CN108841810A

CN108841810A - A kind of multifunctional cellulase gene and its application

Info

Publication number: CN108841810A
Application number: CN201810807008.7A
Authority: CN
Inventors: 江正兵; 宋慧婷; 李华南; 刘艳芳; 肖文静; 刘子璐
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2018-07-21
Filing date: 2018-07-21
Publication date: 2018-11-20
Anticipated expiration: 2038-07-21
Also published as: CN108841810B

Abstract

The present invention relates to a kind of multifunctional cellulase and its applications, it includes endo-glucanase enzyme domains, exoglucanase structural domain and beta-glucosidase enzyme domains, and the endo-glucanase enzyme domains, exoglucanase structural domain and beta-glucosidase enzyme domains are connected by flexible joint；Further relate to the expression vector and expressive host of the gene expression frame of above-mentioned multifunctional cellulase；Further relate to the method using above-mentioned multifunctional cellulase lignocellulose degradation.The present invention constructs tool there are three types of different cellulases (endoglucanase, exoglucanase and beta-glucosidase) active multifunctional cellulase for the first time, and the substrate specificity enzymatic activity and total fiber element enzymatic activity of the multifunctional cellulase are all significantly higher than original single enzyme.

Description

A kind of multifunctional cellulase gene and its application

Technical field

The present invention relates to the field of microbial fermentation of cellulose, more specifically it relates to a kind of multifunctional cellulase gene And its application.

Background technique

With the growth to energy demand and to the worry of fossil fuel drawback, it is abundant, renewable for wood fibre procatarxis, It does not compete and receives significant attention with human demand.Lignocellulosic mainly has four big sources：Forest residues (timber, branch, Leaf etc.), agricultural residue (straw, corn stover etc.), (food is useless for energy crop (giantreed, sugarcane etc.) and cellulose waste Object and municipal solid waste etc.).Lignocellulosic is mainly wooden by 40-50% cellulose, 25-30% hemicellulose, 15-20% Plain and a small amount of pectin, nitrogen compound and inorganic constituents composition.

Cellulase has played huge during cellulose degradation as class of enzymes maximum in lignocellulolytic enzymes Big effect.The enzyme synergistic effect that its degradable process needs three kinds of cleavage sites different, including endoglucanase, circumscribed Portugal Dextranase and beta-glucosidase.With the extensive research to cellulase, these biocatalysts are widely used in numerous Industrial circle.In view of the demand of the synergistic effect to a variety of enzymes, the expression of a variety of enzymes may improve degradation effect in individual cells Rate simultaneously reduces the technological process of production.The method that several genes are expressed in many individual cells frequently relies on a variety of screening modes simultaneously And it is complicated for operation, such as coexpression strategy.Currently, amalgamation and expression strategy, can be used as a kind of biotechnology of substitution coexpression, It is used to express a variety of enzymes simultaneously, and have many advantages, including convenient operation, the high-caliber solubility cloned and converted The catalytic capability of expression, cost-effective purifying and upgrading.There is the report much merged two-by-two about cellulase, however But the not no report about three or more cellulase catalytic domain amalgamation and expression.Therefore, in order to reduce multi-functional fibre Complicated operation during plain expression of enzymes is tieed up, we use convergence strategy building tool for the first time, and there are three types of different cellulase functions Single-gene.The gene is expressed in saccharomyces cerevisiae, screening obtains the bacterial strain with different specific enzymatic activities, and its filter paper Enzymatic activity (total fiber element enzymatic activity) is all remarkably higher than corresponding single enzyme, which can be used for succinct Genetic manipulation.The multifunctional cellulase meets the degradation model of three kinds of cellulase synergistics effect, easy to operate, effect is bright It is aobvious, and there is reference to the process of the enzyme lignocellulose degradation of various other types, there is extensive low cost to answer With value.

Summary of the invention

In order to solve the above problem, we attempt the single base for the encoder block that building has there are three types of different cellulase domains Cause.In research process, the cellulase combination of separate sources is tested, as a result, it has been found that many combinations can not show effective fibre Tie up plain enzymatic activity, thus it is speculated that may be because the structural domain space structure of these combinations, which fails effectively to fold to generate, has catalysis Activated centre.In the present invention, we intercept three kinds of different cellulases (endoglucanase, exoglucanase, the Portugals come Glycosidase) catalyst structure domain, utilize flexible linker sequence design three catalyst structure domains three functional cellulose enzyme single-gene sequences Column have obtained the three functional cellulose enzyme of single-gene of specific domain combination through heterogenous expression, which has different spies Specific enzymatic activity, and Filter paperlyase activity is all remarkably higher than single enzyme.

