CN110734901B

CN110734901B - A kind of enzyme fusion protein and its application

Info

Publication number: CN110734901B
Application number: CN201911038022.6A
Authority: CN
Inventors: 刘森林; 陈伟钊
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2019-10-29
Filing date: 2019-10-29
Publication date: 2021-07-09
Anticipated expiration: 2039-10-29
Also published as: CN110734901A

Abstract

The invention discloses an enzyme fusion protein and an application thereof, belonging to the field of genetic engineering. The present invention utilizes Overlap PCR technology to obtain the fusion enzyme Egh31-CBD composed of endoglucanase Egh31 and alkaline endoglucanase III-3 enzyme protein binding domain (CBD). Compared with the original enzyme Egh31 , the specific activity of the fusion enzyme (154.8U/mg) is about 14% higher than that of the original enzyme (135.7U/mg); the temperature stability of the fusion enzyme is greatly improved at 55 ℃, and the relative enzyme activity of the original enzyme is only Under the condition of 1% SDS, the relative enzyme activity of the original enzyme is only about 20%, and the fusion enzyme is about 60%. To sum up, the experimental results show that the Egh31 endoglucanase with CBD binding domain has better application prospects in the enzymatic washing and finishing of the textile industry and the detergent industry.

Description

Enzyme fusion protein and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to an enzyme fusion protein and application thereof.

Background

Carbohydrate-binding modules CBM (carbohydrate-binding module) are protein domains found in carbohydrate-active enzymes, such as glycoside hydrolases. Most of these domains have carbohydrate binding activity. CBM's were originally derived from cellulose Binding domains and were classified into many families based on conserved amino acid similarity, and by 1 month 2018, 83 CBMs (http:// www.cazy.org/Carbohydrate-Binding-modules. html) already exist in the CAZY database.

The endoglucanase binding domain (CBD, also called CBM) is generally "wedge-shaped" with one side hydrophilic and the other hydrophobic, allowing the endoglucanase to bind to both crystalline and amorphous cellulose. Most of the CBDs of cellulases are located at the amino-or carboxy-terminus, and very few are located in the middle, of the molecular structure of the protein (Tomme et al, 1995).

Most cellulases contain a CBD binding domain and have an effect on the catalytic process of the enzyme. The cellulase for removing CBD has little influence on the degradation effect of a soluble substrate, but the enzyme activity is greatly reduced when the cellulase acts on an insoluble substrate (Wangtianhong et al, 2000).

The CBD adsorption function of the Trichoderma reesei endoglucanase EGI is significantly higher than that of the exoglucanase CBHI, mainly due to the substitution of tyrosine by tryptophan in the critical binding region (Linder et al, 1995). Bolam et al (1998) fused CBD from Pseudomonas fluorescens cellulase and xylanase with Clostridium thermocellum endoglucanase EGE, greatly improved the ability to degrade crystalline cellulose, but did not change much the ability to degrade non-crystalline cellulose. Bae et al (2003) added the CBM of xylanase XylA to the C-terminus of endoglucanase Cel5, and the specific activity of the fusion enzyme degraded filter paper was 5-fold higher than that of the original endoglucanase Cel 5. Gundllapalli et al (2007) add cellulose binding domain (CBD2) of Trichoderma reesei CBHII to N-terminal of beta-glucosidase (BGL1) and successfully express in Saccharomyces cerevisiae, and the hydrolytic activity of fusion enzyme on bacterial microcrystalline cellulose and carboxymethyl cellulose (CMC) is increased by 2-4 times, which shows that adding CBD can enhance affinity to substrate and increase flexibility of enzyme molecule.

However, there are also a few cellulases from microorganisms and higher plants which do not have a binding domain, for example the Trichoderma reesei endoglucanase EG3 lacks the CBD domain (Tommee et al, 1995). Although CBD is not essential for the catalytic activity of cellulases, it may have a positive effect on the increase in catalytic efficiency of the enzyme, as the binding domain allows more enzyme to contact the substrate surface, thereby increasing the effective concentration of enzyme on the substrate surface and increasing the efficiency of enzyme contact with the substrate (Chhabra et al, 2002).

