CN113151326B - Endo-cellulase gene egI, protein coded by same and application of endo-cellulase gene egI - Google Patents

Abstract

The invention discloses an endo-cellulase gene egI, and a protein coded by the same and application thereof. The nucleotide sequence of the endo-cellulase gene egI is shown in SEQ ID NO. 1; the protein coded by the gene is the endo-cellulase EGI. The maximum reaction rate of hydrolyzing CMC can reach 22.2 mg-min according to the analysis of EGI enzymatic reaction kinetics^‑1. The analysis of the results of enzymology shows that: at pH 3-4, the highest value of reducing sugar content produced by hydrolyzing CMC per minute by EGI is up to 21.4 mg/mL^‑1Belonging to acid endo-cellulase; when the temperature reaches 100 ℃, the content of reducing sugar generated by hydrolyzing CMC per minute by EGI can reach 12.4 mg/mL^‑1It still has strong activity at high temp. The invention provides theoretical basis and technical guarantee for resource utilization of agricultural wastes and development of ecological agriculture.

Description

Endo-cellulase gene egI, protein coded by same and application of endo-cellulase gene egI

Technical Field

The invention belongs to the field of enzymology engineering, and particularly relates to an endo-cellulase gene egI, and a protein coded by the same and application of the endo-cellulase gene egI.

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 utilizable 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, has the advantages of low energy consumption, mild reaction conditions, environmental friendliness and the like, and gradually becomes the 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.

The biological mechanism of cellulose hydrolysis by the endo-cellulase is very critical, and the endo-cellulase randomly cuts an amorphous region in a long chain of the cellulose, so that the integrity of the cellulose structure is reduced, and the cellulose is hydrolyzed to generate a certain amount of micromolecular polysaccharide. Wherein the endo-cellulase preferentially acts on amorphous cellulose and hydrolyzes from the inside of cellulose molecule. In the trichoderma cellulase system, a regulation mechanism in a microorganism regulates and controls the ratio of cellulase of each component, and artificial regulation is difficult to complete in modes of regulating and controlling culture conditions and the like. In different industrial productions of cellulase, different requirements are often provided for the proportion of each component, for example, in the textile washing link, endo-cellulase with higher relative activity is required, and under the condition, the cellulase directly synthesized by trichoderma hardly meets the requirement of industrial batch production.

The method realizes the high-efficiency expression of the endo-cellulase gene egI by constructing the recombinant pichia pastoris, has important significance for improving the comprehensive utilization of crop straws, and provides theoretical basis for actual production.

The invention content is as follows:

the invention aims to provide an endo-cellulase gene egI, a protein coded by the same and application thereof in the prior art.

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

an endo-cellulase gene egI, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The present invention also protects the protein encoded by the endo-cellulase gene egI described above, i.e., the endo-cellulase EGI.

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 endo-cellulase gene egI described above.

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

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

Specifically, total RNA is extracted by taking a straw compost sample as a raw material, and cDNA of endoglucanase gene egI is obtained by reverse transcription; using cDNA as template, using PCR amplification method to obtain egI gene complete sequence; the complete egI 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-egI.

The invention also protects the recombinant engineering bacteria containing the endo-cellulase gene egI.

Preferably, the recombinant plasmid pPICZ alpha A-egI described above is transformed into Pichia pastoris to obtain a gene egI capable of expressing the endo-cellulase 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-egI 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 EGI can be secreted.

The invention also protects the application of the endo-cellulase gene egI or the protein in the cellulose decomposition.

Has the advantages that:

with the continuous and intensive research on cellulases, cellulases are increasingly used in production practice. A large amount of waste is generated in the agricultural production process of China every year, the main component of the waste is lignocellulose, if the waste is discarded at will, a large amount of resources are wasted, and different environmental pollutions are caused. In addition to their application to the degradation of various lignocellulosic wastes, cellulases find use in industry, such as in the degradation of plastics. L-lactic acid belongs to organic acids, and the main constituent element of the L-lactic acid is corn starch. Research shows that the L-lactic acid as a substrate and the cellulase have the best effect of degrading plastics, which provides a feasible route for solving the problem of 'white garbage' seriously polluting the environment

The invention aims to comprehensively improve the decomposition efficiency of cellulose, establishes an efficient recombinant protein expression system through a proper genetic engineering technology, obtains a genetic engineering strain of the enzyme, and 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 endo-cellulase EGI;

FIG. 2 Effect of different pH buffers on endo-cellulase EGI;

