CN113736807B

CN113736807B - Cellobiohydrolase, and coding gene and application thereof

Info

Publication number: CN113736807B
Application number: CN202110989903.7A
Authority: CN
Inventors: 李刚; 刘懿德
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2021-08-26
Filing date: 2021-08-26
Publication date: 2023-05-02
Anticipated expiration: 2041-08-26
Also published as: CN113736807A

Abstract

The invention discloses cellobiohydrolase, and a coding gene and application thereof. The invention provides a cellobiohydrolase gene CBH2124 and cellobiohydrolase CBH2124, the nucleotide sequence and the amino acid sequence of which are respectively shown in SEQ ID NO:1 and SEQ ID NO: 2. The cellobiohydrolase of the present invention has the following properties: the activity of almost 100% can be maintained after the reaction is carried out for 5 hours at 30 ℃; incubation at pH 6.2 for 5h still maintains activity above 90%; has better tolerance to partial organic solvents and heavy metal ions and has better industrial application prospect.

Description

Cellobiohydrolase, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to cellobiohydrolase, and a coding gene and application thereof.

Background

Cellulose is widely found in nature and is a major constituent of plant cell walls. Cellulose is a high molecular polymer formed by connecting glucose molecules by beta-1, 4 glucosidic bonds, and the glucose residues contained in each molecule are different from 100 to 10,000. Cellulose can be effectively utilized in industrial production through degradation of cellulose, such as fermentation production of organic chemicals such as ethanol, so that the activity of cellulose and the tolerance to severe reaction conditions can directly influence the degradation of cellulose and the application prospect of cellulose in industry.

Cellulase is a generic term for complex enzymes that decompose cellulose to eventually form glucose, and is mainly composed of three major enzymes, endoglucanase, cellobiohydrolase (exoglucanase), and beta-glucosidase. The endoglucans hydrolyze cellulose into cellulose fragments, the free end generated by the hydrolysis of endoglucanases by cellobiohydrolase further hydrolyzes the cellulose fragments into cellobiose, and finally, the cellobiose is hydrolyzed into glucose by beta-glucosidase, and the three components cooperate to complete the degradation of cellulose, so that the degradation efficiency is reduced due to the lack of any one enzyme. Endoglucanases act mainly on the amorphous regions of cellulose, whereas only cellobiohydrolases are able to degrade in the crystalline regions, and therefore cellobiohydrolases are of great importance for cellulose degradation.

The existing cellobiohydrolase applied in industry has the defects of low enzyme activity, low high temperature resistance, poor stability and the like, and the cellobiohydrolase mutant with improved performances in various aspects is obtained by modifying amino acid of the cellobiohydrolase in the prior art, for example: CN107236719B discloses a thermostable cellobiohydrolase, which is a polypeptide having hydrolytic activity using phosphoric acid-swollen microcrystalline cellulose as a substrate, composed of an amino acid sequence formed by deletion, substitution or addition of amino acids; CN104870642a discloses a mutant of cellobiohydrolase, resulting from substitution and/or deletion of one or more amino acid residues. The natural cellobiohydrolases obtained from natural environments and having high activity and excellent enzymatic properties are still relatively scarce, and therefore, there is a need to provide more cellobiohydrolases for industrial application selection.

Disclosure of Invention

The invention aims to overcome the defects and the shortages in the prior art and provide a novel, natural and high-activity cellobiohydrolase CBH2124 with excellent enzymatic properties, and a coding gene and application thereof.

A first object of the present invention is to provide a cellobiohydrolase gene cbh2124.

A second object of the present invention is to provide a cellobiohydrolase CBH2124.

It is a third object of the present invention to provide a method for producing the cellobiohydrolase.

It is a fourth object of the present invention to provide the use of said cellobiohydrolase.