Disclosure of Invention

In order to overcome the disadvantages and shortcomings of the prior art, the primary object of the present invention is to provide an enzyme fusion protein.

Another object of the present invention is to provide the use of the above enzyme fusion protein.

In order to further improve the catalytic property of the enzyme, the invention carries out enzyme molecular modification on the enzyme on the basis of bioinformatics analysis: according to the structural characteristic that the endoglucanase Egh31 lacks a binding domain, a binding domain CBD of the endoglucanase produced by a Bacillus sp. III-3 strain is added to the C-terminal end of the endoglucanase.

The purpose of the invention is realized by the following technical scheme:

an enzyme fusion protein is formed by endoglucanase and a CBD binding domain of endoglucanase produced by a Bacillus sp.III-3 strain. The amino acid sequence of the enzyme fusion protein is shown as SEQ ID No: 1 is shown.

A gene encoding the above enzyme fusion protein; preferably, the nucleotide sequence of the gene is shown as SEQ ID No: 2, respectively.

The endoglucanase is an endoglucanase derived from Streptomyces sp.H31, and preferably, the amino acid sequence of the endoglucanase is shown in SEQ ID No: 1, and the nucleotide sequence is shown as SEQ ID No: 2 from base 1 to base 759.

The amino acid sequence of the binding domain is shown as SEQ ID No: 1, and the nucleotide sequence is shown as SEQ ID No: 2 at bases 780-1479.

A recombinant plasmid for expressing the enzyme fusion protein in a host cell.

Preferably, the recombinant plasmid is a vector suitable for expression in E.coli.

Preferably, the recombinant plasmid carries the gene, which is marked as pET-28a-egh 31-CBD.

An engineering strain carrying the recombinant plasmid.

The engineering strain is a recombinant strain obtained by taking Escherichia coli E.coli BL21 Star (DE3) as a host strain and transforming a recombinant plasmid into the host strain.

The enzyme fusion protein is applied to washing and paper making industries.

Specifically, the specific activity of the enzyme fusion protein is 154.8U/mg, which is improved by about 14 percent compared with the original recombinant enzyme Egh31 (135.7U/mg); the heat stability of the enzyme fusion protein at 55 ℃ is obviously enhanced, the relative enzyme activity of the original recombinant enzyme Egh31 at 55 ℃ is about 60%, and the relative enzyme activity of the fusion enzyme Egh31-CBD reaches more than 90%; the tolerance of the enzyme fusion protein to SDS is obviously higher than that of endoglucanase, especially 0.5% -2% SDS.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention adds CDB derived from alkaline endoglucanase III-3 enzyme protein to the C-terminal of endoglucanase gene Egh31 by using an Overlap PCR technology to obtain a fusion enzyme Egh31-CBD consisting of endoglucanase Egh31 and alkaline endoglucanase III-3 enzyme protein binding structural domain (CBD).

(2) Compared with the proenzyme Egh31, the specific activity (154.8U/mg) of the enzyme fusion protein Egh31-CBD is improved by about 14 percent compared with the proenzyme (135.7U/mg); the temperature stability of the fusion enzyme is greatly improved at 55 ℃, the relative enzyme activity of the proenzyme is only about 60 percent, and the fusion enzyme is 90 percent; under the condition of 1% SDS, the relative enzyme activity of the proenzyme is only about 20%, and the relative enzyme activity of the fusion enzyme is about 60%. The experimental results can show that: the Egh31 endoglucanase added with the CBD binding structure domain has better application prospect in enzyme washing finishing and detergent industry of textile industry.

Drawings

FIG. 1 is a conserved domain analysis of Egh 31; egh31 belong to glycoside hydrolase family 12, but the CBM is deleted and lacks a binding domain.

FIG. 2 is a schematic diagram of the structure of CBM _17_ 28; CBM _17_28 is composed mainly of beta sheets (blue part of the figure).

FIG. 3 is a schematic representation of fusion enzyme Egh 31-CBD.

