CN113151325B - Beta-glucosidase gene bgI, and encoding protein and application thereof - Google Patents

Abstract

The invention discloses a beta-glucosidase genebgI, and a coding protein and application thereof. Beta-glucosidase Gene of the inventionbgI, the nucleotide sequence of which is shown as SEQ ID NO. 1; the protein coded by the gene is beta-glucosidase BGI. The maximum reaction rate of hydrolyzing the p-NPG reaches 0.648 mu M.min from the analysis of BGI enzymatic reaction kinetics^‑1. The analysis of the enzymology result shows that: at the pH value of 4-6, the highest value of reducing sugar content which can be produced by BGI hydrolysis p-NPG per minute is as high as 0.058 mu M.mL^‑1Belonging to acidic endo-cellulase; when the temperature reaches 100 ℃, the content of reducing sugar generated by BGI hydrolysis p-NPG per minute can reach 0.023 mu M.mL^‑1And has strong activity at high temperature. The invention provides theoretical basis and technical guarantee for resource utilization of agricultural wastes and development of ecological agriculture.

Description

Beta-glucosidase gene bgI and encoding protein and application thereof

Technical Field

The invention belongs to the field of enzymology engineering, and particularly relates to a beta-glucosidase gene bgI as well as a coding protein and application thereof.

Background

Environmental and resource issues are one of the most important and serious crisis facing human beings in today's society. In the global ecological environment, only cellulose is a resource having a renewable capability and a widely distributed and inexpensive characteristic. Global plants produce approximately 200 million tons of dry matter per year through photosynthesis, with a proportion of cellulose content exceeding 50%. The current degradation and utilization of such renewable biological resources is limited by the limitations of lignocellulose. The cellulose is surrounded by a lignin layer, and the high-crystalline structure is insoluble in water, so that it is difficult to hydrolyze the cellulose directly into available glucose, and no suitable utilization method has been found so far. Along with the growing world population, resource shortage and other social problems, the method is increasingly remarkable, and has important practical significance for exploring how to utilize cellulase to develop biological industry in order to solve the current world energy crisis, food shortage, environmental pollution and other hot-spot social problems, and realizing the biological conversion and utilization of cellulose. The research at the present stage shows that the enzymatic hydrolysis method is difficult to completely degrade by only a chemical method and a physical method, and has the advantages of low energy consumption, mild reaction conditions, environmental friendliness and the like, so that the enzymatic hydrolysis method gradually becomes a focus of attention of researchers in various countries. However, the industrial production and market promotion and application of cellulase are not optimistic, and the cellulase still has low enzyme activity utilization rate and high production cost at present. As such, researchers in all circles are paying more attention to research on cellulase and paying more attention to industrial application of cellulase.

Beta-glucosidase plays a key role in the process of cellulose hydrolysis by cellulase. In the cellulase system, the regulation mechanism in the microorganism regulates and controls the cellulase proportion of each component, and the artificial regulation is difficult to complete in the modes of regulating and controlling the culture conditions and the like. However, different requirements are often imposed on the proportion of each component in different industrial productions of cellulase, for example, in the textile washing link, beta-glucosidase with high relative activity is required, and under the condition, the cellulase directly synthesized by trichoderma cannot meet the requirement of industrial batch production. In order to solve these problems, some researchers have tried to design Trichoderma strains by genetic engineering techniques to increase the ratio of a certain cellulase content in cellulase system, and manually intervene in control to achieve the desired effect.

Disclosure of Invention

The invention aims to provide a beta-glucosidase gene bgI, and a coding protein and application thereof, aiming at the prior art.

The purpose of the invention can be realized by the following technical scheme:

a beta-glucosidase gene bgI, the nucleotide sequence is shown in SEQ ID NO. 1.

The invention also protects the protein coded by the beta-glucosidase gene bgI, namely beta-glucosidase BGI.

Preferably, the amino acid sequence of the protein is shown as SEQ ID NO. 2.

The invention also protects a recombinant expression vector containing the beta-glucosidase gene bgI.

Preferably, the recombinant expression vector is a recombinant plasmid pPICZ alpha A-bgI.