The above object of the present invention is achieved by the following technical solutions:

the mangrove in Guangdong area has wide distribution and special environment, the soil is often covered with fallen leaves, the root system of the internal plant is developed, and the mangrove is soaked by seawater, so that the mangrove has the characteristics of salinization, swamp formation and the like, has rich and unique microbial resources, and is likely to have novel cellobiohydrolase which is salt-tolerant, can work under extreme conditions and meets industrial application. The conventional method for obtaining a large amount of cellobiohydrolase needs to obtain strain producing cellobiohydrolase first and then clone and express genes, but microorganisms which can be cultivated in a laboratory only account for less than 1% of all microorganism types, so that the novel cellobiohydrolase obtained by the conventional method is very likely to be omitted when strain is enriched in the early stage, and a large amount of manpower, financial resources and time are required to be consumed. The method for constructing metagenome library and screening functions has the advantages of multiple types of microorganism DNA contained in the library, high screening throughput and the like, and can obtain the gene for encoding the novel cellobiohydrolase from the gene level in a short time and construct the gene on a prokaryotic or eukaryotic expression system for expression, so that the time for separating a large number of strains, culturing strains and cloning genes is saved.

The invention obtains a polypeptide with cellobiohydrolase activity by using a metagenomic function screening method, which is named as cellobiohydrolase CBH2124; the nucleotide sequence of the cellobiohydrolase gene cbh2124 is shown in SEQ ID NO:1, the amino acid sequence of the coded cellobiohydrolase CBH2124 is shown as SEQ ID NO: 2.

It should be noted that, the SEQ ID NO:1 and SEQ ID NO:2 or the amino acid sequence can be synthesized by a biotechnological method, but the cost of the artificial synthesis is far higher than that of the biotechnological method.

The invention also provides a recombinant expression vector which contains the cellobiohydrolase gene cbh2124.

The invention also provides a recombinant bacterium which contains the recombinant expression vector of the cellobiohydrolase gene cbh2124.

Preferably, the recombinant strain adopts escherichia coli strain BL21 (DE 3).

The invention also provides a preparation method of the cellobiohydrolase CBH2124, which comprises the following steps:

s1, inserting the cellobiohydrolase gene cbh2124 of claim 1 into a multi-cloning site of a prokaryotic expression vector to obtain a recombinant expression vector, and then transforming the recombinant expression vector into host bacteria to obtain a recombinant strain;

s2, culturing the recombinant strain, and inducing and expressing recombinant cellobiohydrolase;

s3, after induction expression, collecting thalli, carrying out ultrasonic crushing, and collecting supernatant fluid to obtain the microbial inoculum.

Preferably, the recombinant expression vector is cbh2124-pET32a (+).

More preferably, the conditions for inducing expression in step S2 are: IPTG was added to a final concentration of 0.8mM, and induction was carried out at 22℃and 200rpm for 14 hours.

The invention also provides application of the cellobiohydrolase CBH2124 in cellulose degradation or preparation of products for degrading cellulose.

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

the invention provides a polypeptide CBH2124 with cellobiohydrolase activity. The research of the invention shows that CBH2124 belongs to weak acid cellobiohydrolase, the activity of the weak acid cellobiohydrolase can be maintained at about 100% after the reaction is carried out for 5 hours at 30 ℃, and the activity can be maintained at more than 90% after the incubation is carried out for 5 hours at pH 6.2; and most of the metal ions contribute to their enzymatic activity, while low concentrations of Mn ²⁺ (1 mM) can increase the enzyme activity of CBH2124 to 210%; the cellobiohydrolase CBH2124 has better tolerance to partial organic solvents and heavy metal ions and has good application prospect in industry.

Drawings

FIG. 1 is an electrophoretically detected image of the product of mangrove soil metagenomic DNA extraction.

FIG. 2 is a drawing showing positive clones with cellobiohydrolase activity obtained by functional screening of metagenomic libraries.

FIG. 3 is a graph showing the change in CBH2124 relative to enzyme activity at different IPTG concentrations.