FIG. 4 shows the amplification of the fusion enzyme gene egh31-CBD and the identification of the recombinant plasmid; a: amplification of the fusion enzyme gene egh31-CBD, M: molecular weight markers DL5000, 1: PCR products; b: and (3) carrying out enzyme digestion identification on the recombinant plasmid pET-28a-egh31-CBD, wherein M: molecular weight marker DL 15000; 1, single enzyme digestion by EcoRI; 2, Xho I and EcoR I.

FIG. 5 is a purified electrophoretogram of Egh 31-CBD; m: labeling with molecular weight; 1, purified protein Egh31, 2, purified protein Egh 31-CBD.

FIG. 6 is the effect of pH on the fusion enzyme Egh 31-CBD; a: the optimal reaction pH for the fusion enzyme Egh 31-CBD; b: the pH stability of the fusion enzyme Egh 31-CBD.

FIG. 7 is the effect of temperature on the fusion enzyme Egh 31-CBD; a: the optimal reaction temperature for the fusion enzyme Egh 31-CBD; b: thermal stability of the fusion enzyme Egh 31-CBD.

FIG. 8 shows the effect of SDS on the fusion enzyme Egh 31-CBD.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The experimental procedures for specific experimental conditions not specified in the following examples are generally performed according to conventional experimental conditions or according to experimental conditions recommended by the manufacturers.

The egh31 gene in the embodiment refers to the neutral endoglucanase H31 gene disclosed in the patent 201510036517.0, a neutral endoglucanase and a coding gene and application thereof.

The Bacillus sp.III-3 alkaline endoglucanase gene described in the examples refers to the alkaline endoglucanase gene III-3-a disclosed in patent 200710030788.0, an alkaline endoglucanase gene, a recombinase thereof and applications thereof.

Example 1

1 Material

1.1 strains and vectors

(1) Coli TOP10, E.coli BL21 Star (DE3) were purchased from Invitrogen.

(2) Streptomyces sp.H31 has been disclosed in patent 201510036517.0, a neutral endoglucanase and its coding gene and application.

(3) Coli expression vector pET-28a (+) protein expression vector was purchased from Invitrogen corporation.

(4) Coli ligation T vector was purchased from Takara.

1.2 culture Medium

LB culture medium: 0.5% of yeast powder, 1% of peptone, 1% of NaCl and 2% of Agar added in a solid LB culture medium. When the transformant of the recombinant plasmid is screened, ampicillin (E.coli prokaryotic expression) with the final concentration of 50-100 mug/mL is added.

1.3 Main instrumentation

A PCR instrument: applied Biosystems Inc

A centrifuge: eppendorf Co Ltd

Electrophoresis apparatus: bio-rad Co

Vertical pressure steam sterilization pot: hirayama, Japan Ltd

Gel imaging system: Dolphin-DOC

2 method

2.1 addition of binding Domain (CBD) to H31 endoglucanase Gene Using Overlap PCR technology

2.1.1 bioinformatics analysis methods:

the endoglucanase Egh31 amino acid sequence (see amino acids 1-253 of SEQ ID No: 1) was analyzed using CCD Database (Conserved Domain Database) and found to contain only the cellulase Catalytic Domain (CD) and lack the Cellulase Binding Domain (CBD). The Bacillus sp.III-3 alkaline endoglucanase gene is stored in the laboratory, and the CBD derived from the Bacillus sp.III-3 alkaline endoglucanase is added to the C end of the endoglucanase Egh31 in the part to construct the fusion enzyme Egh 31-CBD.

Bioinformatics analysis tool:

CDD (conserved Domain database) using NCBI (national center for information and intelligence)) (https:// www.ncbi.nlm.nih.gov/cdd) The database was subjected to domain analysis.

SWISS-MODEL: using SWISS-MODEL (https://swissmodel.expasy.org/) The three-dimensional structure of unknown proteins is modeled based on the structure of homologous proteins.