FIG. 3 effect of different metal ions on endo-cellulase EGI; wherein, 1: no metal ion treatment is added; 2: fe³⁺；3：Zn²⁺；4：Co²⁺；5：Mn²⁺；6：Ca²⁺；7：Mg²⁺；8：K⁺；9：Ni²⁺；10：Cu²⁺；

FIG. 4 Effect of endo-cellulase EGI on different substrates;

FIG. 5 kinetic curves of endo-cellulase EGI hydrolysis of CMC;

FIG. 6 shows the results of colony PCR verification of different recombinant yeasts with endo-cellulase EGI;

FIG. 7 is an SDS-PAGE electrophoresis of purified endo-cellulase EGI; wherein, 1 is the unloaded protein, 2 is the EGI crude enzyme liquid, and 3 is the purified EGI.

The specific implementation mode is as follows:

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 egI Gene and construction of engineered Strain thereof

1. egI cloning of Gene

Designing a primer: according to the gene characteristics of the endo-cellulase 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 known sequence information of the bulk signal peptide gene of the endo-cellulase egI gene, and the sequence is shown in table 1.

Table 1: endocellulase gene egI primer sequence 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 egI gene fragment, and a egI 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 egI gene

Cutting egI 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 double enzyme digestion product endo-cellulase gene egI and a cloning vector, and the enzyme-linked reaction system is shown in table 4, wherein the ratio of 10: 1 ratio design the molar ratio of gene fragment to vector.

Table 4: enzyme linked reaction system

5. Competent preparation of Pichia pastoris

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 medium was extracted, transferred to a triangular flask containing 100mL of YPD medium, and cultured overnight at 30 ℃ and 180rpm with shaking until OD was reached₆₀₀Saturation, 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 under a constant temperature of 30 DEGAnd (4) warming, and stopping culturing after single bacterium 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 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 an endo-cellulase gene egI primer.

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. Finely grinding to (NH)₄)₂SO₄Is in powder form, and is convenient for dissolving, and is slowly added into the supernatant of the crude enzyme solution in small amount for multiple times, the adding process needs to be in an ice bath state and continuously stirred, and the mixed solution is centrifugally operated for 10min at 4 ℃ and 10000 rpm. 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 endo-cellulase gene provided by 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, so that the purification purpose is achieved.

EXAMPLE 3 Effect of different temperatures on endo-cellulase EGI

Add 300. mu.L of 1% sodium carboxymethylcellulose substrate and 690. mu.L of acetic acid-sodium acetate buffer (pH 4.0) to a 2mL centrifuge tube, add 10. mu.L of purified diluted enzyme solution containing 1. mu.M enzyme, mix, treat in a water bath for 10min, set the temperature: 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, 100 deg.C. Three replicates of each experimental design were run and enzyme activity was measured using a DNS method with a microplate reader. Data were measured and chart analysis was made. The optimal hydrolysis temperature of the endonuclease EG-I is 50 ℃, but the relative enzyme activity of more than 60 percent is still maintained at the lower temperature of 20 ℃, and the relative enzyme activity of more than 50 percent is maintained at the high temperature of 100 ℃.

Example 4 Effect of different pH buffers on Endocellulase EGI

To a 2mL centrifuge tube was added 300 μ L of 1% sodium carboxymethylcellulose substrate and 690 μ L of different pH buffers with a gradient of: na at pH 2.0, 3.0, 4.0₂HPO₄-citric acid bufferThree replicates per gradient run were set up for solutions of acetate-sodium acetate buffer pH 4.0, 5.0, 6.0, 7.0 and Tris-HCl buffer pH 7.0, 8.0, 9.0. Meanwhile, 10 μ L of the purified diluted enzyme solution containing 1 μ M of the enzyme is added into a 2mL centrifuge tube, and after uniform mixing, the diluted enzyme solution is treated at the water bath temperature for 10min under the known optimal temperature condition, and the enzyme activity is measured by using a DNS method. Data were measured and a chart analysis was made. The optimum pH value of the endo-cellulase EGI is 3-4, belongs to acidophilic cellulase, and the relative enzyme activity is sharply reduced when the pH value is 6.