FIG. 4 is a graph showing the change of CBH2124 relative enzyme activity at different induction temperatures.

FIG. 5 is a graph showing the change of CBH2124 relative to the enzyme activity at different induction times.

FIG. 6 is a graph of the optimal reaction temperature for CBH2124.

FIG. 7 is a graph showing the time course of CBH2124 enzyme activity under different temperature conditions.

FIG. 8 is a graph showing the measurement of the optimal reaction pH for CBH2124.

FIG. 9 is a graph showing the time course of CBH2124 enzyme activity at various pH conditions.

FIG. 10 is a graph showing the effect of different metal ions on CBH2124 enzyme activity.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 preparation of cellobiohydrolase

(1) Extracting total DNA of mangrove soil:

the DNA was extracted from the soil collected from the mangrove in Australian island of Margaritifera at a depth of 10-20 cm by using Hipure Soil DNA Kit, and the quality of the extracted DNA was detected by 1% agarose gel electrophoresis, and the detection result is shown in FIG. 1.

(2) Construction and screening of metagenomic libraries:

the fragment digested with BamHI was ligated into pUC118 and transformed into E.coli DH 5. Alpha. The positive clones were transferred to LB medium containing 0.2g/L of 4-methylumbelliferyl- β -D-cellobiose, cultured for 2 to 3 days, and observed under 365nm ultraviolet light for fluorescence generation. As a result, as shown in FIG. 2, a clone producing a fluorescent ring was obtained. After sequencing, SEQ ID NO is obtained: 1, designated cbh2124, having the amino acid sequence set forth in SEQ ID NO: 2.

(3) The construction method of the cbh2124-pET32a (+) expression vector comprises the following steps:

a pair of PCR primers F1 (CGG) was designedGGTACCATG GCC CCG CGT GAA CGC GCT GCC AAT G) and R1 (CGC)GGATCCAAG ATT GAC TAC GAC GGT CGA GCC GCC CT) are used to amplify the cbh2124 gene from positive clones. The underlined parts in F1 and R1 are the sites for restriction endonucleases KpnI and BamHI, respectively. PCR conditions were 94℃for 1 minute, 60℃for 1 minute, 72℃for 2 minutes. A total of 30 cycles were performed. The PCR product was recovered, digested with KpnI and BamHI, and ligated with the same two digested pET32a vectors using a ligase to construct a successful cbh2124-pET32a (+) expression vector.

(4) The preparation method of cellobiohydrolase comprises the following steps:

and (3) transforming the constructed cbh2124-pET32a (+) expression vector into escherichia coli BL21 (DE 3) to obtain a BL21 (DE 3) transformed strain carrying the cbh2124 gene. The strain was inoculated into 5mL of LB liquid medium (containing 100. Mu.g/mL of Amp) and cultured at 37℃and 220rpm to OD ₆₀₀ =0.8. IPTG was added to the super clean bench to a final concentration of 0.8mM, and induction was performed at 30℃and 200rpm for 18 hours.

And taking the induced bacterial liquid, centrifugally collecting bacterial bodies, washing with ultrapure water, and then resuspending. Crushing the thalli by using an ultrasonic crusher, centrifuging and collecting supernatant, wherein the supernatant is cellobiohydrolase crude enzyme liquid. The prepared cellobiohydrolase can be detected for its enzymatic activity by the following method: the enzymatic reaction substrate used for detection is 4-nitrophenyl-beta-D-cellobioside, the reaction system is shown in Table 1, and 200 mu L of 1M Na is added after 30min of reaction ₂ CO ₃ The reaction was terminated and absorbance at 405nm was measured, defining the maximum enzyme activity as 100%, with three parallel runs and a set of control runs per reaction.