2.1.2 design of Overlap PCR primers

According to the sequence of Bacillus sp.III-3 alkaline cellulase gene and the sequence of egh31 endoglucanase gene, the following primers were designed for Overlap PCR splicing (Table 1).

2.1.3 Overlap PCR in vitro amplification of egh31-CBD Gene

pET-28a-H31 and pET-28a-III-3-a plasmids were extracted. Egh31 upstream and downstream primers in the table 1 are used as primers, and pET-28a-H31 is used as a template; the CBD upstream primer F and the CBD downstream primer R are primers, pET-28a-III-3-a is a template, and the full length of egh31 gene (see the 1 st-759 th base of SEQ ID No: 2) and the CBD of Bacillus sp.III-3 alkaline endoglucanase gene (see the 780 nd 1479 th base of SEQ ID No: 2) are obtained by amplification respectively. The obtained products are mutually used as a template and a primer, and egh31 upstream primer F and CBD downstream primer R are added to amplify to obtain egh31-CBD gene (shown in SEQ ID No: 2).

Wherein, pET-28a-H31 is disclosed in 201510036517.0, a neutral endoglucanase and a coding gene and application thereof; pET-28a-III-3-a is disclosed in "200710030788.0, an alkaline endoglucanase gene, a recombinase thereof and uses thereof".

TABLE 1 Overlap PCR primers

Primer and method for producing the same	Sequence (5 '-3')
		egh31 upstream primer F	CCGgaattcATGCTCCTCGCCGCCGCCACTC
egh31 downstream primer R	GTGTAAATGCACCAGACACTTCCACCACCTGGCAGGCGGGCGC
		CBD upstream primer F	GCGCCCGCCTGCCAGGTGGTGGAAGTGTCTGGTGCATTTACAC
CBD downstream primer R	CCGctcgagTTATTTTGGATTATGAACAA

2.1.4 construction of recombinant plasmid pET-28a-egh31-CBD

The egh31-CBD gene obtained by amplification was digested simultaneously with EcoRI and XhoI, ligated with the similarly digested pET-28a (+) expression plasmid at 16 ℃ for 12 hours, and transformed into E.coli BL21 Star (DE 3). And (4) sequencing and identifying the transformant.

2.1.5 inducible expression of recombinant E.coli BL21 Star (DE3)

(1) Escherichia coli E.coli BL21 Star (DE3) (E.coli BL21 egh31-CBD for short), which was successfully transferred into pET-28a-egh31-CBD, was inoculated into an appropriate amount of LB medium for overnight culture.

(2) The overnight cultured E.coli BL21 egh31-CBD broth was inoculated into LB medium at 1% inoculum size.

(3) And measuring the OD value of the bacterial liquid at intervals, and adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 1mM for induction expression when the OD is 0.5-0.6.

(4) After culturing for a proper time, taking the bacterial liquid, centrifuging, adding a buffer solution, cracking cells by using an ultrasonic wall-breaking method or a liquid nitrogen repeated freeze-thaw method, centrifuging the sample at a high speed of 12000r/min for 5min, taking the supernatant, and carrying out the next determination.

2.2 isolation and purification of the enzyme and study of the enzymatic Properties

2.2.1 separation and purification of endoglucanase protein

The recombinant endoglucanase (Egh 31 or rEgh31) expressed in E.coli and the recombinant fusion endoglucanase (Egh 31-CBD) expressed in E.coli used both contained 6 XHis-tag, and thus the protein could be isolated and purified by affinity chromatography. The method comprises the following steps:

(1) cell lysates (E.coli expression system) were dissolved in equilibration buffer. The cell lysate referred to herein is 201510036517.0, which is a crude enzyme solution obtained in step (ii) 10 of example 2.

(2) Subjecting the Ni affinity chromatography column HisTrap^TMEquilibrate to baseline with equilibration buffer.

(3) The sample was loaded onto a Ni affinity chromatography column at a flow rate of 1mL/min by a constant flow pump.

(3) Gradient elution is carried out on imidazole elution solution, elution peaks are collected to determine enzyme activity, and after overnight dialysis, the imidazole elution solution is concentrated by 10 times for standby.