Preferably, the sequence of the coding region of the beta-glucosidase gene bgI is obtained by amplification, and then the recombinant plasmid pPICZ alpha A-bgI is constructed by using a double enzyme digestion and enzyme ligation method.

Specifically, a straw compost sample is taken as a raw material to extract total RNA, and cDNA of a beta-glucosidase gene bgI is obtained through reverse transcription; using cDNA as template, using PCR amplification method to obtain bgI gene complete sequence; the complete bgI gene is inserted into the pPICZ alpha A expression vector by adopting a double enzyme digestion and enzyme linking method to obtain a recombinant expression plasmid pPICZ alpha A-bgI.

The invention also protects the recombinant engineering bacteria containing the beta-glucosidase gene bgI.

Preferably, the recombinant plasmid pPICZ α A-bgI is transformed into Pichia pastoris to obtain a gene bgI capable of expressing the β -glucosidase gene described above. Preferably, high yields of protein are obtained under induction medium conditions.

Specifically, an electric shock transformation method is adopted to transform the recombinant expression plasmid pPICZ alpha A-bgI into pichia pastoris competence, and high-efficiency engineering bacteria transformants are screened; after the engineering bacteria are induced and cultured by a liquid fermentation culture medium, a large amount of endo-cellulase BGI can be secreted.

The invention also protects the beta-glucosidase gene bgI or the application of the beta-glucosidase in cellulose decomposition.

Advantageous effects

With the continuous and intensive research on cellulases, cellulases are increasingly used in production practice. For example, a large amount of waste in our lives is made of cellulose, and if the waste is discarded randomly and naturally degraded, the waste is too long and the surrounding environment is damaged. Therefore, the cellulase is usually utilized for degradation, ethanol fuel and the like can be obtained, the use of non-renewable energy sources can be reduced, and the environmental pollution can be avoided. In addition to the use of cellulases to degrade fiber waste, their use in industry is also ubiquitous, such as degrading plastics. L-lactic acid belongs to organic acids, and the main constituent element of the L-lactic acid is corn starch. Research shows that L-lactic acid as a substrate and cellulase have the best effect of degrading plastics, which provides a feasible route for solving the problem of 'white garbage' seriously polluting the environment at present.

The invention aims to comprehensively improve the decomposition efficiency of cellulose, and establishes an efficient recombinant protein expression system through a proper genetic engineering technology; and the activity of the enzyme is improved by modifying the molecular structure of the enzyme, so that the method has important significance for improving the cellulose degradation by improving the cellulase activity in trichoderma and improving the resource utilization of agricultural solid wastes.

Drawings

FIG. 1 effect of different temperatures on beta-glucosidase BGI;

FIG. 2 Effect of different pH buffers on beta-glucosidase BGI;

FIG. 3 Effect of different metal ions on β -glucosidase BGI, wherein 1 is not treated with metal ions; 2Fe³⁺；3Cu²⁺；4Mg²⁺；5Ca²⁺；6K⁺；7Mn²⁺；8Co²⁺；9Zn²⁺；

Figure 4 pH stability of β -glucosidase BGI;

FIG. 5 kinetics curves of hydrolysis of p-NPG by beta-glucosidase BGI;

FIG. 6 PCR verification results of different recombinant yeast colonies of beta-glucosidase BGI;

FIG. 7 is an SDS-PAGE electrophoresis of purified β -glucosidase BGI, with no-load protein 1; 2 is BGI crude enzyme solution; 3 is purified BGI.

Detailed Description

The present invention is further illustrated by the following examples, in which experimental procedures not specifically identified are generally performed by means well known in the art.

Example 1 cloning of 1 bgI Gene and construction of engineered Strain thereof

1. bgI cloning of Gene

Designing a primer: according to the gene characteristics of the beta-glucosidase gene and the known different polyclonal locating points on the expression vector, the primer sequence capable of heterologously expressing the gene in pichia pastoris is designed by combining the gene sequence information of the known body signal peptide of the beta-glucosidase gene, and the sequence is shown in table 1.