TABLE 1 reaction System for measuring enzyme Activity

EXAMPLE 2 screening of optimal IPTG Induction concentration

BL21 strain with cbh2124-pET32a (+) was inoculated into 5mL of LB liquid medium (100. Mu.g/mL Amp) and cultured at 37℃and 220rpm to OD ₆₀₀ =0.8. IPTG was added to the super clean bench to give final concentrations of 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM and 1.2mM, respectively, and induction was carried out at 30℃for 18 hours at 200 rpm.

And taking the induced bacterial liquid, centrifugally collecting bacterial bodies, washing with ultrapure water, and then resuspending. The cells were disrupted by an ultrasonic disrupter, and the supernatant was collected after centrifugation to determine the enzyme activity. The enzymatic reaction substrate used for detection is 4-nitrophenyl-beta-D-cellobioside, the reaction system is shown in Table 1, and 200 mu L of 1M Na is added after 30min of reaction ₂ CO ₃ The reaction was terminated and absorbance at 405nm was measured, defining the maximum enzyme activity as 100%, with three parallel runs and a set of control runs per reaction.

As a result, as shown in FIG. 3, the relative enzyme activity was highest at the final IPTG concentration of 0.8mM, and thus the optimal IPTG induction concentration was determined to be 0.8mM.

EXAMPLE 3 screening of optimal Induction temperature

BL21 (DE 3) strain inoculated with cbh2124-pET32a (+) expression vector was cultured to OD at 37℃and 220rpm in 5mL LB liquid medium (100. Mu.g/mL Amp) ₆₀₀ =0.8. The induction was carried out at 200rpm at 20℃at 22℃at 24℃at 26℃at 28℃at 30℃for 18h, respectively, at the optimum IPTG induction concentration. The method for measuring the enzyme activity was the same as in example 1.

As a result, as shown in FIG. 4, the relative enzyme activity was highest at an induction temperature of 22℃and thus the optimum induction temperature was determined to be 22 ℃.

EXAMPLE 4 screening for optimal Induction time

BL21 strain with cbh2124-pET32a (+) was inoculated into 5mL of LB liquid medium (100. Mu.g/mL Amp) and cultured at 37℃and 220rpm to OD ₆₀₀ =0.8. At best IThe induction concentration of PTG and the optimal induction temperature are respectively 10h, 12h, 14h, 16h and 18h. The method for measuring the enzyme activity was the same as in example 1.

As a result, as shown in FIG. 5, the induction time was 14h, and the relative enzyme activity was the highest, so that the optimal induction time was 14h.

Example 5 Effect of temperature on enzyme Activity and stability

Under the conditions of the reaction system in Table 1, the reaction was carried out in a water bath at 20℃and 30℃and 40℃and 50℃and 60℃for 30 minutes, 200. Mu.L of 1M Na was added ₂ CO ₃ Terminating the reaction, setting 3 groups of repetition and 1 group of control group in each reaction, detecting the light absorption value of 405nm wavelength, defining the highest enzyme activity value as 100%, gradually narrowing the temperature interval relative to the enzyme activity result, and finally determining the optimal reaction temperature.

The influence of temperature on the stability of cellobiohydrolase is that the cellobiohydrolase is added into the reaction system of the table 1 after water bath for 1h, 2h, 3h, 4h and 5h at 20 ℃, 30 ℃ and 40 ℃ respectively, and then reacted for 30min at the optimal reaction temperature, 200 mu L of 1M Na is added ₂ CO ₃ The reactions were terminated, 3 replicates per reaction and 1 control were set, absorbance at 405nm was measured, and the highest enzyme activity was defined as 100%.

As can be seen from fig. 6 and 7, the optimal reaction temperature is 22 ℃; the enzyme activity is not obviously reduced when the polypeptide is placed for 5 hours at 20 ℃ and 30 ℃ and 40 ℃ compared with that of 1 hour, which shows that the polypeptide has better stability in the reaction temperature range of 20 ℃ to 60 ℃ and can react for a long time and keep the enzyme activity.