2.2.2 enzyme activity determination method

Adding a proper amount of enzyme solution into 1% CMC-Na solution, reacting at 50 deg.C for 30min, adding DNS solution into boiling water bath for 10min, measuring absorbance at 540nm, and calculating reducing sugar by standard curve. The amount of enzyme required to produce 1.0mg of glucose per hour under the above conditions was defined as one unit of enzyme activity, expressed in U/mL.

Definition of specific activity: enzyme activity per mg of protein (U/mg).

2.2.3 electrophoretic analysis of proteins

(1) SDS-PAGE gel formulation

The specific method is shown in Table 2.

(2) Step of electrophoresis

1. Centrifuging the sample at 12000r/min to remove precipitate, adding 5 Xsample buffer solution, boiling in boiling water for 10min, centrifuging to remove precipitate, and collecting the supernatant.

2. Carefully loading the sample by using a sample loader, wherein the sample loading amount is selected according to actual needs, and the sample does not overflow the sample loading hole.

3. Electrophoresis was started immediately after the end of the loading. The sample is in the gel concentration stage, generally low voltage is selected, the voltage is increased after the sample enters the separation gel until the bromophenol blue indicator band is positioned at the bottom of the gel, and the electrophoresis is finished.

4. The gel is carefully removed and stained for a period of time depending on the stain concentration.

5. And after dyeing is finished, pouring out the dyeing solution, washing the dyeing solution for multiple times by using deionized water, and then adding the decoloring solution.

6. And (4) after the decoloration is finished, photographing and analyzing by using a gel imaging system.

The specific steps refer to patent 201510036517.0, neutral endoglucanase and its coding gene and application, step (II) 9 of example 2.

TABLE 2 SDS-PAGE gel formulation

Components	Concentrated gum (5%) (5mL)	Separation gel (12%) (10mL)
			H₂O	3.6	3.3
30% acrylamide	0.83	4.0
			1M Tris-HCL(pH 6.8)	0.42	Is free of
1.5M Tris-HCL(pH 8.8)	Is free of	2.5
			10％SDS	0.05	0.1
10％AP	0.05	0.1
			TEMED	0.005	0.004

2.2.4 method for measuring protein concentration

Protein concentration was determined using the BCA protein quantification assay kit.

(1) 5mL of Solution A and 0.1mL of Solution B were used for each reaction to prepare an A + B mixture, which was mixed well.

(2) 0.1mL of sample to be tested is added into 2mL of A + B mixed solution, and the reaction is carried out for 30 minutes at 37 ℃.

(3) After the reaction, the absorbance of light was measured at 562 nm.

(4) Protein concentration was calculated by standard curve.

2.2.5 optimum temperature and thermostability of endoglucanases

Each data in the course of the enzymatic property analyses herein was obtained by taking the average of three replicates.

(1) Optimum reaction temperature

The citric acid buffer solution with pH 7.0 is used as a buffer system, 1% CMC is used as a substrate, the reaction system is 1100 mu L, and the reaction time is 30 min. And (3) measuring the enzyme activity of the endoglucanase Egh31 or Egh31-CBD within the temperature range of 30-80 ℃, calculating the highest enzyme activity to be 100%, further calculating the relative enzyme activity within other temperature ranges, and fitting into a curve for analysis.

(2) Thermal stability

Respectively taking 100 mu L of enzyme solution, preserving the heat for 60min at the temperature of 30-80 ℃, taking a citric acid buffer solution with the pH of 7.0 as a buffer system, taking 1% CMC as a substrate, taking a reaction system as 1100 mu L, measuring the enzyme activity of endoglucanase Egh31 or Egh31-CBD, calculating the highest enzyme activity as 100%, further calculating the relative enzyme activity in other temperature ranges, and fitting into a curve for analysis.

2.2.6 optimum pH and pH stability of endoglucanases

(1) Optimum reaction pH value

The reaction temperature is 60 ℃, 1% CMC is used as a substrate, the reaction system is 1100 mu L, and the reaction time is 30 min. And (3) measuring the enzyme activity of the endoglucanase Egh31 or Egh31-CBD under the condition of pH 3-11, calculating the highest enzyme activity to be 100%, further calculating the relative enzyme activity of other reaction pH values, and fitting a curve to analyze.