Table 1: primer sequence of beta-glucosaccharase gene bgI heterologously expressed in pichia pastoris

Total RNA was extracted from the straw compost sample using Trizol reagent (Qiagen), and cDNA was synthesized by reverse transcription using RT-PCR kit (Takara). The cDNA synthesized by the reverse transcription is used as a template, primers in the table 1 are used for PCR amplification to obtain a complete bgI gene fragment, and a bgI gene without a signal peptide sequence is obtained after sequencing. The PCR system is shown in Table 2, and the procedure for PCR is shown in Table 3.

Table 2: PCR reaction system

Table 3: PCR procedure

2. Recovering the cDNA product of bgI gene

Cutting bgI gene DNA gel under ultraviolet lamp, absorbing water, drying, cutting into pieces, transferring to 1.5mL centrifuge tube, weighing, according to formula: calculate gel volume, 100 μ Ι _ to 100mg one gel volume;

adding Buffer DE-A with the volume twice that of the gel, uniformly mixing under a vortex condition, and preserving in a water bath kettle at a constant temperature of 75 ℃ until the gel is molten; adding Buffer DE-B with the total volume of 1/2 of the Buffer DE-A, and uniformly mixing under a vortex condition; transferring the mixed solution into a tube of a centrifugal device for two times, centrifuging for 1min at 12000rpm, and discarding the filtrate; adding 500 μ L Buffer W1 into DNA column, centrifuging at 14000rpm for 30s, and discarding the filtrate; adding 700 μ L Buffer W2 into DNA column, centrifuging at 14000rpm for 30s, and discarding the filtrate; adding 400 μ L Buffer W2 into DNA column, centrifuging at 14000rpm for 30s, and discarding the filtrate; centrifuging at 12000rpm, and idling for 1 min; transfer of the column from a 1.5mL centrifuge tube and addition of 15. mu.L of sterilized ddH₂O onto a column matrix which had been sterilized and passed through a water bath at 65 ℃ and left to stand at room temperature for 3min, and centrifuged at 13000rpm for 1 min.

3. Double enzyme digestion

The endocellulase gene heterologously expressed by pichia pastoris and a vector plasmid are subjected to XbaI and EcoRI step-by-step double enzyme digestion operation, and the designed enzyme digestion total system is 20 mu L. The total system included 2. mu.L of 10 Xbuffer, 1. mu.L of XbaI, 1. mu.L of EcoRI, 6.75. mu.L of the clean gene of interest recovered from the gel cut and 9.25. mu.L of ddH₂O。

Carrying out double enzyme digestion on the recovered gel-cutting cDNA and pPICZ alpha A by using corresponding restriction enzymes; carrying out clean recovery on the enzyme digestion product obtained in the last step by using a PCR recovery kit; carrying out secondary enzyme digestion operation on the recovered product, wherein the method is carried out according to the designed enzyme digestion reaction total system, and the enzyme digestion temperature is controlled and kept at 38 ℃; and (4) carrying out clean recovery on the secondary enzyme digestion product by using a PCR recovery kit.

4. Enzyme linked to

Enzyme-linked operation is carried out between the beta-glucosidase gene bgI of the double enzyme digestion product and the cloning vector, and the enzyme-linked reaction system is shown in the table 4, wherein the ratio of the enzyme-linked reaction system to the cloning vector is 10: 1 ratio design the molar ratio of gene fragment to vector.

Table 4: enzyme linked reaction system

5. Pichia competent preparation

1) Streaking and selecting 10 mu L of a Pichia pastoris X33 single colony on a YPD plate, inoculating the single colony into 5mL of a YPD culture medium, and performing shake culture at 30 ℃ and 180rpm to obtain dispersed single colonies;

2) mu.L of the single colony mixed culture solution was extracted, transferred to a triangular flask containing 100mL of YPD medium, and subjected to shaking culture at 30 ℃ and 180rpm overnight to OD₆₀₀Saturation, i.e. OD₆₀₀1.3 to 1.5;

3) inoculating liquid culture medium into pre-cooled 50mL sterilized centrifuge tube, centrifuging at 3000rpm and 4 deg.C for 10min, draining supernatant culture medium, adding 30mL pre-cooled ddH₂O heavy suspension; centrifuging at 4 deg.C and 3000rpm for 10min, and repeating the process twice to ensure that suspended thallus is completely precipitated;

4) adding 20mL of ice bath-cooled 1M sorbitol solution, resuspending the precipitated thallus, centrifuging at 4 deg.C and 3000rpm for 10min, decanting the supernatant culture solution, adding 1mL of ice bath-cooled 1M sorbitol solution, and resuspending the precipitated thallus; the final volume was made to 1.5 mL.