Example 6 influence of pH on enzyme Activity and stability

Under the conditions of the reaction system in Table 1, 200. Mu.L of 1M Na was added after water-bath reaction at optimum temperatures for 30min under the conditions of pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0, respectively ₂ CO ₃ Terminating the reaction, setting 3 groups of repetition and 1 group of control group in each reaction, detecting the light absorption value of 405nm wavelength, defining the highest enzyme activity value as 100%, gradually narrowing the temperature interval relative to the enzyme activity result, and finally determining the optimal reaction pH.

The influence of temperature on the stability of cellobiohydrolase is divided intoDifferent from the water bath under the conditions of pH of 5.6, 6.2 and 7.0 for 1h, 2h, 3h, 4h and 5h, the reaction system of the table 1 is added, the reaction is carried out for 30min at the optimal reaction temperature, and 200 mu L of 1M Na is added ₂ CO ₃ The reactions were terminated, 3 replicates per reaction and 1 control were set, absorbance at 405nm was measured, and the highest enzyme activity was defined as 100%.

As can be seen from fig. 8 and 9, cbh 124 belongs to the weak acid cellobiohydrolase with an optimum pH of 6.2; the enzyme activity is not obviously reduced when the enzyme is placed for 5 hours under three pH conditions compared with that of the enzyme is placed for 1 hour, and the activity is improved when the enzyme is placed for 3 hours under the pH of 6.2.

The optimal enzyme activity was determined to be 18.2U/mL at an optimal IPTG induction concentration of 0.8mM, an optimal induction temperature of 22℃and an optimal induction time of 14h, and an optimal pH of 6.2.

EXAMPLE 7 Effect of Metal ions on enzyme Activity

Under the reaction system of Table 1, metal ions (K ⁺ 、Ca ²⁺ 、Mg ²⁺ 、Cu ²⁺ 、Fe ²⁺ 、Ni ⁺ 、Zn ²⁺ 、Mn ²⁺ 、Co ² ⁺ ) At a final concentration of 1mM, 5mM, 15mM, at an optimum temperature and pH for 30min, 200. Mu.L of 1M Na was added ₂ CO ₃ The reactions were terminated, 3 replicates per reaction and 1 control were set, absorbance at 405nm was measured, and the highest enzyme activity was defined as 100%.

As can be seen from FIG. 10, most of the ions have a promoting effect on the enzyme activity, wherein Mn is present at a low concentration ²⁺ (1 mM) the promotion effect on the enzyme activity is most obvious, and the enzyme activity is improved to 210%; medium and high concentration Fe ² 、Zn ²⁺ The enzyme activity inhibition effect is obvious, the enzyme activity is respectively reduced to 58 percent and 46 percent at the medium concentration (5 mM), and the enzyme activity is reduced to 22 percent and 13 percent at the high concentration (15 mM); cu (Cu) ²⁺ The inhibition of the enzyme activity was most pronounced, and the enzyme activity was reduced to 14% at low concentrations and undetectable at high concentrations.

Example 8 Effect of organic solvents on enzyme Activity

Under the reaction system of Table 1, respectively adding organic solvent acetonitrile, methanol,Ethanol, isopropanol, DMSO (final concentration 1%, 15%, 30%), metal ion chelating agent EDTA (final concentration 10mM, 25mM, 50 mM), surfactant SDS (1%, 5%, 10%) at optimum temperature and optimum pH for 30min, adding 200 μL 1M Na ₂ CO ₃ The reactions were terminated, 3 replicates per reaction and 1 control were set, absorbance at 405nm was measured, and the highest enzyme activity was defined as 100% and the results are shown in Table 2.

