ID NO: 1.

2. A DNA molecule encoding the β -glucosidase of claim 1, wherein the DNA molecule is (a) or (b):

(a) The nucleotide sequence is shown as SEQ ID NO:2 is shown in the figure;

(b) SEQ ID NO:2 by replacing one or more nucleotides with a nucleotide sequence encoding a beta-glucosidase according to claim 1.

3. A recombinant vector comprising the DNA molecule of claim 2 and regulatory sequences for expression operably linked to the DNA molecule.

4. A host cell comprising the DNA molecule of claim 2 or the recombinant vector of claim 3.

5. Use of the beta-glucosidase according to claim 1 for degrading cellulose.