6. Electrotransformation of Pichia pastoris

5 mu g of linear DNA and 100 mu L of yeast competent cells are mixed uniformly, transferred into a sterilized electric conversion cup precooled at the temperature of 2mm-20 ℃, and electrically converted for 4-10ms under the setting of 1500V, 200 omega and 20 mu F in the electric conversion cup. After the electric shock was completed, 1mL of sorbitol solution cooled in an ice bath was immediately added to the clean bench, shaken gently and homogenized, and the mixture was transferred to a 1.5mL centrifuge tube. Resuscitated at 30 ℃ for 1h at 100rpm, and pipetted 100. mu.L of the suspension onto YPDS plates (containing 100. mu.g. mL)^-1Zeocin) in an incubator, setting the temperature to be constant at 30 ℃, and stopping culturing after single fungus appears.

7. Screening for multicopy transformants

Selecting suitable single colony from the single colony plate culture medium to inoculate 200 mug. multidot.mL mixed in advance^-1The YPDS plates of Zeocin (K, K) were incubated at constant temperature, and when the growth of colonies on the plates was excellent and did not overlap each other, the colonies were again selected and inoculated into pre-mixed 1000. mu.g.mL plates with the proposed numbers^-1The culture was continued on YPDS plates from Zeocin.

8. Colony PCR validation

Mixing the mixture with 1000. mu.g/mL^-1And (3) selecting a proper single colony on a YPDS culture medium plate of Zeocin for preparing a PCR reaction template, wherein the PCR verification needs to be carried out together with a primer of beta-glucosidase gene bgI.

9. Enzyme production induced fermentation

The obtained yeast transformant single colony was inoculated into a 2.5L Erlenmeyer flask containing 1L of BMGY, shake-cultured at 180rpm, and incubated at 30 ℃ to OD₆₀₀Methanol was added to a 1L Erlenmeyer flask at a rate of about 1.0 at intervals of 24 hours in a volume of 1% of the total volume of the induction medium (10mL), and the culture was continued for 7 days. Centrifuging at 10000rpm for 5min with a Beckmann centrifuge, collecting supernatant, discarding precipitate to obtain crude enzyme solution, measuring enzyme activity, and storing at-80 deg.C.

10. SDS-PAGE analysis of enzyme-producing fermentation broth

1) And (3) crude enzyme sample treatment: according to the following steps: 1, mixing the treated sample with a Tris-HCl buffer solution with the pH value of 6.8 and the concentration of 100mM, a beta-mercaptoethanol solution with the concentration of 200mM, a bromophenol blue solution with the concentration of 0.2%, an SDS solution with the concentration of 4% and a glycerol solution with the concentration of 20%, transferring the mixture into a 1mL centrifugal tube, shaking the mixture evenly, sealing the opening of the centrifugal tube tightly by using a preservative film, and carrying out boiling water bath for 5 min;

2) electrophoresis: selecting an SDS-PAGE method which is a commonly used extracellular protein detection method for genetic engineering, wherein the specific operation steps refer to protein electrophoresis experimental technology;

3) examination glue: the film is dyed and decolored by Coomassie brilliant blue staining solution according to protein electrophoresis experimental technology until clear protein bands are obtained.

