CN109486782B

CN109486782B - Method for improving sucrose phosphorylase expression efficiency through molecular chaperone co-expression

Info

Publication number: CN109486782B
Application number: CN201811415908.3A
Authority: CN
Inventors: 韩瑞枝; 姚栋; 倪晔; 肖静; 王克芬
Original assignee: Shandong Lonct Enzymes Co ltd; Jiangnan University
Current assignee: Shandong Lonct Enzymes Co ltd; Jiangnan University
Priority date: 2018-11-26
Filing date: 2018-11-26
Publication date: 2020-06-09
Anticipated expiration: 2038-11-26
Also published as: CN109486782A

Abstract

The invention discloses a method for improving the expression efficiency of sucrose phosphorylase by molecular chaperone co-expression, belonging to the technical field of biological engineering and enzyme engineering. According to the invention, the recombinant plasmid pET-20b-SPase and pGro7 are co-expressed, so that the molecular chaperone protein expressed by pGro7 can effectively reduce the formation of inclusion bodies, and the improvement of the soluble expression level and activity of the SPase is promoted. By optimizing the expression conditions and using a molecular chaperone co-expression system, the intracellular enzyme activity of the SPase reaches 24.33U/mL, and the specific enzyme activity reaches 6.58U/mg.

Description

Method for improving sucrose phosphorylase expression efficiency through molecular chaperone co-expression

Technical Field

The invention relates to a method for improving the expression efficiency of sucrose phosphorylase by molecular chaperone co-expression, belonging to the technical field of biological engineering and enzyme engineering.

Background

Sucrose phosphorylase (SPase, EC 2.4.1.7) belongs to GH13 family, mainly exists in Bifidobacterium longum, Leuconostoc mesenteroides, Pseudomonas saccharophila, has wide application in food, cosmetics and medicine industries due to its wide substrate specificity, mainly comprises three aspects of (1) taking sucrose as a donor and rhamnose, xylose, fructose and galactose as an acceptor, catalyzing to obtain corresponding oligosaccharide which has one more glucose group, and (2) being capable of catalyzing certain substances which are not stable to synthesize stable derivatives thereof, such as sucrose phosphorylase catalyzing 2-O- α -glucosylation of L-ascorbic acid in sucrose solution with high efficiency and excellent site selectivity to synthesize 2-O- α -D-glucoside (AA-2G) which is a highly stable derivative of vitamin C, has important industrial application in cosmetics, foods and medicines, and modified and hydroxyl group, and phenol-catalyzed to synthesize hydroxyl group, and phenol-glycosyl-phenol-containing compound, has wide application value in the medical industries, and is used for synthesizing arbutin, and has good anti-inflammatory activity, and is widely used for medical purposes.

However, since there are complex metabolic regulation mechanisms in the wild type strains, the content of sucrose phosphorylase in these strains is not high. The sucrose phosphorylase is produced by fermentation of natural strains, the yield and the production efficiency are low, and the requirements of industrial application are difficult to meet. Recently, many researchers have constructed recombinant strains by using genetic engineering principles to realize overexpression of the SPase, but the overexpression of the SPase still has the defects of poor soluble expression effect, low enzyme activity and the like. In 2006, Jin-Ha Lee et al cloned the SPase gene from Leuconostoc mesenteroides NRRL B-742 on E.coli BL21(DE3) pLysS with enzyme production of 1.8U/mL. In 2018, Wang, MM et al expressed Spase from Bifidobacterium adolescentis in Bacillus subtilis, and in the absence of a signal peptide, the SPase was efficiently secreted into the extracellular medium. After the recombinant strain is cultured in a 3L bioreactor, the yield and activity of the crude SPase reach 7.5g/L and 5.3U/mL respectively, which are the highest levels of the yield and activity of the SPase reported at present. So far, no report that the expression efficiency of the SPase is improved by the co-expression of molecular chaperones appears. Therefore, it is very important to provide a method for improving the expression efficiency of sucrose phosphorylase for the industrial production of SPase.

Disclosure of Invention

The first purpose of the invention is to provide a method for improving the expression efficiency of sucrose phosphorylase, which is to co-express sucrose phosphorylase and pGro7 in Escherichia coli.