Based on the above research, the present invention provides a kind of multifunctional cellulases comprising endo-glucanase enzyme domains, Exoglucanase structural domain and beta-glucosidase enzyme domains, the endo-glucanase enzyme domains, exoglucanase knot Structure domain is connected with beta-glucosidase enzyme domains by flexible joint.

In a specific embodiment, the flexible joint is such as SEQ ID NO:Shown in 4.

In a preferred embodiment, the endo-glucanase enzyme domains such as SEQ ID NO:It is described circumscribed shown in 8 Dextranase structural domain such as SEQ ID NO:Shown in 9, the beta-glucosidase enzyme domains such as SEQ ID NO:Shown in 10.

The present invention also provides a kind of multifunctional cellulase expression vectors, and it includes above-mentioned multifunctional cellulase genes Expression cassette.

The present invention also provides a kind of host for expressing multifunctional cellulase, contain above-mentioned multi-functional fibre in genome Tie up plain enzyme gene expression frame.

In a specific embodiment, the host is S. cervisiae host.

The present invention also provides application of the above-mentioned multifunctional cellulase in decomposition of cellulose.

The present invention also provides it is a kind of degradation object in cellulose method comprising by the object be added to containing The step of being reacted in the solution of above-mentioned multifunctional cellulase.

In a preferred embodiment, reaction temperature is 40-65 DEG C, pH 5.0-7.5.

In a preferred embodiment, reaction temperature is 55 DEG C, pH 6.5.

The present invention constructs tool for the first time, and there are three types of different cellulases (endoglucanase, exoglucanase and β-Portugals Polyglycoside enzyme) active multifunctional cellulase, the substrate specificity enzymatic activity and total fiber element enzyme of the multifunctional cellulase Activity is all significantly higher than original single enzyme.

Detailed description of the invention

Fig. 1 is the building flow chart of fusion bce；

Fig. 2 is the gel electrophoresis images of PCR amplification fusion bce；

Fig. 3 is the plasmid schematic diagram of the saccharomyces cerevisiae expression of multifunctional cellulase BCE；

Fig. 4 is the recombinant Saccharomyces cerevisiae bacterial strain flat board screening figure for expressing BCE；

Fig. 5 is the specific substrate enzymatic activity of multifunctional cellulase BCE compared with three kinds of single function enzymes；

Fig. 6 is the Filter paperlyase activity of multifunctional cellulase BCE compared with three kinds of single function enzymes；

Fig. 7 is influence of the pH to the filter paper enzyme activity of multifunctional cellulase BCE；

Fig. 8 is influence of the temperature to the filter paper enzyme activity of multifunctional cellulase BCE.

Specific embodiment

The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and It is non-to be used to limit the scope of the invention.

1. the building of fusion bce

The building process of fusion bce is as shown in Figure 1, be respectively provided with endo-glucanase for building this subject group early period Enzyme (EG) gene, exoglucanase (cellobiohydrolase；CBH) the plasmid of gene and beta-glucosidase (BG) PHBM368-pgk-bg, pHBM368-pgk-cbh and pHBM368-pgk-eg are expanded as template by PCR reaction respectively To encoding cellulase BG catalyst structure domain BG CD (SEQ ID NO:3, corresponding nucleic acid sequence is SEQ ID NO:7), CBH catalyst structure domain CBH CD (SEQ ID NO:2, corresponding nucleic acid sequence is SEQ ID NO:And EG catalyst structure domain 6) EG CD(SEQ ID NO:1, corresponding nucleic acid sequence is SEQ ID NO:5) gene order.By the method for fusion DNA vaccine, CD segment is cascaded, centre is with flexible joint (G₄S)₃(SEQ ID NO:4) it connects.Primer sequence is as shown in table 1.

Table 1 is used to expand the primer of catalyst structure domain

Note：Overstriking is restriction enzyme site；Sequences in italics is flexible joint (G₄S)₃(i.e.：GGGGSGGGGSGGGGS reversed volume) Code sequence.

After PCR product is carried out ethanol precipitation recycling, with corresponding restriction enzyme enzymatic treatment.With Xba I digestion BG CD, with Xba I and EcoR I digestion CBH CD, with EcoR I digestion CBH CD.Gel electrophoresis recycles the segment after digestion, presses According to etc. amount of substance concentration mixing be added coupled reaction system.Using enzyme connection mixture as template, using primer BG-SnaB I-F and EG-Eco81I-R, after PCR amplification, agarose gel electrophoresis shows 3 band (Fig. 2), wherein most bright band is about for length The segment of 3.0kb recycles the band and obtains bce segment.

2. the conversion of saccharomyces cerevisiae and the screening of recon

Fusion bce is cloned between SnaB I and the Eco81I restriction enzyme site of pHBM368 plasmid, is opened in pgk Under the control of mover, bce expression plasmid carrier (Fig. 3) is obtained.After the Hpa I digestion of bce expression plasmid carrier, recycling is used for Electrotransformation saccharomyces cerevisiae INVSc.