Example 2 concentration and purification of enzyme

1. Concentration of the enzyme

By ammonium sulfate precipitation method, (NH)₄)₂SO₄Powder, the mass at 0 ℃ at which the saturation of the sample is 50% is (NH)₄)₂SO₄And (4) adding the amount. Fine grinding to (NH)₄)₂SO₄Is in powder form, and is easy to dissolve, and can be slowly added into the supernatant of the crude enzyme solution in small amount for multiple times, the addition process needs to be carried out in ice bath state and stirring is not stopped, and the mixed solution is centrifuged at 4 deg.C and 10000rpm for 10 min. The supernatant medium was decanted and the protein pellet was further resuspended in 50mM phosphate buffer.

2. Purification of enzymes

The nickel ion in the Ni column can be bound to a protein containing a His (histidine) tag, and can also be bound to imidazole. The crude enzyme solution passes through a Ni column, the beta-glucosidase gene of the invention is provided with a His label and can be adsorbed on the Ni column, and then is eluted by imidazole with different concentrations, thereby achieving the purpose of purification.

Example 3 Effect of different temperatures on beta-glucosidase BGI

Taking 50 μ L of diluted enzyme solution containing 1 μ M enzyme, adding 450 μ L of acetic acid-sodium acetate buffer solution with pH of 5.020 mM into 2mL centrifuge tube, preheating in water bath at 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C and 100 deg.C for 10min, adding 10mM p-NPG solution 500 μ L preheated at each temperature for 10min, mixing, placing in water bath at corresponding temperature for 10min, immediately adding 1mL of 1mol/L Na₂CO₃The reaction was terminated, and the reaction mixture was left at room temperature for 5min, and the absorbance OD was measured at 410 nm. Meanwhile, the enzyme solution which is heated and inactivated is treated by the same method to be used as a blank control. Three replicates were set for each treatment. A graphical analysis is made from the measured data. The optimal hydrolysis temperature of the beta-glucosidase BGI is 50 ℃, the beta-glucosidase BGI has stable enzyme activity at 40-80 ℃, the relative enzyme activity is sharply reduced after 80 ℃, and more than 40% of the relative enzyme activity is still maintained at the high temperature of 100 ℃.

Example 4 Effect of different pH buffers on beta-glucosidase BGI

50. mu.L of diluted enzyme solution containing 1. mu.M of the enzyme was taken, and 450. mu.L of Na having pH of 3.0, 4.0, 5.0 was added₂HPO₄Citric acid buffer, Na at pH 5.0, 6.0, 7.0₂HPO₄-KH₂PO₄Buffer solution, Tris-HCl buffer solution with pH 7.0, 8.0 and 9.0 is put in a 2mL centrifuge tube, preheated for 10min in 50 ℃ water bath, added with 500 mu L of p-NPG solution with concentration of 10mM preheated for 10min, mixed evenly, put in a water bath kettle again for 10min, and immediately added with 1mL of 1 mol.L^-1Na (b) of₂CO₃The reaction was terminated, and the reaction mixture was left at room temperature for 5min, and the absorbance OD was measured at 410 nm. Meanwhile, the enzyme solution which is heated and inactivated is treated by the same method to be used as a blank control. Three replicates were set for each treatment. A graphical analysis is made from the measured data. The optimum pH value of the beta-glucosidase BGI is 4-6, the beta-glucosidase BGI belongs to acidophilic cellulase, and the relative enzyme activity is sharply reduced when the pH value is 8.

Example 5 Effect of different Metal ions on beta-glucosidase BGI

To a 2mL centrifuge tube, 50. mu.L of diluted enzyme solution containing 1. mu.M of enzyme was added, 350. mu.L of acetic acid-sodium acetate buffer solution of pH 5.020 mM and 100. mu.L of a solution of different metal ions of 100mM concentration, the different metal ions being: fe³⁺，Cu^{2 +}，Mg²⁺，Ca²⁺，K⁺，Mn²⁺，Co²⁺，Zn²⁺Preheating in 50 deg.C water bath for 10min, adding preheated 10min p-NPG solution with concentration of 10mM 500 μ L, mixing, placing in water bath again for 10min, and immediately adding 1mL1 mol. L^-1Na of (2)₂CO₃The reaction was terminated, and the reaction mixture was left at room temperature for 5min, and the absorbance OD was measured at 410 nm. Meanwhile, the enzyme solution which is heated and inactivated is treated by the same method to be used as a blank control. Three replicates were set for each treatment. A graphical analysis is made from the measured data. Has no metal ion to obviously promote beta-glucosidase BGI, Fe³⁺And Cu²⁺Has obvious BGI inhibiting effect.