(2) Stability of pH value

100 μ L of the enzyme solution was added to 1mL of a buffer solution having a pH of 3 to 11, and the mixture was incubated at 37 ℃ for 60 min. Then adding a reaction substrate CMC-Na solution (the final concentration of the substrate is 1 percent) to react for 30min at 60 ℃, measuring the enzyme activity of the endoglucanase Egh31 or Egh31-CBD, calculating the highest enzyme activity to be 100 percent, further calculating the relative enzyme activities of other pH values, and fitting a curve to analyze.

2.2.7 Effect of surfactant SDS on enzyme Activity

SDS with different concentrations is added into a reaction system containing endoglucanase Egh31 or Egh31-CBD, so that the final concentration of the SDS is 0, 0.5 percent, 1 percent and 2 percent respectively, after being preserved for 60min in a warm bath at 37 ℃, the enzyme activity of the endoglucanase Egh31 or Egh31-CBD is respectively measured according to a conventional method, and the highest enzyme activity is calculated as 100 percent.

3 results and analysis of the experiments

3.1 endoglucanase Egh 31C-terminal addition of heterologous CBD

3.1.1 analysis of endoglucanase Egh 31C-terminal addition of CBD

The amino acid sequence of Egh31 protein was imported into the NCBI CCD Database (Conserved Domain Database), and based on the analysis of this Database (FIG. 1), endoglucanase Egh31 belongs to glycoside hydrolase family 12, but lacks its carbohydrate-binding module (CBM), indicating that the enzyme lacks a binding Domain.

Bacillus sp.III-3 alkaline endoglucanase gene is stored in the laboratory, the encoded alkaline endoglucanase belongs to glycoside hydrolase family 5 (Liu et al, 2011), and the C terminal is a tandem fiber binding domain of CBM17 and 28 families. Families of CBM17 and 28 often form the tandem domain CBM _17_28(Boraston et al, 2002). The structure of the device is schematically shown in figure 2. The fusion enzyme Egh31-CBD was constructed by adding the Bacillus sp.iii-3 alkaline endoglucanase gene encoding binding domain portion to the C-terminus of endoglucanase Egh31, as schematically shown in fig. 3.

3.1.2Overlap PCR in vitro amplification of egh31-CBD Gene

A single-band DNA product was successfully amplified by the experimental procedure (FIG. 4-A), the sequence was sequenced to match the predicted result, and the fusion gene was named egh 31-CBD.

3.1.3 construction of recombinant plasmid pET-28a-egh31-CBD

The egh31-CBD obtained by amplification is extracted and constructed into a pET-28a (+) expression plasmid and transformed into Escherichia coli E.coli BL21 Star (DE 3). Plasmid double-restriction enzyme digestion verification is carried out on transformant extraction, and the result is in accordance with expectation (FIG. 4-B). The recombinant plasmid pET-28a-egh31-CBD is successfully constructed through sequencing verification.

3.1.4 expression of the fusion enzyme Egh31-CBD

And performing induction culture on the recombinant bacteria, repeatedly freezing and thawing the recombinant bacteria by liquid nitrogen, performing centrifugation on the crude enzyme solution to obtain supernatant, purifying the supernatant by affinity chromatography, performing protein electrophoresis after dialysis and concentration by 10 times, and measuring the activity of the CMC enzyme. As can be seen from FIG. 5, Egh31-CBD has been successfully expressed in E.coli and the molecular weight of the fusion enzyme is about 52 kDa. The specific activity of the fusion enzyme Egh31-CBD was 154.8U/mg, which is about 14% higher than that of endoglucanase Egh31(135.7U/mg) without CBD.