In one embodiment of the invention, the gene sequence encoding the sucrose phosphorylase gene is shown in SEQ ID NO. 2.

In one embodiment of the invention, the vector for expressing the sucrose phosphorylase comprises pET-20b, and the Escherichia coli comprises E.coli BL 21.

In one embodiment of the invention, E.coli after co-expression is induced with L-arabinose at a final concentration of 0.5-1g/L and IPTG at a final concentration of 0.05-0.1mM for 8-20 h.

The second purpose of the invention is to provide a genetically engineered bacterium which expresses the sucrose phosphorylase shown in SEQ ID NO.1 and the molecular chaperone plasmid pGro7 together.

In one embodiment of the invention, the genetically engineered bacterium expresses a sucrose phosphorylase gene shown by SEQ ID No.2 by using Escherichia coli BL21 as a host and pET series plasmids as a vector.

The third purpose of the invention is to provide a sucrose phosphorylase gene, which is characterized in that the nucleotide sequence is shown as SEQ ID NO. 2.

A fourth object of the invention is to provide the use of the above method in the food, cosmetic or pharmaceutical field.

The fifth purpose of the invention is to provide the application of the genetically engineered bacteria in the fields of food, cosmetics or pharmacy.

The sixth purpose of the invention is to provide the application of the genetic engineering bacteria in the preparation of products containing sucrose phosphorylase.

The invention has the beneficial effects that:

the expression efficiency of sucrose phosphorylase (SPase, EC 2.4.1.7) is improved by means of molecular chaperone co-expression, and the recombinant plasmid pET-20b-SPase and pGro7 are co-expressed, so that the molecular chaperone protein expressed by pGro7 can effectively reduce the formation of inclusion bodies and promote the improvement of the soluble expression level and activity of the SPase. By optimizing expression conditions and using a molecular chaperone co-expression system, the intracellular enzyme activity of the SPase reaches 24.33U/mL, and compared with the method of expressing the SPase in bacillus subtilis by Wang, MM and the like, the enzyme activity of the recombinant sucrose phosphorylase obtained by the invention is improved by 3.5 times. When the concentration of the inducer L-arabinose is 0.5g/L, the final concentration of IPTG is 0.05mM, and the molecular chaperone pGro7 and pET-20b-SPase are co-expressed for 8 hours, the specific enzyme activity can reach 6.58U/mg.

Drawings

FIG. 1: and (4) drawing a glucose standard curve.

FIG. 2: SDS-PAGE analysis of supernatant and pellet from the co-expression of pET-20b-SPase-pGro7 and the expression of pET-20b-SPase alone, M: blue Plus II Protein Marker; 1: co-expressing pET-20b-SPase-pGro7, and then collecting the supernatant of the bacterial liquid; 2: after coexpression of pET-20b-SPase-pGro7, the part of the bacterial liquid is precipitated; 3: only expressing the supernatant part of the bacterial liquid after pET-20 b-SPase; 4: only the portion of the bacterial liquid precipitate after expression of pET-20 b-SPase.

Detailed Description

Protein purification step:

A. required reagent solution:

binding liquid: 0.5mol/L NaCl, 20mmol/L imidazole, 20mmol/L PBS, 1% glycerol, pH 7.4.

Eluent: 0.5mmol/L NaCl, 500mmol/L imidazole, 20mmol/L PBS, 1% glycerol, pH 7.0.

Lysis solution: 0.5mmol/L NaCl, 20mmol/L PBS, 50mmol/L EDTA, pH 7.0.

NiSO₄：100mmol/L NiSO₄。

B. The operation is as follows:

regeneration: a1 mL Ni-NTA pre-loaded gravity column was selected for protein purification by first washing the column with 10mL of binding solution, followed by 10mL lysis solution, followed by 10mL water, followed by 10mL NiSO4, followed by 10mL water, followed by 10mL binding solution, followed by 20% ethanol.

Combining: the column was washed with 20% ethanol, then 20mL of binding solution, and then the sample was applied to the column, followed by washing with 10mL of binding solution.

And (3) elution: firstly washing with 10mL of binding solution, collecting the flow-through solution by using an EP tube, then washing with imidazoles with different concentrations (50 mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, 250mmol/L and 300mmol/L respectively) and collecting the liquid by using the EP tube, namely obtaining eluents of imidazoles with different concentrations, then washing with 10mL of eluents, and then washing with 20% ethanol.