Example 6 pH stability of beta-glucosidase BGI

50 μ L of diluted enzyme solution containing 1 μ M of enzyme, pH 3.0, 4.0 Na₂HPO₄Citric acid buffer, Na at pH 5.0, 6.0, 7.0₂HPO₄-KH₂PO₄Buffer, pHPlacing 8.0, 9.0 Tris-HCl buffer solution in 2mL centrifuge tube, mixing uniformly at 4 deg.C, standing for 4h, preheating in 50 deg.C water bath for 10min, adding 10mM p-NPG solution 500 μ L, mixing uniformly, placing in water bath for 10min, immediately adding 1mL1 mol. L^-1Na of (2)₂CO₃Terminating the reaction, standing at room temperature for 5min, measuring absorbance OD at 410nm, and treating the enzyme solution with the same method to obtain blank control. Three replicates were set for each treatment. A graphical analysis is made from the measured data. The beta-glucosidase BGI has stability at pH4-7, is almost inactivated at pH 3 and pH 9, and is not suitable for long-time reaction under peracid or over-alkali conditions.

Example 7 kinetic Curve of hydrolysis of p-NPG by beta-glucosidase

Adding 50 mu L of diluted enzyme solution containing 1 mu M of enzyme and 450 mu L of acetic acid-sodium acetate buffer solution with pH 5.0 and 20mM into a 2mL centrifuge tube, preheating in a water bath at 50 ℃ for 10min, adding p-NPG with different concentrations which are preheated for 10min, and setting the gradient of the p-NPG with different concentrations as follows: 0.2 mM. mL^-1、0.4mM·mL^-1、0.6mM·mL^-1、0.8mM·mL^-1、1.0mM·mL^-1、1.2mM·mL^-1After mixing, putting into water bath again for 10min, immediately adding 1mL of 1 mol.L^-1Na (b) of₂CO₃The reaction was terminated, and the reaction mixture was left at room temperature for 5min, and the absorbance OD was measured at 410 nm. Meanwhile, the enzyme solution which is heated and inactivated is treated by the same method to be used as a blank control. Three replicates were set for each treatment. A graphical analysis is made from the measured data. The kinetic equation of the hydrolysis of p-NPG by beta-glucosidase BGI obtained by nonlinear regression analysis is as follows: y ═ Vmax × Xn/(Kn + Xn), we finally found: vmax is 0.648. mu.M.min^-1；Km＝0.215μM·mL^-1. Wherein, the lower Km represents the stronger affinity with the substrate, and the data show that the beta-glucosidase BGI has stronger affinity with the substrate.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be protected.

Sequence listing

<110> Nanjing university of agriculture

<120> beta-glucosidase gene bgI, and coding protein and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

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<211> 2148

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gacagcaaag tcgtaccccc tgcaggtact ccttggggcg tcgcatacgg caaggcacag 60