3.2 enzymatic Property analysis of the fusion enzyme Egh31-CBD

3.2.1 optimum reaction pH and pH stability

The optimum reaction pH and pH stability of endoglucanases Egh31 and Egh31-CBD expressed by escherichia coli are measured by taking CMC as a substrate at the temperature of 60 ℃ and within the pH range of 3-11. The optimum pH reaction results are shown in FIG. 6-A, which shows that the optimum reaction pH of the fusion enzyme Egh31-CBD was Egh31, which was approximately pH 7. However, the relative activity of the fusion enzyme is slightly reduced under acidic conditions. As can be seen from FIG. 6-B, the tolerance of the fusion enzyme was decreased under acidic conditions (pH3-5), indicating that the pH stability of the fusion enzyme Egh31-CBD was slightly inferior to that of the original recombinant enzyme Egh31 under acidic conditions. As can be seen from the experimental results, the addition of the binding domain had no significant effect on the pH optimum response and pH stability of endoglucanase Egh 31.

3.2.2 optimum reaction temperature and thermal stability

The optimum reaction temperature and the thermal stability of endoglucanases Egh31 and Egh31-CBD expressed by Escherichia coli were determined at pH 7.0 using CMC as a substrate. As shown in FIG. 7-A, the optimal reaction temperatures of the two enzymes are substantially the same, but the relative enzyme activities of the fusion enzymes are greatly improved at 50 ℃, which suggests that the addition of the binding domain can improve the enzyme activity of the endoglucanase Egh31 under the condition of medium temperature, so that the temperature range of the enzymes for playing the role is widened. In the temperature stability study, compared with the original recombinant enzyme Egh31, the thermal stability of the fusion enzyme Egh31-CBD at 55 ℃ is obviously enhanced, the relative enzyme activity of the original recombinant enzyme Egh31 at 55 ℃ is about 60%, and the relative enzyme activity of the fusion enzyme Egh31-CBD is more than 90%. The results of the experiment show that the addition of the binding domain at the C-terminus of endoglucanase Egh31 enhances its thermostability (FIG. 7-B).

3.2.3 tolerance to SDS

In the SDS tolerance assay, the fusion enzyme Egh31-CBD showed higher tolerance. When the final concentration of SDS is 0.5%, the residual enzyme activity is up to above 60%, and when the concentration of SDS is 1%, the enzyme activity is slowly reduced, and in the 2% SDS solution, the relative enzyme activity still exceeds 30%. Whereas the relative enzyme activity of the original recombinant enzyme in 0.5% SDS was less than 40%, the activity decreased faster as the concentration of SDS increased (FIG. 8).

4 small knot

A fusion enzyme Egh31-CBD comprising endoglucanase Egh31 and alkaline endoglucanase III-3 enzyme protein binding domain (CBD) was obtained.

CDB derived from alkaline endoglucanase III-3 enzyme protein was added to the C-terminus of endoglucanase gene Egh31 by the Overlap PCR technique. The specific activity of the fusion enzyme is improved by about 14 percent compared with that of the original Egh 31; the temperature stability of the fusion enzyme is greatly improved at 55 ℃, the relative enzyme activity of the original recombinant enzyme is only about 60 percent, and the fusion enzyme is 90 percent; under the condition of 1% SDS, the relative enzyme activity of the original recombinant enzyme is only about 20%, and the relative enzyme activity of the fusion enzyme is about 60%. The experimental results can show that: the Egh31 endoglucanase added with the CBD binding structure domain has better application prospect in enzyme washing finishing and detergent industry of textile industry.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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<223> nucleotide sequence of enzyme fusion protein

