CN112695006B

CN112695006B - Recombinant bacillus subtilis for expressing D-psicose-3-epimerase

Info

Publication number: CN112695006B
Application number: CN202110163604.8A
Authority: CN
Inventors: 饶志明; 魏玉霞; 张显; 胡孟凯; 杨套伟; 徐美娟; 邵明龙
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-02-05
Filing date: 2021-02-05
Publication date: 2023-07-18
Anticipated expiration: 2041-02-05
Also published as: CN112695006A

Abstract

The invention discloses a recombinant bacillus subtilis for expressing D-psicose-3-epimerase, belonging to the technical field of bioengineering. The invention realizes the expression of the D-psicose 3-epimerase obtained from Dorea sp.CAG317 and Clostridium cellulolyticum H by constructing recombinant bacillus subtilis by taking bacillus subtilis 168 as a chassis cell and pMA5 as an expression vector. Through strong constitutive promoter screening, the catalytic efficiency of the recombinant strain is successfully improved to 12-13 times of that of a single-enzyme recombinant strain under weak acid condition, and the recombinant strain B.subtilis 168/pMA5-spovG-DS-srfA-RC can generate 244.3g/L of D-psicose within 30 minutes, the conversion rate is 32.6%, and the browning phenomenon of a reaction solution is eliminated.

Description

Recombinant bacillus subtilis for expressing D-psicose-3-epimerase

Technical Field

The invention relates to a recombinant bacillus subtilis for expressing D-psicose-3-epimerase, belonging to the technical field of bioengineering.

Background

D-psicose (D-allose) is an epimer of D-fructose at the C3 position, and has a melting point of 96 ℃ and high solubility in water: 291g of D-psicose can be dissolved in 100g of water at 25 ℃, the mass percentage is 74% (w/w), and the solution density reaches 1.35kg/L; the solubility at 50 ℃ is further increased, and 489g of D-psicose can be dissolved in 100g of water, and the mass percentage is 83% (w/w). D-psicose is a novel functional monosaccharide with special health care function, and its sweetness phase is discovered in recent yearsWhen the calorie of the food is 70% of the sucrose, the calorie of the food is 0.3% of the sucrose, the food has the taste and volume characteristics similar to those of the sucrose, and the food can also have Maillard reaction with amino acid or protein in the food, so that the food can be used as an ideal substitute of the sucrose in the food. Toxicology experiments demonstrated that half the Lethal Dose (LD) of D-psicose ₅₀ ) LD of 16.3g/kg compared to fructose ₅₀ LD of erythritol at 14.7g/kg ₅₀ At 15.3g/kg, D-psicose belongs to the "relatively harmless" category (lowest toxicity rating) according to toxicity rating. In 2014, the U.S. food and drug administration (U.S. food and Drug Administration, FDA) officially approved D-psicose as generally recognized as safe (Generally Recognized as Safe, GRAS), allowing its use in foods, dietary supplements, and pharmaceutical preparations.

D-psicose has various health care effects, can resist hyperglycemia, prevent or treat obesity and inflammation caused by obesity, and is beneficial to regulating cholesterol metabolism, and the biological effect is exerted by regulating intestinal microbiota. At a maximum dose of 2000mg/kg, D-psicose had no adverse effect on the reproductive toxicity of rats. D-psicose belongs to natural rare sugar, and can be synthesized by a chemical method and a microbial method. Because the chemical method is not friendly to the environment, the biological method for synthesizing the D-psicose is widely focused on green environmental protection; whereas biological processes typically produce D-psicose under the catalysis of D-psicose 3-epimerase or D-tagatose 3-epimerase, non-enzymatic browning is susceptible to occurring during the production process, also known as maillard Maillard reaction reaction.