Example 5 Effect of different Metal ions on Endocellulase EGI

To a 2mL centrifuge tube was added 300. mu.L of 1% sodium carboxymethylcellulose substrate, 590. mu.L of acetic acid-sodium acetate buffer at pH (pH 4.0), and 100. mu.L of a 100mM solution of different metal ions: fe³⁺，Zn²⁺，Co²⁺，Mn^{2 +}，Ca²⁺，Mg²⁺，K⁺，Ni²⁺，Cu²⁺Three replicates were set up for each metal ion treatment. Meanwhile, 10 μ L of the purified diluted enzyme solution containing 1 μ M of the enzyme is added into a 2mL centrifuge tube, and after uniform mixing, the diluted enzyme solution is treated at the water bath temperature for 10min under the known optimal temperature condition, and the enzyme activity is measured by using a DNS method. Data were measured and a chart analysis was made. Different metal ions have certain influence on the endonuclease EGI, actually Co²⁺The promotion effect is most remarkable.

Example 6 Effect of endo-cellulase EGI on different substrates

The following were added to 2mL centrifuge tubes:

treatment of 1: 300. mu.L of 1% sodium carboxymethylcellulose substrate, 690. mu.L of acetic acid-sodium acetate buffer (pH 4.0), 10. mu.L of a diluted enzyme solution after purification containing 1. mu.M of the enzyme;

treatment 2, 10mg of crystalline cellulose, 990. mu.L of acetic acid-sodium acetate buffer (pH 4.0), and 10. mu.L of a purified diluted enzyme solution containing 1. mu.M of the enzyme;

treatment 3:10mg of corncob powder, 990. mu.L of acetic acid-sodium acetate buffer (pH 4.0), 10. mu.L of purified diluted enzyme solution containing 1. mu.M of enzyme;

treating 4:10mg straw powder, 990 μ L acetic acid-sodium acetate buffer (pH 4.0), 10 μ L purified diluted enzyme solution containing 1 μ M enzyme;

treatment 5:10mg of filter paper, 990. mu.L of acetic acid-sodium acetate buffer (pH 4.0), 10. mu.L of the diluted enzyme solution after purification containing 1. mu.M of enzyme;

treatment 6: 300. mu.L 100 mg/mL^-1Phosphoric acid swollen cellulose, 690. mu.L of an acetic acid-sodium acetate buffer (pH 4.0), 10. mu.L of a purified diluted enzyme solution containing 1. mu.M of the enzyme;

mixing the above solutions, treating at water bath temperature for 10min under known optimum temperature condition, and measuring enzyme activity by DNS method. Three replicates of each substrate treatment were set up. Data were measured and chart analysis was made. The endo-EGI has strong hydrolysis effect on CMC, but has little influence on straws and filter paper.

Example 7 kinetics curves of endo-cellulase EGI hydrolysis of CMC

100 μ L of sodium carboxymethylcellulose substrate of different concentrations was added to a 2mL centrifuge tube, the concentration gradient of the different substrates being: 10 mg/mL^-1、20mg·mL^-1、30mg·mL^-1、40mg·mL^-1、50mg·mL^-1、60mg·mL^-1Three replicates were set up for each substrate concentration treatment. Meanwhile, 890. mu.L of acetic acid-sodium acetate buffer (pH 4.0) and 10. mu.L of the purified diluted enzyme solution containing 1. mu.M of the enzyme were added to a 2mL centrifuge tube, and after uniform mixing, the mixture was treated with water bath at a known optimum temperature for 10min, and the enzyme activity was measured by the DNS method. The kinetic equation of the endonuclease EGI hydrolysis CMC obtained by nonlinear regression analysis is as follows: y Vmax Xn/(Kn + Xn), which gives: vmax is 22.2 mg/min^-1；Km＝1.03mg·mL^-1. Wherein the lower the Km, the stronger the affinity with the substrate, and from this data, it can be seen that the endonuclease EGI has a 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> endo-cellulase gene egI, protein coded by same and application thereof

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cagcaaactg tttgggggca atgcggaggt caaggatgga gcggcccgac tagttgcgtt 60