<400> 2

atgctcctcg ccgccgccac tcccgcccgg gcggacacca cgatctgcga gcccttcggg 60

tcgaccgtga tccagggtcg ctacgtcgtc cagaacaacc gctggggcac cggcgccccc 120

cagtgcgtca ccgcgacgga caccggcttc cgggtcatcc aggccgacgg ctcggtgccc 180

accgacggcg ctcccaagtc gtacccgtcg gtcttcaacg gctgccacta caccaactgt 240

tcgcccggga ccaggctccc cgcacggatc agcaccatct ccagcgcgcc cagcagcatc 300

tcctacggct acgtgccggg cggtgtgtac aacgccgcgt acgacatctg gctggacccg 360

acgccccgca ccgacggtgt caaccggacc gagatcatga tctggttcaa ccgggtcggc 420

ccggtccagc cgatcggctc tccggtcgcc accgcaaccg tcggtgggcg cacctgggag 480

gtgtggacgg gcagcaacgg caccaacgac gtgatctcct tcgtcgcccc gtcgaccatc 540

acgagctgga gcttcgacgt catggacttc gtcgaccagg ccgtcaaccg gggcctggcg 600

cagcgcgact ggtacctgac gagcgttcag gccggcttcg aaccgtggcg ggacggcgtc 660

ggactggcgg tgcactcctt ctcctccacc gtgaacgtcg gcggtgaccc cggcgggccg 720

ggcgggccgg gtgccccggc gcccgcctgc caggtggtgg aagtgtctgg tgcatttaca 780

ccattcgagt taggaaaatc taacgcaact aatcttgacc caggtccaga ccatgtttgg 840

gcacctgaag agttaagtct ttctggagaa tatgtacgtg ctcgtattaa aggtgtgaac 900

tatgagccaa tcgaccgtac taaatacacg aaagtacttt gggactttaa tgatggaacg 960

aagcaaggat ttggagtgaa tggagattct ccaaataaag agcttattgc agttgataat 1020

gaaaacaaca ctttgaaaat ttcggggtta gatgtaagta acgatgtttc tgatggcaac 1080

tactgggcta atgctcgtct ttcagccaac ggttggggga aaagtgttga tatattaggt 1140

gctgaaaaac taactatgga tgttattgtt gatgagccga cgacggtagc gattgctgca 1200

attccacaag gtccatcagc aaattggatt aatccaattt gcgcagttaa ggttgagcca 1260

actgatttcg tgccgtttgg agataagttt aaagcagaat taactataac tacagcggac 1320

tctccagcga tagaagcgat tgcgatgcat gctgaaaata acaatatgaa caacatcatt 1380

ctgttcgtag gaactgatgc agctgacgtt atttatttag ataacattaa agtaattgga 1440

acagaagttg aaattccagt tgttcataat ccaaaataa 1479

<210> 3

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> egh31 upstream primer F

<400> 3

ccggaattca tgctcctcgc cgccgccact c 31

<210> 4

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> egh31 downstream primer R

<400> 4

gtgtaaatgc accagacact tccaccacct ggcaggcggg cgc 43

<210> 5

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CBD upstream primer F

<400> 5

gcgcccgcct gccaggtggt ggaagtgtct ggtgcattta cac 43

<210> 6

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CBD downstream primer R

<400> 6

ccgctcgagt tattttggat tatgaacaa 29

Claims

1. an enzyme fusion protein is characterized in that: this enzyme fusion protein is a fusion protein formed by the binding domain CBD of endoglucanase and Bacillus sp.III-3 bacterial strain produced endoglucanase; The amino acid sequence of the enzyme fusion protein is shown in SEQ ID No: 1.

2. A gene encoding the enzyme fusion protein of claim 1.

3 . The gene according to claim 2 , wherein the nucleotide sequence of the gene is shown in SEQ ID No: 2. 4 .

4. A recombinant plasmid for expressing the enzyme fusion protein of claim 1 in a host cell.

5. The recombinant plasmid according to claim 4, wherein the recombinant plasmid is a vector suitable for expression in Escherichia coli.

6 . The recombinant plasmid according to claim 4 or 5 , wherein the recombinant plasmid carries the gene according to claim 2 or 3 . 7 .

7. An engineering strain, characterized in that: the engineering strain carries the recombinant plasmid according to claim 4, 5 or 6.

8. engineering strain according to claim 7, is characterized in that:

The engineering strain is a recombinant strain obtained by transforming the recombinant plasmid into the host strain with Escherichia coli E.coliBL21 Star (DE3) as the host strain.

9. the application of the enzyme fusion protein of claim 1 in washing and papermaking industry.