The maillard reaction can affect the manufacture and storage of many foods, can occur on almost all foods, including the coking of sugars and other carbohydrates, and maillard reactions that occur after heat treatment or storage of carbohydrates and proteins, amino acids, etc., and both of these chemical reactions can produce brown items. Although some foods are purposely prepared using the color produced by the maillard reaction, the maillard reaction in the bioconversion process for preparing D-psicose consumes sugar resulting in the formation of byproducts and darkening of the product color, thereby burdening the downstream purification process.

In addition, in the prior art, D-psicose 3-epimerase is an enzyme capable of maintaining higher enzyme activity under alkaline conditions, and the enzyme activity is insufficient under weak acid conditions; in the previous study, the enzyme activities of D-psicose 3-epimerase DPease derived from Dorea sp.CAG317 and RCDPease derived from Clostridium cellulolyticum H10D-psicose 3-epimerase were compared, respectively, and the results are shown in Table 1:

table 1: enzyme activity of different D-psicose 3-epimerase under different pH conditions

pH	DSDPease relative enzyme Activity (%)	Relative enzyme Activity of RCDPease (%)
			4.5 acetic acid/sodium acetate	0	3.24
5 acetic acid/sodium acetate	12.30902	11.27
			5.5 acetic acid/sodium acetate	29.71851	35.86
6 acetic acid/sodium acetate	56.23458	48
			Disodium hydrogen phosphate/sodium dihydrogen phosphate	20.92	25.7
6.5 disodium hydrogen phosphate/sodium dihydrogen phosphate	31.51	34.24
			Disodium hydrogen phosphate/sodium dihydrogen phosphate	56.36	41.23
7.5 disodium hydrogen phosphate/sodium dihydrogen phosphate	71.78	39.93
			7.5 Tris/HCl	73.06	51.63
8 Tris/hydrochloric acid	82.39	49.93
			8.5 Tris/HCl	100	100
9 Tris/hydrochloric acid	89.99	86.43

It can be seen that the enzyme activity of D-psicose 3-epimerase is low at pH 6-7, whereas in many industrial reactions, pH 6-7 is a common reaction condition where the enzyme activity of D-psicose 3-epimerase is low, and therefore how the enzyme activity of D-psicose 3-epimerase under weak acid conditions can be improved becomes a hot spot for research.

The bacillus subtilis is a GRAS strain approved by the FDA, has clear genetic background, good operability and safety, and is an important industrial production strain. Bacillus subtilis has abundant expression elements and great excavation potential, has various endogenous strong constitutive promoters and self-inducible promoters, and does not need to add extra substances in the culture process to induce protein expression.

Therefore, how to successfully express the D-psicose 3-epimerase by using bacillus subtilis weakens the Maillard reaction between a sugar solution and a biological enzyme in a catalytic reaction, and keeps high enzyme activity under weak acid conditions is a difficult problem in industrial production.

Disclosure of Invention

Technical problems:

the invention aims to provide a recombinant bacillus subtilis capable of successfully expressing D-psicose 3-epimerase which can weaken Maillard effect in the reaction process and can keep higher enzyme activity under weak acid condition and application thereof.

The technical scheme is as follows:

the present invention co-expresses D-psicose 3-epimerase genes (DSDPease and RCDPease) from Dorea sp.CAG317 and Clostridium cellulolyticum H10 in Bacillus subtilis 168; by constructing an expression system of B.subtilis 168/pMA5-DS-RC, inserting the promoters HpaII, P43, srfA and spovG into the 5 'end of the gene segment DSDPease and the 5' end of the gene segment RCDPease respectively, and expressing the recombinant bacteria under corresponding culture conditions.

In order to solve the above problems, the present invention provides a recombinant Bacillus subtilis expressing a D-psicose 3-epimerase derived from Dorea sp.CAG317 and a D-psicose 3-epimerase derived from Clostridium cellulolyticum H.

In one embodiment of the present invention, the recombinant bacillus subtilis is an expression host of bacillus subtilis 168.

In one embodiment of the invention, the recombinant bacillus subtilis is an expression vector of pMA 5.