gcagcactgg caaaactcac cctccaagac aaggtcggca tcgtgtctgg cgttggctgg 120

aaccaggggc cctgcgttgg aaacacgtct ccagtctcaa agattggcta tccttcgtta 180

tgtcttcaag atggacctct cggtgttcgc ttctcatctg gcagcacggc ctttacacca 240

ggcgttcagg ccgcttcaac ttgggatctg aacctaatcc gcgaacgtgg acaattcatc 300

ggacaagaag ttagagatac gggaattcac gtcactctcg gtcccgtagc tggacctctg 360

ggaaagacgc ctcagggagg tcgcaactgg gaaggcttca gtgttgatcc gtacctcacg 420

ggattagcta tggctcagac aattaacggc attcagtctg tgggagtgca agctacagcc 480

aagcactaca ttcttaatga acaagaacgc aaccgagaga caatgtcgag caatgccgat 540

gatcgaactc ttcacgagct ttatgcttgg ccctttgccg acgctgtaca ggcaaatgtc 600

gcctctgtca tgtgctctta caacaagatc aacacaactt gggcgtgtga agataagaac 660

acgctacaaa ctctgctcaa agatcagctg ggattcccgg gatacgtcat gacagactgg 720

aacgctcaac actcgacggt gcaggccgcc actgctgggc tcgatatgtc aatgcccggc 780

actgacttta acggcaacaa caggctatgg ggcccagctc tcactaacgc agtcaacagc 840

aaccaggtgc cctcgagcag agttgatgat atggtgacgc gaatcttagc tgcatggtat 900

ctaacaggcc aagatgccgc aggctatccg tcgttgagtc tcagcaggaa cgttcaagga 960

actcacaaaa ccaacgtgag atcaattgcc agagacggca ttgttctcct caagaacgac 1020

ggcaacatcc tgccgctgaa gaagcccgca agcattgctg tcatcgggtc tgccgctatc 1080

attggtgctc acgccagcaa ctcaggttca tgcggtgata agggctgtga caatggcgcc 1140

ttgggcatgg gctggggatc cggcgccgtc aactacccat actttgtagc accctatgat 1200

gccatcaata ctagagccac ctcgcaaggc accagagtta ccttgagtaa cacggacaac 1260

acttcttcgg gtgcatctgc agcaagtgga aaagacgtcg ccattgtctt catcactgcc 1320

gattcaggag aagggtacat caccgtggag ggcaacgctg gtgaccgcaa cgacttgaac 1380

gcctggcaca acggaaatgc tctcgttcag gcagtggcag gttccaacca aaatgtcatt 1440

gttgtcgttc actcggtagg cgccatcatc ctcgagcaaa tcattgcttt gccccaggtc 1500

aaggctattg tctgggcagg tctcccatcg caggagagtg gcaatgcgct tgtcgacgtg 1560

ctgtggggag atgtcagccc ctctggcaag ctcgtgtaca ccattgcaaa gagtccaaac 1620

gactacaaca cacgcattac ttccggcgac gacagtttca gcgaaggact gtttatcgac 1680

tacaagcatt tcgacgacgc aggtatcacg cctcgttacg agtttggctt tggactatct 1740

tacaccaagt tcaactactc tcgcctctct gtcttgtcca ctgcaaagtc tggccccgcg 1800

accggagcag tggtacctgg aggcccaagt gatttgttcc agaatgtcgc aaccatcacc 1860

gtggacatta cgaattccgg tgcagtgact ggagctgaag ttgctcagct gtatctcact 1920

tacccgtctt cagcgcccag aacaccacct aagcaactgc gtggctttgc gaagctgagt 1980

ctcacggcag gccagactag cacagcgacg ttcaacattc gaagaaggga tctgagctac 2040

tgggatacga gttcgcagaa gtgggtggtg ccttcgggat cgtttggtat cagcgtggga 2100

gcaagcagca gggatatccg gctgacgggc agtctgtcgg tatcatga 2148

<210> 2

<211> 715

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Asp Ser Lys Val Val Pro Pro Ala Gly Thr Pro Trp Gly Val Ala Tyr