Transformant is screened with the SC solid medium without uracil, obtained transformant is inoculated into YPC- platform phenol indigo plant solid On culture medium, 28 DEG C are cultivated about 4 days, screen the recombinant bacterial strain with cellulase activity.As a result as shown in figure 4, original bacteria INVSc1 growing way on the YPC- platform phenol indigo plant solid medium using CMC-Na as sole carbon source is very poor, and recombinant bacterial strain then can be normal Growth illustrates that recombinant bacterium has cellulase (endoglucanase) activity.Since platform phenol indigo plant is in combination with macromolecular polysaccharide class Substance, and can not be in conjunction with micromolecular polysaccharide, if recombinant bacterial strain table decomposition of cellulose generates small molecule carbohydrate, periphery of bacterial colonies will go out Now hydrolysis circle.Wherein the periphery of bacterial colonies of No. 1, No. 2 and No. 3 bacterial strain can see apparent hydrolysis circle, illustrate these recombinant bacterial strains reality The secreting, expressing of cellulase is showed.

No. 3 bacterial strains are chosen further to be verified.The total DNA of No. 3 bacterial strains is extracted as template, uses primer BG-SnaB I-F and EG-Eco81I-R has carried out PCR verifying, there is the band that apparent prediction size is 3.0kb as the result is shown.In addition, carrying out SDS-PAGE detection, bacterial strain have apparent destination protein band, and size is about 119.0kDa, with predict it is in the same size.Therefore, The saccharomyces cerevisiae that No. 3 bacterial strains are chosen as single-gene multifunctional cellulase BCE expresses bacterial strain, is named as INVSc1-BCE.

3. the cellulase activity of recombinant bacterial strain I NVSc1-BCE is analyzed

The saccharomyces cerevisiae recombinant bacterium of saccharomyces cerevisiae original strain INVSc1, three kinds of single expression cellulases has been activated simultaneously Strain (INVSc1-BG, I NVSc1-CBH and I NVSc1-EG) and three functions merge cellulase recombinant bacterial strain INVSc1-BCE. Control each group inoculum concentration it is identical after be transferred in new YPD fluid nutrient medium, culture two days later the extracellular enzyme solution of centrifuging and taking supernatant into Row purifying, determines three classes cellulase specific substrate enzymatic activity and total fiber element enzymatic activity, is analyzed compared with.

3.1 cellulase specific substrate activity ratios compared with

Cellulase specific substrate activity ratio measures BG enzymatic activity compared with by substrate of PNPG, measures by substrate of MCC CBH enzymatic activity measures EG enzymatic activity by substrate of CMC-Na, while determining the fusion cellulose for constructing and expressing in this research These three types of cellulase activities of enzyme BCE.

Dilution 50 μ L of enzyme solution is taken to be separately added into the corresponding substrate of 950 μ L (5mM PNPG, 1% (W/V) MCC, 1% (W/V) CMC), after 45 DEG C are reacted 30 minutes, takes 200 μ L to be added in the centrifuge tube containing 400 μ L DNS reagents immediately and be sufficiently mixed, 100 DEG C metal bath 10 minutes, cooled on ice 5 minutes, measure OD₅₄₀.The definition of enzyme-activity unit：Reaction per hour generates 1 μm of o l reduction The amount of enzyme required for sugar is 1U.

As a result as shown in figure 5, the activity of beta-glucosidase of cellulase BCE, exoglucanase activity and inscribe Portugal Enzyme respectively reaches 1008.0U/mg, 135.6U/mg and 1240.2U/mg, wherein β-Portugal of cellulase BCE Polyglycoside enzymatic activity and endo-glucanase are respectively than BG and EG high 46.27% and 46.20%, exoglucanase activity and CBH Almost.It is improved positioned at the activity of activity ratio's single expression of the CBH in fusion protein middle position few, thus it is speculated that possible cause It is the middle part that the area exoglucanase CD is located at fusion protein, steric hindrance is larger, and cause enzyme-to-substrate contact to have obstacle, thus Leading to the hydrolysis efficiency of enzyme reduces.

3.2 total fiber element enzymatic activitys

The comparison of total fiber element enzymatic activity determines the total fiber of single expression and amalgamation and expression using filter paper as substrate respectively Plain enzymatic activity (also referred to as Filter paperlyase activity).

It takes dilution 50 μ L of enzyme solution that 950 μ L citrate buffer solutions are added, while two small pieces Whatman NO.1 filter paper is added (about 0.5mg), 45 DEG C reaction 1 hour after, take immediately 200 μ L be added the centrifuge tube containing 400 μ L DNS reagents in be sufficiently mixed, 100 DEG C metal bath 10 minutes, cooled on ice 5 minutes, measure OD540.The definition of enzyme-activity unit：Reaction generates 1 μm of o l also per hour The amount of enzyme required for raw sugar is 1U.