In one embodiment of the invention, the recombinant bacillus subtilis is enhanced in expression of D-psicose 3-epimerase using one or more of promoters HpaII, P43, srfA, spovG.

In one embodiment of the present invention, promoters HpaII, P43, srfA and spovG are inserted into the 5 'end of the gene fragment DSDPease and the 5' end of the gene fragment RCDPease, respectively, and the recombinant bacteria are expressed under corresponding culture conditions.

In one embodiment of the invention, the amino acid sequence of the D-psicose 3-epimerase derived from Dorea sp.CAG317 is shown in SEQ ID NO 1 and the amino acid sequence of the D-psicose 3-epimerase derived from Clostridium cellulolyticum H is shown in SEQ ID NO 2.

In one embodiment of the invention, the nucleotide sequence of the gene encoding the D-psicose 3-epimerase derived from Dorea sp.CAG317 is shown in SEQ ID NO 7 and the nucleotide sequence of the gene encoding the D-psicose 3-epimerase derived from Clostridium cellulolyticum H is shown in SEQ ID NO 8.

In one embodiment of the invention, the nucleotide sequence of the promoter HpaII is shown as SEQ ID NO 3; the nucleotide sequence of the promoter P43 is shown as SEQ ID NO 4; the nucleotide sequence of the promoter srfA is shown in SEQ ID NO 5; the nucleotide sequence of the promoter spovG is shown in SEQ ID NO 6.

The invention also provides a method for constructing the recombinant bacillus subtilis, which comprises the following steps:

(1) Constructing a recombinant plasmid: d-psicose 3-epimerase DS derived from Dorea sp.CAG317 and D-psicose 3-epimerase RC derived from Clostridium cellulolyticum H are respectively connected with a pMA5 plasmid after enzyme digestion to obtain a recombinant plasmid pMA5-DS-RC;

(2) Constructing recombinant bacillus subtilis: and (3) converting the recombinant plasmid pMA5-DS-RC obtained in the step (1) into bacillus subtilis 168 to obtain recombinant bacillus subtilis.

The invention also provides a method for weakening browning reaction, which is to add the recombinant bacillus subtilis into a reaction system containing D-fructose for reaction.

In one embodiment of the invention, the OD of the recombinant Bacillus subtilis is added ₆₀₀ At least 15.

In one embodiment of the present invention, the reaction is carried out at a pH of 6.0 to 7.0 and a temperature of 55 to 65 ℃.

The invention also provides an application of the recombinant bacillus subtilis in reducing browning reaction in food or in preparing high-sweetness low-calorie food.

The invention also provides the recombinant bacillus subtilis, which has the advantages that the enzyme activity is improved under the acidic condition, and the Maillard reaction browning phenomenon in the bioconversion process of the D-psicose is weakened.

Advantageous effects

(1) The invention successfully introduces D-psicose 3-epimerase genes (DSDPease and RCDPease) derived from Dorea sp.CAG317 and Clostridium cellulolyticum H into bacillus subtilis 168 and coexpresses the genes, and recombinant strains B.sub.168/pMA 5-DS-RC, B.sub.168/pMA 5-DS-HpaII-RC, B.sub.168/pMA 5-DS-P43-RC, B.sub.168/pMA 5-DS-srfA-RC, B.sub.168/pMA 5-DS-spovG-RC, B.sub.168/pMA 5-DS-spP 43-srfA-RC and B.sub.sub.168/pMA 5-DS-srfA-RC are constructed by replacing an endogenous strong promoter.

(2) The recombinant strain B.subtilis 168/pMA5-spovG-DS-srfA-RC with highest whole cell enzyme activity obtained by the invention has no browning reaction under the condition of pH6.5, the enzyme activity is improved by 12-13 times compared with that of single enzyme expression strain, the enzyme activity of the repeated utilization batch for 13 times is still more than 50%, and the problem of insufficient enzyme activity under weak acid condition is solved.