TABLE 2 influence of organic solvents on enzyme Activity

Note that: ND represents no detected activity

The results are shown in Table 2, which shows the effect of the organic solvent on the enzyme activity: in the organic solvent, the enzyme activity is reduced along with the increase of the concentration of methanol, ethanol, acetonitrile and isopropanol, and the reduction trend is similar, and the enzyme activity is reduced along with the increase of the concentration of the organic solvent, but the enzyme activity still shows better enzyme activity and shows good tolerance. While increasing DMSO concentration had no effect on enzyme activity. While the enzyme activity was reduced to 76% at low concentration in the increase of EDTA concentration, the enzyme activity was not further reduced at the increase of EDTA concentration. SDS has the most pronounced effect on enzyme activity, and CBH2124 has been inactivated at low concentrations.

Sequence listing

<110> university of Zhongshan

<120> cellobiohydrolase, and coding gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2124

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atggccccgc gtgaacgcgc tgccaatgcg ctgccgttcg gaaccgtgct gggtcggcga 60

aatcgctccc agaaaagcgg gcacctcgag cgaggcggga ggccgatttt ttcgcagatt 120

cccgtcgtgc aggctctgct tctcctctac attcggcgtg cgtctcgggg tatgagacgc 180

gggcggaggg cgaaatgccc tcgaaggtca gcaggatcgg aggcaggttt ggcacacgcc 240

ggcaagttgt ggctcggttt cattctcgcg gcagcgtcgc tgacgtcgtg catcaagcgc 300

gagccgcggc cggaggcgcc cgtcgaagca gcgcccgccg tcgtcgagga cgactcgagc 360

agcaagtcga ttcaggagat ggcggcgctc cccgctaccg ggaagttcgg gcaagtgcaa 420

ggggtcgaca tgctcggcgg caaaggcgtc cgcgccttcc agctgcaggg caacgtggag 480

ggcgtggagg cgaagctgct gccggtcgag gggcagccgt tttcggagat gatccgaacc 540

aagaccacga aggcgacgca gaacacctgg gacgtccaga tccgcgccag cagcgctgct 600

ccggtggagc ggggggacgc gctgctgttc acgatgtact ttcgcacgga ggcgtcgctc 660

gaggagagtg ggcaagggca gtcggagttc gtgttcgagc aggcgacgga cccatggacg 720

aagtccgtga cgtatccggt gggggctgcc gccgagtgga cgaagatgta cgtgcccttc 780

ctcgctcagg agagctacga ggccggggca gcacagctgg cgttccgcct cggctacgcg 840

ccgcagacga tcgagttcgc gggcctccag ctcgtgaact acggcaagga tctcacgctc 900

gccgacctgc cgcgcacgca ggtcacgtat ccgggctcgg cagaagacgc gccgtggcgc 960

gccgaggccc aagcgcggat cgagaagatc cgcaaagctc cgctgacgat ccgtgtccag 1020

gacgcgtcgg gcaagcccgt cgccggggcc accgtccgag cccagctcgt gcgcaacgat 1080

ttcgacttcg gcacgtgcgc gcccgcagcg ctgctgctcg atcccaagca gaagcagttt 1140

caggaggtga tccctcgcct gttcaacatg gtgaccttgg agaacgatct caagtggcag 1200

ccgctggcgg gggaatgggg cggccagttc acgctggatc gcgcgaaggc agcggtgaag 1260

gcggtcgagg ggtgggggct cgacgtgcgc gggcacgtgc tggtgtggcc cgggtggcag 1320

aatctacccg ccaagctcaa gctgctcgag aaagacaagc cgcggctgcg caaggaggtc 1380

gccgaccaca tccgcgaggt cgcgggagcg gtgaagggca acgtcgtcga ctgggacgtc 1440

gtcaacgagc cgttcaccaa ccacgacctg ctcgacatcc tcggaccgga agtgatggtc 1500

gactggttca agctcacgcg gaagatcgac ccgaaggcgc ggctgttcat caacgatttt 1560

gcgattctga gcgggggcgg tggggatacg gcgcaccgcg accactacga gaagatgatt 1620

cagctgctca gcgaccagca cgcgccgttc gacggtatcg gcatgcaggg gcactttgcc 1680

gacagcctga cgggcccgga agacatgttg aagatcctgg accgcttcgc caagttcggc 1740

aaaccgatcc tcatcaccga gtacgacgtg gtgaccgacg atgaagactt ggcggcgaag 1800

ttcactcggg acttctacct cacgatgttc agccacgaag ccgtacgcgg gatcgtgatg 1860

tggggcttct gggacgcggt gcactggaag aagaacgcgc cgatctatcg ccaggactgg 1920

agcgagaagc cttccggcaa ggtctacgaa gagctcttga aggagtggac cacggatgcc 1980

agcggtcaga gcgacgcgca gggcgtgatg accgtcaacg gattcctggg cagctatgag 2040

ctctccgtta cgcacgcagg ggcgacgaag aaggtcaaag gcgtgctgaa gaagggcggc 2100

tcgaccgtcg tagtcaatct ttag 2124

<210> 2

<211> 707

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Ala Pro Arg Glu Arg Ala Ala Asn Ala Leu Pro Phe Gly Thr Val

