阅读说明：本技术 一种重组β-1,4-内切葡聚糖酶固定化细胞的制备与应用 (Preparation and application of recombinant beta-1, 4-endoglucanase immobilized cell ) 是由 侯进慧 戴鹏飞 于 2021-10-20 设计创作，主要内容包括：本发明公开了一种重组β-1,4-内切葡聚糖酶固定化细胞的制备与应用,属于酶工程技术领域。该重组β-1,4-内切葡聚糖酶固定化细胞是通过重组β-1,4-内切葡聚糖酶菌株固定于海藻酸钠和钙盐形成的凝胶中制备得到的,该固定化细胞具有较好的稳定性,制备工艺合理,内切酶活性高、温度耐受性和pH稳定性好,实现了β-1,4-内切葡聚糖酶的重复、高效利用。(The invention discloses preparation and application of a recombinant beta-1, 4-endoglucanase immobilized cell, belonging to the technical field of enzyme engineering. The recombinant beta-1, 4-endoglucanase immobilized cell is prepared by immobilizing a recombinant beta-1, 4-endoglucanase strain in a gel formed by sodium alginate and calcium salt, has good stability, reasonable preparation process, high endoglucanase activity, good temperature tolerance and good pH stability, and realizes the repeated and efficient utilization of the beta-1, 4-endoglucanase.)

1. A recombinant beta-1, 4-endoglucanase immobilized cell, wherein the recombinant beta-1, 4-endoglucanase strain is immobilized in a gel formed by sodium alginate and calcium salt.

2. The recombinant β -1, 4-endoglucanase immobilized cell of claim 1, wherein the recombinant β -1, 4-endoglucanase strain is an EG/BL21 strain; the calcium salt is CaCl₂。

3. A method for producing a recombinant β -1, 4-endoglucanase-immobilized cell according to any one of claims 1-2, comprising the steps of:

(1) activating and culturing the EG/BL21 strain, and collecting bacterial liquid;

(2) centrifuging the bacterial liquid, transferring the centrifuged thallus into sodium alginate solution with mass percent concentration of 2-6% by using phosphoric acid buffer solution, uniformly mixing, taking the sodium alginate thallus mixed solution, and dropwise adding CaCl with mass percent concentration of 1-3%₂Forming gel beads in the solution, standing, filtering, washing the gel beads with deionized water for 2-3 times to obtain the recombinant beta-1, 4-endoglucanase immobilized cells.

4. The method according to claim 3, wherein the activation is specifically inoculating EG/BL21 strain to a liquid medium for overnight culture; the culture is specifically to culture the activated EG/BL21 strain to OD₆₀₀Is 1.0.

5. The preparation method of claim 3, wherein the sodium alginate solution has a concentration of 3% by mass.

6. The method of claim 3, wherein the CaCl is₂The mass percentage concentration of the solution is 2%.

7. The method according to claim 3, wherein the volume ratio of the centrifuged microbial cells to the sodium alginate solution in step (2) is 1: 40.

8. Use of the recombinant β -1, 4-endoglucanase-immobilized cell of any one of claims 1-2 in a cellulose saccharification process.

9. The use according to claim 8, wherein the recombinant β -1, 4-endoglucanase-immobilized cell is cultured at a pH of 7.0.

10. The use according to claim 8, wherein the recombinant β -1, 4-endoglucanase-immobilized cell is cultured at a temperature of 45 ℃.

Technical Field

The invention relates to the technical field of enzyme engineering, in particular to preparation and application of a recombinant beta-1, 4-endoglucanase immobilized cell.

Background

In the natural ecosystem, cellulose is the most abundant organic compound and is also the major constituent in plant tissue cells, typically accounting for 30% -50% of the dry weight of the plant. The utilization rate of abundant substances containing cellulose, such as a large amount of straws, rice stalks and the like, is low, and most of the substances are treated by adopting a combustion method, so that the environmental pollution is caused, the physicochemical property of soil is damaged, and organic matter components are lost. The cellulose resource is fully utilized and effectively converted into the available resource, and the method has great significance for solving the current worldwide problems of energy crisis, food shortage, environmental pollution and the like. If the technology for converting cellulose to glucose is successfully developed on an industrial scale, the cellulose resource can become a new source of human food, animal feed, fermentation industry raw materials, and energy. However, the main obstacle of effectively utilizing cellulose biomass resources at present is that the enzymolysis efficiency of cellulase is very low, and the difference is more than 2 orders of magnitude compared with amylase, so that the cost of the cellulase in the cellulose enzymolysis process is over high and accounts for about more than 40% of the cellulose saccharification process, and the wide application of the cellulase in the cellulose saccharification is seriously hindered.