And (3) storage: stored in 20% ethanol.

Loading: 40 μ L of the flow-through solution and the eluate were taken, 10 μ L of SDS-PAGE protein loading buffer was added thereto, and after heating at 100 ℃ for 5min and centrifugation, 5 μ L of the eluate was subjected to SDS-PAGE protein electrophoresis analysis.

And (3) analysis: according to SDS-PAGE electrophoresis picture, the sample with higher protein concentration is concentrated and desalted to obtain pure enzyme solution.

SDS-PAGE electrophoretic analysis:

sampling samples of 80 μ L each, adding 20 μ L mercaptoethanol, mixing, heating at 100 deg.C for 10min, 12000 r.min^-1Centrifuging for 5min, and sampling the supernatant 10 μ L; the sample loading electrophoresis conditions are respectively 130V and 15min for flattening strips, and 200V and 45min for separating protein; after electrophoresis is finished, taking out the albumin glue, washing the albumin glue clean with water, immersing the albumin glue in Coomassie brilliant blue staining solution, and placing the Coomassie brilliant blue staining solution in a universal shaking table for staining; pouring out the staining solution, washing the albumin glue, immersing the albumin glue in the Coomassie brilliant blue destaining solution, and placing the Coomassie brilliant blue destaining solution in a universal shaking table for destaining; the decolorized protein gel was placed in a gel imager and the electrophoretogram protein bands were observed.

(II) an enzyme activity determination method:

the reducing sugar method by DNS: the reaction system contained 900. mu.L of PBS buffer (pH 6.5), 400. mu.L of 1.48M sucrose solution, 200. mu.L of SPase solution, and was reacted in 55 ℃ water bath for 10min in 1.5mL of the reaction system. The reaction was terminated by heating in a boiling water bath, and then the amount of fructose produced was measured by DNS.

Definition of enzyme activity: at 55 deg.C, pH 6.5, 1min free enzyme hydrolyzed sucrose to produce 1. mu. mol fructose as one unit of activity (U).

Definition of specific enzyme activity: the number of units of enzyme activity possessed by a unit weight (mg) of the protein.

Drawing a standard curve: and taking 7 graduated test tubes, and respectively adding glucose standard solution with different volumes and concentration of 1mg/mL, DNS reagent and distilled water. Specifically, the results are shown in Table 1.

TABLE 1 content of ingredients in different test tubes

Shaking the tubes, heating in boiling water bath for 10min, cooling to room temperature, adding distilled water to constant volume of 10mL, reversing the stopper, calibrating with test tube No.1 as control, and measuring absorbance at wavelength 540nm with spectrophotometer. By OD₅₄₀Plotted as ordinate and glucose content as abscissa, a standard curve is plotted, as shown in FIG. 1.

Preparing a glucose standard solution: weighing 100g of glucose which is dried at 70 ℃ to constant weight, placing the glucose in a small beaker, adding a small amount of water to dissolve the glucose, then fixing the volume of the glucose to 100mL by a volumetric flask, and uniformly mixing the glucose and the water.

Preparation of 3, 5-dinitrosalicylic acid (DNS): 10.6g DNS and 19.8g NaOH were added to a volume of hot aqueous solution containing 306g potassium sodium tartrate, and 7.6mL phenol and 8.3g sodium metabisulfite were added and stored in a brown bottle for seven days before use.

The enzyme activity calculation formula is as follows: and calculating the glucose content m according to the glucose standard curve. Unit volume enzyme activity U/mL-m/180 gamma 1000/10/V

Specific enzyme activity U/mg ═ unit volume enzyme activity/protein concentration

(III) culture Medium

LB medium (g/L): 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl

TB medium (g/L): 24g/L yeast powder, 12g/L peptone, 4g/L glycerol and KH₂PO₄17mM，K₂H PO₄·3H₂O72mM。

(IV) thermal conversion Process

Adding the recombinant plasmid (30-100ng) or the ligation reaction product (10 mu L) into E.coli BL21(DE3) competent cells melted on ice, embedding the cells on ice for 25min, immersing the cells in a water bath at 42 ℃ for 90s, quickly placing the cells in the ice bath for cooling for 15min, adding an antibiotic-free LB liquid culture medium in a sterile environment, culturing at the constant temperature of 37 ℃ for 15min, and then placing the cells in a shaking table at 37 ℃ for shake culture for 1 h. After the culture is finished, a proper amount of culture solution is taken to be coated on an LB solid plate with resistance, and the plate is placed upside down in a thermostat at 37 ℃ for overnight culture for 12-16 h.