1 5 10 15

Gly Lys Ala Gln Ala Ala Leu Ala Lys Leu Thr Leu Gln Asp Lys Val

20 25 30

Gly Ile Val Ser Gly Val Gly Trp Asn Gln Gly Pro Cys Val Gly Asn

35 40 45

Thr Ser Pro Val Ser Lys Ile Gly Tyr Pro Ser Leu Cys Leu Gln Asp

50 55 60

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

65 70 75 80

Gly Val Gln Ala Ala Ser Thr Trp Asp Leu Asn Leu Ile Arg Glu Arg

85 90 95

Gly Gln Phe Ile Gly Gln Glu Val Arg Asp Thr Gly Ile His Val Thr

100 105 110

Leu Gly Pro Val Ala Gly Pro Leu Gly Lys Thr Pro Gln Gly Gly Arg

115 120 125

Asn Trp Glu Gly Phe Ser Val Asp Pro Tyr Leu Thr Gly Leu Ala Met

130 135 140

Ala Gln Thr Ile Asn Gly Ile Gln Ser Val Gly Val Gln Ala Thr Ala

145 150 155 160

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

165 170 175

Ser Asn Ala Asp Asp Arg Thr Leu His Glu Leu Tyr Ala Trp Pro Phe

180 185 190

Ala Asp Ala Val Gln Ala Asn Val Ala Ser Val Met Cys Ser Tyr Asn

195 200 205

Lys Ile Asn Thr Thr Trp Ala Cys Glu Asp Lys Asn Thr Leu Gln Thr

210 215 220

Leu Leu Lys Asp Gln Leu Gly Phe Pro Gly Tyr Val Met Thr Asp Trp

225 230 235 240

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

245 250 255

Ser Met Pro Gly Thr Asp Phe Asn Gly Asn Asn Arg Leu Trp Gly Pro

260 265 270

Ala Leu Thr Asn Ala Val Asn Ser Asn Gln Val Pro Ser Ser Arg Val

275 280 285

Asp Asp Met Val Thr Arg Ile Leu Ala Ala Trp Tyr Leu Thr Gly Gln

290 295 300

Asp Ala Ala Gly Tyr Pro Ser Leu Ser Leu Ser Arg Asn Val Gln Gly

305 310 315 320

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

325 330 335

Leu Lys Asn Asp Gly Asn Ile Leu Pro Leu Lys Lys Pro Ala Ser Ile

340 345 350

Ala Val Ile Gly Ser Ala Ala Ile Ile Gly Ala His Ala Ser Asn Ser

355 360 365

Gly Ser Cys Gly Asp Lys Gly Cys Asp Asn Gly Ala Leu Gly Met Gly

370 375 380

Trp Gly Ser Gly Ala Val Asn Tyr Pro Tyr Phe Val Ala Pro Tyr Asp

385 390 395 400

Ala Ile Asn Thr Arg Ala Thr Ser Gln Gly Thr Arg Val Thr Leu Ser

405 410 415

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

420 425 430

Val Ala Ile Val Phe Ile Thr Ala Asp Ser Gly Glu Gly Tyr Ile Thr

435 440 445

Val Glu Gly Asn Ala Gly Asp Arg Asn Asp Leu Asn Ala Trp His Asn

450 455 460

Gly Asn Ala Leu Val Gln Ala Val Ala Gly Ser Asn Gln Asn Val Ile

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Arg Ile Thr Ser Gly Asp Asp Ser Phe Ser Glu Gly Leu Phe Ile Asp

545 550 555 560

Tyr Lys His Phe Asp Asp Ala Gly Ile Thr Pro Arg Tyr Glu Phe Gly

565 570 575

Phe Gly Leu Ser Tyr Thr Lys Phe Asn Tyr Ser Arg Leu Ser Val Leu

580 585 590

Ser Thr Ala Lys Ser Gly Pro Ala Thr Gly Ala Val Val Pro Gly Gly

595 600 605

Pro Ser Asp Leu Phe Gln Asn Val Ala Thr Ile Thr Val Asp Ile Thr

610 615 620

Asn Ser Gly Ala Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Leu Thr

625 630 635 640

Tyr Pro Ser Ser Ala Pro Arg Thr Pro Pro Lys Gln Leu Arg Gly Phe

645 650 655

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

660 665 670

Ile Arg Arg Arg Asp Leu Ser Tyr Trp Asp Thr Ser Ser Gln Lys Trp

675 680 685

Val Val Pro Ser Gly Ser Phe Gly Ile Ser Val Gly Ala Ser Ser Arg

690 695 700

Asp Ile Arg Leu Thr Gly Ser Leu Ser Val Ser

705 710 715

Claims (1)

1. Beta-glucosidase genebgI. Beta-glucosidase genebgI the use of the protein coded by I in the decomposition of cellulose, wherein, the beta-glucosidase genebgThe nucleotide sequence of I is shown as SEQ ID NO. 1; the amino acid sequence of the protein is shown as SEQ ID NO. 2.

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