Cellulases are a generic term for a class of enzymes that can be classified as endoglucanases (endo-1,4- β -D-glucanases, EC3.2.1.4, i.e., the C1 enzyme), exoglucanases (1,4- β -D-glucancellobilase or exo-1,4- β -D-glucanase, EC 3.2.1.91), and β -glucanases (β -1, 4-glucanase, BG, EC 3.2.1.21). Among them, endoglucanase is the most important enzyme in cellulase system, and can act on amorphous region in cellulose molecule, hydrolyze long-chain cellulose molecule into short-chain, produce small-molecule cellulose, provide a large amount of reaction end for exonuclease, and hydrolyze small-molecule cellulose oligosaccharide. In recent years, many studies have been made on the molecular structure of cellulase, functional amino acids, and cloning of enzyme genes. Related studies have shown that cellulase molecules have a similar structure, generally consisting of three parts, a globular Cellulose Catalytic Domain (CD), a Cellulose Binding Domain (CBD) and a Linker bridge (Linker).

Immobilization of enzymes refers to a technique in which the enzymes are bound or confined to a certain area by some carrier material, and the enzymes still perform their own catalytic reactions and can be recovered and reused. The immobilized enzyme is an enzyme which is in a closed state in a certain space, can still retain its catalytic activity, and can be continuously or repeatedly used. The immobilized cell technology is similar to that of immobilized enzyme, and after being immobilized, the cell can continue to survive and maintain the whole cell catalytic characteristic. In addition, the immobilized cells have lower cost than immobilized enzymes, can grow and use repeatedly, and are easy to separate from a reaction system.

However, no report has been made so far on the immobilization of recombinant β -1, 4-endoglucanase strains by the immobilized cell technology.

Disclosure of Invention

The invention aims to provide preparation and application of a recombinant beta-1, 4-endoglucanase immobilized cell, which aims to solve the problems in the prior art, the prepared recombinant beta-1, 4-endoglucanase immobilized cell has good stability, reasonable preparation process, high endoglucanase activity, good temperature tolerance and good pH stability, and realizes the repeated and efficient utilization of beta-1, 4-endoglucanase.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a recombinant beta-1, 4-endoglucanase immobilized cell, wherein a recombinant beta-1, 4-endoglucanase strain is fixed in a gel formed by sodium alginate and calcium salt.

Further, the recombinant beta-1, 4-endoglucanase strain is EG/BL21 strain; the calcium salt is CaCl₂。

The invention also provides a preparation method of the recombinant beta-1, 4-endoglucanase immobilized cell, which comprises the following steps:

(1) activating and culturing the EG/BL21 strain, and collecting bacterial liquid;

(2) centrifuging the bacterial liquid, transferring the centrifuged thallus into sodium alginate solution with mass percent concentration of 2-6% by using phosphoric acid buffer solution, uniformly mixing, taking the sodium alginate thallus mixed solution, and dropwise adding CaCl with mass percent concentration of 1-3%₂Forming gel beads in the solution, standing, filtering, washing the gel beads with deionized water for 2-3 times to obtain the recombinant beta-1, 4-endoglucanase immobilized cells.

Further, the activation is specifically inoculating EG/BL21 strain to liquid mediumCulturing overnight; the culture is specifically to culture the activated EG/BL21 strain to OD₆₀₀Is 1.0.

Further, the mass percentage concentration of the sodium alginate solution is 3%.

Further, the CaCl₂The mass percentage concentration of the solution is 2%.

Further, the volume ratio of the centrifuged thallus to the sodium alginate solution in the step (2) is 1: 40.

The invention also provides an application of the recombinant beta-1, 4-endoglucanase immobilized cell or the preparation method of the recombinant beta-1, 4-endoglucanase immobilized cell in a cellulose saccharification process.

Further, the culture pH of the recombinant beta-1, 4-endoglucanase immobilized cell is 7.0.

Further, the culture temperature of the recombinant beta-1, 4-endoglucanase immobilized cells is 45 ℃.