Example 1 construction method of chaperone Co-expression System

1. Cloning of sucrose phosphorylase Gene

The sucrose phosphorylase gene is synthesized by a chemical total synthesis method, and the nucleotide sequence of the sucrose phosphorylase gene is shown as SEQ ID NO. 2.

2. Construction of recombinant plasmid pET-20b-SPase

pET-20b was first double-digested with NcoI and XhoI, and then the synthetic gene fragment was inserted between the NcoI and XhoI cleavage sites of pET-20 b.

3.CaCl₂Preparation of competent cells of Escherichia coli and thermal transformation procedure

The glycerol deposited strain of e.coli BL21(DE3) was streaked on an LB solid plate without an anti-plate, and the plate was inverted and cultured in a 37 ℃ incubator for about 12 hours to isolate a single colony. Picking single colony with regular edge and moderate size in fresh culture medium, and culturing at 37 deg.C in 180rpm shaking table to OD₆₀₀Stopping culturing when the culture time is 0.3, and placing the bacterial liquid on ice for ice bath for 30 min; subpackaging the precooled bacterial liquid into 50mL centrifuge tubes, centrifuging at 4 ℃ and 4000rpm for 10min, and discarding the supernatant; add approximately 10mL of 0.1mol/L MgCl to the centrifuge tube₂·CaCl₂(precooling on ice), rotating on ice gently until the thalli are fully dissolved, and continuously adding 30mL of MgCl₂·CaCl₂Centrifuging the solution at 4 ℃ and 4000rpm for 10min, and removing the supernatant; repeating the previous step once; 0.5mL of 0.1mol/L CaCl was added to the centrifuged tube₂(precooling on ice) and 0.5mL of 30% glycerol (precooling on ice), and the mixture is gently and fully mixed by a pipette gun, and then the mixture is subpackaged into 1.5mL of EP tubes (precooling on ice) by 100 mu L/tube and stored in a refrigerator at the temperature of minus 80 ℃.

4. Induced expression purification and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis of target protein

Recombinant plasmid pET-20b-SPase (ampicillin resistance)Sex) and the molecular chaperone plasmid pGro7 (chloramphenicol resistance), were added simultaneously to an e.coli BL21(DE3) competent cell for heat transformation at a molar concentration of 1: 1. When a single colony grows on the plate, the colony PCR verification is carried out by using the primers, and positive clones are obtained only if the verification result is positive. A single strain of positive clone was picked and dropped into 25mL of fresh LB medium containing both Amp and Cm resistance, and shake-cultured at 37 ℃ for 10 hours. Transferred to a medium containing 40mL of TB (40. mu.L Amp/Cm) at 1% (v/v) and cultured at 37 ℃ and 180rpm to OD₆₀₀0.8-0.9, and performing an induction condition optimization experiment.

(1) Culturing the recombinant bacterium to OD₆₀₀0.8 to 0.9, IPTG at a final concentration of 0.05mM and L-arabinose at a concentration of 0.25g/L, 0.5g/L, 0.75g/L, 1.0g/L, respectively, were added, induction-cultured at 18 ℃ for 20 hours, and then centrifuged at 8000rpm for 15 minutes at 4 ℃ to collect wet cells. Using 50mM K₂HPO₄-KH₂PO₄(pH 6.5) after suspending the cells in the buffer, the cells were disrupted by ultrasonication, and the disrupted solution was centrifuged and assayed for enzyme activity as shown in Table 2. The result shows that when the concentration of the arabinose is 0.5g/L, the induction effect is good, and the enzyme activity per unit volume can reach 20.88U/mL.