As a result as shown in fig. 6, the Filter paperlyase activity of BCE reaches 122.4U/mg, it is higher than BG (33.0U/mg) and EG (67.8U/mg), it is suitable with CBH (120.6U/mg).Experiment shows that four kinds of cellulases all have degradation complex substrate (filter paper) Ability.In addition, Filter paperlyase activity is not whole high in this research, it may be possible to since these cellulases lack CBM (cellulose Binding structural domain), the degradation of insoluble cellulosic substrate this to filter paper is more out of strength.Accordingly, it may be desirable to the knot of enzyme Structure domain is further modified.

4. the optimum reaction conditions of cellulase BCE

When above measuring the filter paper enzyme activity of BCE, since it is considered that compare with the cellulase of three kinds of single expressions Compared with, and selected pH 6.0, temperature 45 C as reaction condition.In order to determine the optimal reaction item of BCE total fiber element enzymatic activity Part determines pH and temperature to the active influence of BCE Filter paperlyase respectively.

4.1pH is on the active influence of BCE Filter paperlyase

Other reaction conditions are constant, and the filter paper of BCE is determined under conditions of pH is 4.5,5,5.5,6,6.5,7 and 7.5 Enzymatic activity, as a result as shown in Figure 7.Optimal pH is 6.5, in the citrate buffer solution of pH5.0-7.0, the Filter paperlyase activity of BCE It is maintained at 80% or more, it is best with pH 5.5 and 6.5.

4.2 temperature are on the active influence of BCE Filter paperlyase

Other reaction conditions are constant, and pH is set as 6.5 according to 4.1 result, temperature be 30,35,40,45,50, 55, the filter paper enzyme activity of BCE is measured under conditions of 60 and 65 DEG C.As a result as shown in figure 8, within the temperature range of 40-65 DEG C, recombination The Filter paperlyase activity of fusion cellulase is maintained at 60% or more, within the temperature range of 30-35 DEG C, recombination fusion cellulase Filter paperlyase activity be lower than 50%.Optimum temperature is 55 DEG C.And under conditions of pH 6.5 and 55 DEG C, the filter paper enzyme activity of BCE It can reach 198.96U/mg, be higher by 62.6% than Filter paperlyase activity under the conditions of pH6.0, temperature 45 C.The result shows that melting The optimization of synthase pH and temperature condition significantly improves the Filter paperlyase activity of fusion enzyme.

The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Sequence table

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<210> 10

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

acttctagac agggaaatca ggatttc 27

<210> 11

<211> 71

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

agaattccga gccaccgcca cccgacccac caccgcccga gccaccgcca ccataagtgc 60

tatcaatcgg a 71

<210> 12

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

atcgaattcc agtcgctttg cgaccaat 28

<210> 13

<211> 31

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 13

actcctgagg ctagttgttt tgttgggcgg a 31

Claims

1. a kind of multifunctional cellulase, which is characterized in that including endo-glucanase enzyme domains, exoglucanase structural domain With beta-glucosidase enzyme domains, the endo-glucanase enzyme domains, exoglucanase structural domain and beta-glucosidase Structural domain is connected by flexible joint.

2. multifunctional cellulase according to claim 1, which is characterized in that the flexible joint is such as SEQ ID NO: Shown in 4.

3. multifunctional cellulase according to claim 1 or 2, which is characterized in that the endo-glucanase enzyme domains Such as SEQ ID NO:Shown in 1, the exoglucanase structural domain such as SEQ ID NO:Shown in 2, the beta-glucosidase knot Structure domain such as SEQ ID NO:Shown in 3.

4. a kind of multifunctional cellulase expression vector, which is characterized in that include more function of any of claims 1-3 The expression cassette of energy cellulose enzyme gene.

5. a kind of host for expressing multifunctional cellulase, which is characterized in that contain any one of claim 1-3 in genome The gene expression frame of the multifunctional cellulase.

6. the host of expression multifunctional cellulase according to claim 5, which is characterized in that the host is wine brewing ferment Female host.

7. application of the multifunctional cellulase of any of claims 1-3 in decomposition of cellulose.

8. a kind of method of lignocellulose degradation, which is characterized in that have the right to want including the lignocellulosic to be added to contain Ask the step of reacting in the solution of multifunctional cellulase described in any one of 1-3.

9. according to the method described in claim 8, it is characterized in that, reaction temperature be 40-65 DEG C, pH 5.0-7.5.

10. according to the method described in claim 9, it is characterized in that, reaction temperature be 55 DEG C, pH 6.5.