The invention discloses the following technical effects:

the invention uses sodium alginate as a main matrix to immobilize the recombinant beta-1, 4-endoglucanase strain, and the prepared recombinant beta-1, 4-endoglucanase immobilized cell has better stability, reasonable preparation process, high endoglucanase activity, good temperature tolerance and pH stability, realizes the repeated and efficient utilization of the beta-1, 4-endoglucanase, and lays an important foundation for the application of the recombinant beta-1, 4-endoglucanase strain in cellulose hydrolysis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a schematic diagram of the composition of recombinant plasmid pETEG;

FIG. 2 is a gel bead formed by preparing immobilized cells in example 1;

FIG. 3 is a graph showing the effect of temperature on the activity of immobilized cell enzymes;

FIG. 4 is a graph showing the effect of pH on the activity of immobilized cell enzymes;

FIG. 5 shows CaCl₂Influence of concentration on the activity of immobilized cell enzyme;

FIG. 6 shows the effect of sodium alginate concentration on the activity of immobilized cell enzymes.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1 immobilization of recombinant beta-1, 4-endoglucanase cells

1.1 Experimental materials

The recombinant strain EG/BL21 for expressing beta-1, 4-endoglucanase is constructed in the subject group, and the strain and the plasmid are stored in key laboratories for developing food resources and saving quality safety in Jiangsu province of Xuzhou engineering academy. Sodium carboxymethylcellulose and 3, 5-dinitrosalicylic acid were purchased from Fochen chemical reagent works, Tianjin. Yeast extract, peptone and other microbial culture reagents, sodium alginate and CaCl₂Such immobilized reagents are available from chemical reagents of national drug group, ltd. Molecular biology reagents were purchased from Tiangen Biochemical technology (Beijing) Ltd.

1.2 laboratory instruments and apparatus

Model AL204 electronic analytical balance: METSILER TOEDO; 723C visible spectrophotometer: shanghai Xinmao instruments, Inc.; HYG type shake culture cabinet: shanghai Xinxin Automation Equipment, Inc.; sigma3-16pk type high speed refrigerated centrifuge: SIGMA corporation, Germany; MDF-382E type ultra-low temperature refrigerator: SANYO electronics, Inc., Japan; water bath HH-4: guo hua electric appliances limited; superclean bench: shanghai Boxun industries, Inc.

1.3 recombinant bacterial cell construction

Firstly, a recombinant plasmid pETEG is constructed, wherein EG is beta-1, 4-endoglucanase gene (shown in figure 1) constructed on a pET vector, the recombinant plasmid pETEG can express beta-1, 4-endoglucanase, then the recombinant plasmid is transformed into a BL21 strain, and the engineering bacteria carrying the positive plasmid is recombinant bacteria EG/BL 21.

1.4 preparation method of immobilized cells

(1) Inoculating EG/BL21 strain stored in a freezing tube into 50mL LB liquid medium (containing 30. mu.g/mL kanamycin), shaking and culturing at 37 ℃ overnight, and activating;

(2) inoculating to 50mL LB (containing 30. mu.g/mL kanamycin) liquid medium according to 1% inoculum size, and shake-culturing at 37 ℃ until OD is reached₆₀₀When the concentration is 1.0, collecting bacterial liquid and carrying out cell immobilization;

(3) taking 100mL of cultured bacterial liquid for centrifugation, transferring the centrifuged thallus into 10mL of sodium alginate solution with the concentration of 3% by using phosphate buffer solution with the pH value of 7.0, wherein the volume ratio of the engineering bacteria to the sodium alginate is 1: 40, uniformly mixing, sucking the sodium alginate thallus mixed solution by using a 20mL syringe, and dropwise adding CaCl with the mass percentage concentration of 2%₂Forming gel beads in the solution, standing for 0.5h, filtering out the beads, washing the gel beads with deionized water for 2-3 times, and then placing in a refrigerator at 4 ℃ for later use.

1.5 preparation of glucose Standard Curve

Preparing a 1.0mg/mL glucose standard solution: 0.1g of anhydrous glucose is weighed and made up to 100mL with distilled water.

The glucose standard curve is a graph prepared by measuring absorbance at 540nm with glucose as a standard and DNS as a color developing agent. Taking 6 test tubes with scales, washing and drying for later use, and numbering 1 to 6. Adding standard glucose solution with corresponding volume into test tubes numbered 1-6 according to standards of 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL respectively, adding distilled water to constant volume of 1mL, mixing well, and preparing into glucose solution with different concentrations. Respectively adding 2mL of DNS solution into each test tube, fully and uniformly mixing, carrying out boiling water bath for 5min, taking out, standing at room temperature for cooling, finally adding distilled water to a constant volume of 25mL, and uniformly mixing. The absorbance of each test tube solution was measured at a wavelength of 540nm using test tube No. 1 as a blank. And finally, drawing a glucose standard curve by taking the glucose content (mg) as an abscissa and the OD value of the solution as an ordinate according to the measured result.

1.6 method for measuring enzyme activity of immobilized cell

5g of the immobilized cell gel beads (shown in FIG. 2) were placed in a triangular flask containing 195mL of LB liquid medium (containing 30. mu.g/mL kanamycin), shake-cultured at 120rpm at 37 ℃ for 5 hours, and 5. mu.L of IPTG was added to the bacterial solution to induce 3 hours, and the absorbance at 540nm was measured.