TABLE 2 enzyme Activity measured by expression under different arabinose concentrations

(2) Culturing the recombinant bacterium to OD₆₀₀0.8-0.9, adding 0.5 g/L-arabinose and IPTG with a final concentration of 0.05mM, inducing and culturing at 18 ℃, respectively sampling for 4h, 8h, 12h, 16h and 20h, and centrifuging at 4 ℃ and 8000rpm for 15 minutes to collect wet cells. Using 50mM K₂HPO₄-KH₂PO₄(pH 6.5) after suspending the cells in the buffer, the cells were disrupted by ultrasonication, and the disrupted solution was centrifuged and assayed for enzyme activity as shown in Table 3.

SDS-PAGE results of supernatant and pellet of the bacterial suspension obtained by co-expressing pET-20b-SPase-pGro7 and expressing pET-20b-SPase alone are shown in FIG. 2, and the band of the pellet fraction co-expressing pET-20b-SPase-pGro7 is significantly brighter than that of the pellet fraction expressing pET-20b-SPase alone.

TABLE 3 enzyme Activity measured by expression under different Induction time conditions

Induction time (h)	Unit volume enzyme activity U/mL	Specific activity (U/mg)
			4	4.24	2.91
8	12.93	6.58
			12	12.31	5.96
16	24.33	5.56
			20	22.43	4.99

In conclusion, compared with the expression of the SPase in the bacillus subtilis by Wang, MM and the like, the pET-20b-SPase and the molecular chaperone pGro7 are transferred into escherichia coli for co-expression, the intracellular enzyme activity of the SPase reaches 24.33U/mL and is improved by 3.5 times, and meanwhile, when the concentration of an inducer L-arabinose is 0.5g/L and the final concentration of IPTG is 0.05mM, the co-expression is carried out for 8 hours, and the specific enzyme activity also reaches 6.58U/mg. If pET-20b-SPase is expressed only in Escherichia coli, the intracellular enzyme activity of SPase is only 3.24U/mL.

Comparative example 1 pET-20b-SPase and chaperone PKJE7 or PG-TF2 were transformed into E.coli to be co-expressed, and the other conditions were the same as in example 1

After pET-20b-SPase and molecular chaperone PKJE7 or PG-TF2 are transferred into escherichia coli for co-expression, the obtained recombinant bacteria are cultured to OD₆₀₀0.8-0.9, adding 0.5 g/L-arabinose or tetracycline and IPTG at the final concentration of 0.05mM, inducing culture at 18 ℃, sampling after 16h, and centrifuging at 8000rpm for 15 minutes at 4 ℃ to collect wet cells. Using 50mM K₂HPO₄-KH₂PO₄(pH 6.5) after suspending the cells in a buffer, the cells were disrupted by ultrasonication, and the disrupted solution was centrifuged and assayed for enzyme activity. As shown in Table 4, the intracellular activities of the SPase were 3.72U/mL and 2.36U/mL, respectively.

TABLE 4 recombinase enzyme activity obtained by coexpression of pET-20b-SPase and molecular chaperone PKJE7 or PG-TF2

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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SHANDONG LONCT ENZYMES Co.,Ltd.

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Claims

1. A method for improving the expression efficiency of sucrose phosphorylase is characterized in that the sucrose phosphorylase and pGro7 are co-expressed in Escherichia coli, and the amino acid sequence of the sucrose phosphorylase is shown as SEQ ID No. 1.

2. The method of claim 1, wherein the vector for expressing the sucrose phosphorylase comprises pET-20b and the e.coli comprises e.coli bl21.

3. The method of claim 1 or 2, wherein the co-expressed E.coli is induced with L-arabinose at a final concentration of 0.5-1g/L and IPTG at 0.05-0.1mM for 8-20 h.

4. A gene engineering bacterium is characterized in that sucrose phosphorylase shown in SEQ ID NO.1 and molecular chaperone plasmid pGro7 are expressed; the genetic engineering bacteria take escherichia coli BL21 as a host and pET series plasmids as a vector.

5. Use of the method according to any one of claims 1 to 3 in the food, cosmetic or pharmaceutical field.

6. The genetically engineered bacterium of claim 4 for use in the fields of food, cosmetics or pharmaceuticals.

7. The use of the genetically engineered bacteria of claim 4 in the preparation of sucrose phosphorylase containing products.