gctggatctg cttgttctac tctcaacccc tactacgctc aatgcattcc tggagccacc 120

accatgtcta ccacaaccaa gccgacctcc gtttcagcat caacgaccag ggcgagtgca 180

acatcgtccg ctactccgcc acctagctct ggcctcacca ggtttgctgg agttaatatt 240

gccggattcg attttggctg tgggactgat ggaacctgcg tcacttcgaa ggtgtaccca 300

ccgctgaaga actatgctgg cacaaacaac taccctgatg gcgttggtca aatgcagcac 360

tttgtcaacg atgataaatt aaccattttc cgcctacctg tggggtggca gtaccttgtg 420

aacaataact tgggtggaac tctggattca aacaactttg gtaaatacga ccagctggtt 480

caggcttgcc tctctctggg cgtatactgc attgttgata tacacaacta tgcacgctgg 540

aatggcggga ttattggcca aggtgggcct acaaatgatc agtttactag tctttggtca 600

cagttggcgc agaagtacgc ctctcagtcg aaggtttggt tcggaatcat gaacgagcca 660

catgatgtga atattaacac atgggctact actgttcaag ctgttgtcac tgctatccgt 720

aatgccggcg ctacctcaca atttatttcc ttgccaggaa atgattggca atctgctgga 780

gcgtttattt ctgacggaag tgcagccgct ttgtctcagg tcaagaaccc tgatggttcc 840

acaaccaatc tgattttcga tctacacaag tacctggatt cggataactc tggcactcac 900

gccgactgtg tcacaaataa cgttaatgat gctttctcac ctgttgccac ttggctccgt 960

caaaacaacc gccaagctat cctgactgag acaggcggcg gtaacactca atcatgcatt 1020

caataccttt gccaacagtt ccaatatata aaccaaaact ccgacgtcta ccttggctac 1080

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agtggaagct cgtggactga tacgtctctt gtaagctcat gcctttcccg gaaatag 1197

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Gln Gln Thr Val Trp Gly Gln Cys Gly Gly Gln Gly Trp Ser Gly Pro

1 5 10 15

Thr Ser Cys Val Ala Gly Ser Ala Cys Ser Thr Leu Asn Pro Tyr Tyr

20 25 30

Ala Gln Cys Ile Pro Gly Ala Thr Thr Met Ser Thr Thr Thr Lys Pro

35 40 45

Thr Ser Val Ser Ala Ser Thr Thr Arg Ala Ser Ala Thr Ser Ser Ala

50 55 60

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

65 70 75 80

Ala Gly Phe Asp Phe Gly Cys Gly Thr Asp Gly Thr Cys Val Thr Ser

85 90 95

Lys Val Tyr Pro Pro Leu Lys Asn Tyr Ala Gly Thr Asn Asn Tyr Pro

100 105 110

Asp Gly Val Gly Gln Met Gln His Phe Val Asn Asp Asp Lys Leu Thr

115 120 125

Ile Phe Arg Leu Pro Val Gly Trp Gln Tyr Leu Val Asn Asn Asn Leu

130 135 140

Gly Gly Thr Leu Asp Ser Asn Asn Phe Gly Lys Tyr Asp Gln Leu Val

145 150 155 160

Gln Ala Cys Leu Ser Leu Gly Val Tyr Cys Ile Val Asp Ile His Asn

165 170 175

Tyr Ala Arg Trp Asn Gly Gly Ile Ile Gly Gln Gly Gly Pro Thr Asn

180 185 190

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

195 200 205

Gln Ser Lys Val Trp Phe Gly Ile Met Asn Glu Pro His Asp Val Asn

210 215 220

Ile Asn Thr Trp Ala Thr Thr Val Gln Ala Val Val Thr Ala Ile Arg

225 230 235 240

Asn Ala Gly Ala Thr Ser Gln Phe Ile Ser Leu Pro Gly Asn Asp Trp

245 250 255

Gln Ser Ala Gly Ala Phe Ile Ser Asp Gly Ser Ala Ala Ala Leu Ser

260 265 270

Gln Val Lys Asn Pro Asp Gly Ser Thr Thr Asn Leu Ile Phe Asp Leu

275 280 285

His Lys Tyr Leu Asp Ser Asp Asn Ser Gly Thr His Ala Asp Cys Val

290 295 300

Thr Asn Asn Val Asn Asp Ala Phe Ser Pro Val Ala Thr Trp Leu Arg

305 310 315 320

Gln Asn Asn Arg Gln Ala Ile Leu Thr Glu Thr Gly Gly Gly Asn Thr

325 330 335

Gln Ser Cys Ile Gln Tyr Leu Cys Gln Gln Phe Gln Tyr Ile Asn Gln

340 345 350

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

355 360 365

Asp Ser Thr Tyr Ile Leu Thr Glu Thr Pro Thr Gly Ser Gly Ser Ser

370 375 380

Trp Thr Asp Thr Ser Leu Val Ser Ser Cys Leu Ser Arg Lys

385 390 395

Claims (1)

1. Endo-cellulase geneegI. Endo-cellulase geneegI the use of the protein coded by I in the decomposition of cellulose, wherein, the endocellulase geneegThe 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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