Calculating enzyme activity: the amount of the crude enzyme solution required for hydrolyzing the substrate to 1. mu. mol of reducing sugar per unit time at 50 ℃ was determined as one enzyme activity unit (U/mL). The formula for calculating the enzyme activity is shown as the following formula:

the enzyme activity (U/mL) ═ OD × k × n × 1000)/(180 × t × V) in the formula:

slope of k-glucose Standard Curve

n-dilution of enzyme

1000-conversion of mg to. mu.g

180-glucose Standard molecular weight

t-reaction time (min)

V-volume of crude enzyme solution (mL) required for reaction

1.7 Single-factor experiment of immobilized cells

1.7.1 Effect of temperature on the Activity of immobilized cell enzymes

The enzyme activity determination method of the immobilized cells comprises the following steps: 10 erlenmeyer flasks numbered a0-a9 were used. 100ml of LB liquid medium was added to each 250ml Erlenmeyer flask, 100. mu.l of kanamycin and 100. mu.l of IPTG were added, and the gel beads obtained in 1.4 above were added to a No. a1-a9 Erlenmeyer flask and cultured in an incubator at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃ for 20 hours, respectively. Taking another 10 test tubes, numbering 0-9 respectively, adding 2ml of sodium carboxymethylcellulose solution with the concentration of 2% into the 10 test tubes, adding 2ml of immobilized cell culture solution cultured in an a1-a9 conical flask into the test tubes 1-9 respectively, adding 2ml of LB liquid culture medium into the test tubes 0, placing the 10 test tubes in a water bath at 50 ℃ for 15 minutes, taking out, adding 2ml of DNS reagent into the test tubes 0-9 respectively, immediately placing the test tubes in a boiling water bath for 10 minutes, taking out, fixing the volume to 25ml by using deionized water, shaking the test tubes uniformly, and measuring the absorbance by taking the test tube 0 as a reference. Each set of experiments was performed in 3 replicates.

And (3) evaluating the influence of different temperatures on the activity of the immobilized cell enzyme by taking the relative enzyme activity as an index. The results are shown in FIG. 3.

1.7.2 Effect of pH on immobilized cell enzyme Activity

The enzyme activity determination method of the immobilized cells comprises the following steps: 5 erlenmeyer flasks numbered b1-b5 were used. 100ml of LB liquid medium was added to each 250ml Erlenmeyer flask, the pH of the liquid medium in Erlenmeyer flask b1-b5 was adjusted to 5, 6, 7, 8, 9 with a phosphoric acid buffer solution, 100ul of kanamycin and 100ul of IPTG were added, and finally the gel beads prepared above 1.4 were added, and the mixture was incubated at 37 ℃ for 20 hours. Taking another 6 test tubes, numbering 0-5 respectively, adding 2ml of sodium carboxymethylcellulose solution with the concentration of 2% into the 6 test tubes, adding 2ml of immobilized cell culture solution in a conical flask b1-b5 into the test tubes 1-5 respectively, adding 2ml of LB liquid culture medium into the test tubes 0, placing the 6 test tubes in a water bath at 50 ℃ for 15 minutes, taking out, adding 2ml of DNS reagent into the test tubes 0-5 respectively, immediately placing the test tubes in a boiling water bath, carrying out the boiling water bath for 10 minutes, taking out, fixing the volume to 25ml by deionized water, shaking the test tubes uniformly, and measuring the absorbance by taking the test tube 0 as a reference. Each set of experiments was performed in 3 replicates.

And (3) evaluating the influence of different pH values on the activity of the immobilized cell enzyme by taking the relative enzyme activity as an index. The results are shown in FIG. 4.

1.7.3 CaCl₂Effect of concentration on the Activity of immobilized cell enzymes

The preparation method of the immobilized cell comprises the following steps: centrifuging 100mL of cultured bacterial solution, transferring the centrifuged thallus into 10mL of 3% sodium alginate solution with pH7.0 phosphate buffer solution, mixing well, sucking the mixed solution of sodium alginate thallus with 20mL syringe, and respectively dropwise adding 1.0%, 1.5%, 2.0%, 2.5% and 3.0% CaCl₂Forming gel beads in the solution, standing for 0.5h, filtering out the beads, washing the gel beads with deionized water for 2-3 times, and then placing in a refrigerator at 4 ℃ for later use.