1 5 10 15

Leu Gly Arg Arg Asn Arg Ser Gln Lys Ser Gly His Leu Glu Arg Gly

20 25 30

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

35 40 45

Leu Tyr Ile Arg Arg Ala Ser Arg Gly Met Arg Arg Gly Arg Arg Ala

50 55 60

Lys Cys Pro Arg Arg Ser Ala Gly Ser Glu Ala Gly Leu Ala His Ala

65 70 75 80

Gly Lys Leu Trp Leu Gly Phe Ile Leu Ala Ala Ala Ser Leu Thr Ser

85 90 95

Cys Ile Lys Arg Glu Pro Arg Pro Glu Ala Pro Val Glu Ala Ala Pro

100 105 110

Ala Val Val Glu Asp Asp Ser Ser Ser Lys Ser Ile Gln Glu Met Ala

115 120 125

Ala Leu Pro Ala Thr Gly Lys Phe Gly Gln Val Gln Gly Val Asp Met

130 135 140

Leu Gly Gly Lys Gly Val Arg Ala Phe Gln Leu Gln Gly Asn Val Glu

145 150 155 160

Gly Val Glu Ala Lys Leu Leu Pro Val Glu Gly Gln Pro Phe Ser Glu

165 170 175

Met Ile Arg Thr Lys Thr Thr Lys Ala Thr Gln Asn Thr Trp Asp Val

180 185 190

Gln Ile Arg Ala Ser Ser Ala Ala Pro Val Glu Arg Gly Asp Ala Leu

195 200 205

Leu Phe Thr Met Tyr Phe Arg Thr Glu Ala Ser Leu Glu Glu Ser Gly

210 215 220

Gln Gly Gln Ser Glu Phe Val Phe Glu Gln Ala Thr Asp Pro Trp Thr

225 230 235 240

Lys Ser Val Thr Tyr Pro Val Gly Ala Ala Ala Glu Trp Thr Lys Met

245 250 255

Tyr Val Pro Phe Leu Ala Gln Glu Ser Tyr Glu Ala Gly Ala Ala Gln

260 265 270

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

275 280 285

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

290 295 300

Arg Thr Gln Val Thr Tyr Pro Gly Ser Ala Glu Asp Ala Pro Trp Arg

305 310 315 320

Ala Glu Ala Gln Ala Arg Ile Glu Lys Ile Arg Lys Ala Pro Leu Thr

325 330 335

Ile Arg Val Gln Asp Ala Ser Gly Lys Pro Val Ala Gly Ala Thr Val

340 345 350

Arg Ala Gln Leu Val Arg Asn Asp Phe Asp Phe Gly Thr Cys Ala Pro

355 360 365

Ala Ala Leu Leu Leu Asp Pro Lys Gln Lys Gln Phe Gln Glu Val Ile

370 375 380

Pro Arg Leu Phe Asn Met Val Thr Leu Glu Asn Asp Leu Lys Trp Gln

385 390 395 400

Pro Leu Ala Gly Glu Trp Gly Gly Gln Phe Thr Leu Asp Arg Ala Lys

405 410 415

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

420 425 430

Val Leu Val Trp Pro Gly Trp Gln Asn Leu Pro Ala Lys Leu Lys Leu

435 440 445

Leu Glu Lys Asp Lys Pro Arg Leu Arg Lys Glu Val Ala Asp His Ile

450 455 460

Arg Glu Val Ala Gly Ala Val Lys Gly Asn Val Val Asp Trp Asp Val

465 470 475 480

Val Asn Glu Pro Phe Thr Asn His Asp Leu Leu Asp Ile Leu Gly Pro

485 490 495

Glu Val Met Val Asp Trp Phe Lys Leu Thr Arg Lys Ile Asp Pro Lys

500 505 510

Ala Arg Leu Phe Ile Asn Asp Phe Ala Ile Leu Ser Gly Gly Gly Gly

515 520 525

Asp Thr Ala His Arg Asp His Tyr Glu Lys Met Ile Gln Leu Leu Ser

530 535 540

Asp Gln His Ala Pro Phe Asp Gly Ile Gly Met Gln Gly His Phe Ala

545 550 555 560

Asp Ser Leu Thr Gly Pro Glu Asp Met Leu Lys Ile Leu Asp Arg Phe

565 570 575

Ala Lys Phe Gly Lys Pro Ile Leu Ile Thr Glu Tyr Asp Val Val Thr

580 585 590

Asp Asp Glu Asp Leu Ala Ala Lys Phe Thr Arg Asp Phe Tyr Leu Thr

595 600 605

Met Phe Ser His Glu Ala Val Arg Gly Ile Val Met Trp Gly Phe Trp

610 615 620

Asp Ala Val His Trp Lys Lys Asn Ala Pro Ile Tyr Arg Gln Asp Trp

625 630 635 640

Ser Glu Lys Pro Ser Gly Lys Val Tyr Glu Glu Leu Leu Lys Glu Trp

645 650 655

Thr Thr Asp Ala Ser Gly Gln Ser Asp Ala Gln Gly Val Met Thr Val

660 665 670

Asn Gly Phe Leu Gly Ser Tyr Glu Leu Ser Val Thr His Ala Gly Ala

675 680 685

Thr Lys Lys Val Lys Gly Val Leu Lys Lys Gly Gly Ser Thr Val Val

690 695 700

Val Asn Leu

705

Claims

1. Cellobiohydrolase genecbh2124The nucleotide sequence of the polypeptide is shown as SEQ ID NO: 1.

2. A cellobiohydrolase CBH2124, comprising an amino acid sequence as set forth in SEQ ID NO: 2.

3. A recombinant expression vector comprising the cellobiohydrolase gene of claim 1cbh2124。

4. A recombinant bacterium comprising the recombinant expression vector of claim 3.

5. The recombinant bacterium according to claim 4, wherein the recombinant bacterium is E.coli BL21.

6. A method for preparing cellobiohydrolase CBH2124, comprising the steps of:

s1. Insertion of the cellobiohydrolase gene of claim 1 into a prokaryotic expression vector at the multiple cloning sitecbh2124Obtaining a recombinant expression vector, and then transforming the recombinant expression vector into host bacteria to obtain a recombinant strain;

and S3, collecting thalli after induced expression, performing ultrasonic crushing, and collecting supernatant.

7. The method of claim 6, wherein the conditions for inducing expression in step S2 are: IPTG was added to a final concentration of 0.8mM, 22℃and induction of 14h at 200 rpm.

8. Use of the cellobiohydrolase CBH2124 of claim 2 for cellulose degradation or in the preparation of a product for degrading cellulose.