The enzyme activity determination method of the immobilized cells comprises the following steps: 5 erlenmeyer flasks numbered c1-c5 were used. 100mL of LB liquid medium, 100uL of kanamycin, and 100uL of IPTG were added to each 250mL Erlenmeyer flask. The CaCl with different concentrations are respectively added into conical flasks c1-c5₂The prepared gel beads were cultured in an incubator at 37 ℃ for 20 hours. Another 6 test tubes are respectively numbered with 0-5 and 62mL of sodium carboxymethylcellulose solution with the concentration of 2% is respectively added into the test tubes, 2mL of immobilized cell culture solution prepared in conical flasks c1-c5 is respectively added into test tubes No. 1-5, 2mL of LB liquid culture medium is added into test tube No. 0, 6 test tubes are placed in a water bath at 50 ℃ for 15 minutes, after the test tubes are taken out, 2mL of DNS reagent is respectively added into test tubes No. 0-5, the test tubes are immediately placed in a boiling water bath for 10 minutes, deionized water is used for fixing the volume to 25mL after the test tubes are taken out, all the test tubes are shaken up, and the absorbance is measured by taking test tube No. 0 as a reference. Each set of experiments was performed in 3 replicates.

Evaluating CaCl with different concentrations by using relative enzyme activity as index₂Influence of the solution on the activity of the immobilized cell enzyme. The results are shown in FIG. 5.

1.7.4 Effect of sodium alginate solution concentration on the Activity of immobilized cells

The preparation method of the immobilized enzyme comprises the following steps: the preparation method of the immobilized enzyme comprises the following steps: centrifuging 100mL of cultured bacterial solution, transferring the centrifuged thallus into 10mL of sodium alginate solution with the concentration of 2.0%, 3.0%, 40%, 5.0% and 6.0% respectively by using a phosphate buffer solution with the pH of 7.0, uniformly mixing, sucking the sodium alginate thallus mixed solution by using a syringe with the capacity of 20mL, and dropwise adding CaCl with the concentration of 2.0%₂Forming gel beads in the solution, standing for 0.5h, filtering out the beads, washing the gel beads with deionized water for 2-3 times, and then placing in a refrigerator at 4 ℃ for later use.

The enzyme activity determination method of the immobilized cells comprises the following steps: 5 erlenmeyer flasks numbered d1-d5 were used. 100mL of LB liquid medium, 100uL of kanamycin, and 100uL of IPTG were added to each 250mL Erlenmeyer flask. Adding the gel beads prepared by different sodium alginate solutions into conical flasks d1-d5 respectively, and culturing in an incubator at 37 ℃ for 20 h. Taking another 6 test tubes, numbering 0-5 respectively, adding 2mL of sodium carboxymethylcellulose solution with the concentration of 2.0% into the 6 test tubes, adding 2mL of immobilized cell culture solution prepared in a conical flask d1-d5 into the test tubes 1-5 respectively, adding 2mL of LB liquid culture medium into the test tube 0, placing the 6 test tubes in a water bath at 50 ℃ for 15 minutes, taking out, adding 2mL of DNS reagent into the test tubes 0-5 respectively, immediately placing the test tubes in a boiling water bath for 10 minutes, taking out, fixing the volume to 25mL by deionized water, shaking the test tubes uniformly, and measuring the absorbance by taking the test tube 0 as a reference. Each set of experiments was performed in 3 replicates.

And (3) evaluating the influence of sodium alginate solutions with different concentrations on the activity of the immobilized cell enzyme by taking the relative enzyme activity as an index. The results are shown in FIG. 6.

1.7.5 orthogonal experiments with fixed recombinant cells

After the optimal immobilization method is determined, different factors and levels are selected from the experimental result of the single-factor experiment to carry out orthogonal experiment so as to obtain the optimal immobilization effect. The factors selected in the experiment are temperature, pH, sodium alginate concentration and CaCl₂The concentration was analyzed. The results are shown in tables 1-2.

TABLE 1 orthogonal experimental factor Table for immobilized recombinant beta-1, 4-endoglucanase strains

TABLE 2 results of orthogonal experiments with immobilized recombinant β -1, 4-endoglucanase strains

As can be seen from the experimental results of the orthogonal experiment, the factors influencing the enzymatic activity of the immobilized cells are sequentially as follows according to the order of the influence of the factors: temperature of>CaCl₂Concentration of>Sodium alginate concentration>The pH value. Temperature has the greatest effect on immobilized cell enzyme activity. The optimal conditions for immobilized cell enzyme activity from orthogonal experiments were: at a temperature of 45 ℃ and CaCl₂Has a concentration of 2%, a concentration of sodium alginate of 3% and a pH of 7. Using the immobilized cells as the immobilization conditions, the enzyme activity of the immobilized cells was found to reach 32.88U/ml.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.