(3) The method provided by the invention can prevent Maillard reaction from releasing melanin when being used for reaction under weak acid condition. Recombinant strain b.subilis 168/pMA5-spovG-DS-srfA-RC produced 244.3g/L of D-psicose within 30 minutes at a substrate fructose concentration of 750g/L at weak acid (ph=6.5), with a conversion of 32.6%.

Drawings

Fig. 1: SDS-PAGE gel electrophoresis of crude enzyme solution; wherein, lane 1: subtilis 168/pMA5 empty control; lane 2: subilis 168/pMA5-DS supernatant; lane 3: subilis 168/pMA5-RC supernatant; lane 4: subilis 168/pMA5-DS-RC supernatant; lane 5: subilis 168/pMA5-DS-HpaII-RC supernatant; lane 6: subilis 168/pMA5-DS-P43-RC supernatant; lane 7: subilis 168/pMA5-DS-srfA-RC supernatant; lane 8: subtilis 168/pMA5-DS-spovG-RC supernatant.

Fig. 2: SDS-PAGE gel electrophoresis of crude enzyme solution; wherein, b.subtilis 168/pMA5 empty control; lane 2: subilis 168/pMA5-DS-srfA-RC supernatant; lane 3: subilis 168/pMA5-spovG-DS-srfA-RC supernatant; lane 4: subtilis 168/pMA5-P43-DS-srfA-RC supernatant.

Fig. 3: influence of different pH on browning of the reaction solution.

Fig. 4: biomass and specific enzyme activity in a continuous fermentation process.

Detailed Description

The following examples relate to the following media:

LB liquid medium: 10g/L peptone, 10g/L sodium chloride and 5g/L yeast powder.

LB solid medium: 10g/L peptone, 10g/L sodium chloride, 5g/L yeast powder and 20g/L agar powder.

Super Rich medium: 25g/L of peptone, 20g/L of yeast powder, 3g/L of dipotassium hydrogen phosphate and 30g/L of glucose.

Seed culture medium: 15g/L sucrose and 20g/L, na yeast extract powder ₂ HPO ₄ ·12H ₂ O 1g/L、Mn ²⁺ 0.05mmol/L, 8g/L sodium chloride.

Fermentation medium: 15g/L of sucrose, 20g/L of yeast powder, 4g/L of sodium dihydrogen phosphate, 1g/L of sodium dihydrogen phosphate and 8g/L of sodium chloride.

The detection method involved in the following examples is as follows:

the method for measuring the enzyme activity of D-psicose comprises the following steps:

(1) Determination of pure enzyme Activity

The cells were collected and incubated with pH 7.0.50 mmol.L ^-1 Na ₂ HPO ₄ -NaH ₂ PO ₄ Suspending with buffer solution, centrifuging, washing for 3 times, re-suspending cells, adding lysozyme with a final concentration of 1mg/mL, performing ultrasonic disruption of cells after a period of time, and centrifuging to obtain a supernatant, namely crude enzyme solution. Purifying by Ni column affinity chromatography to obtain DPease pure enzyme for detecting the enzyme activity of the pure enzyme. Enzyme activity measurement reaction system (overall 1 mL): 900 mu L pH 8.5 50 mmol.L ^-1 Na ₂ HPO ₄ -NaH ₂ PO ₄ Buffer-dissolved 100mg of D-fructose and 2. Mu.L of 70mM Co ²⁺ The solution is placed in a water bath kettle at 60 ℃ and preheated for 5min, then 100 mu L of pure enzyme is added, and 100 mu L of 50 mmol.L is directly added in contrast reaction ^-1 Na ₂ HPO ₄ -NaH ₂ PO ₄ The buffer was reacted in a constant temperature water bath at 60℃for 30min, and the reaction was terminated in a rapid boiling water bath. Placing enzyme reaction solution into a centrifuge at 10000 r.min ^-1 Centrifuging for 10min under the condition of removing protein, collecting supernatant, and diluting the product concentration of the reaction solution to 5g.L ^-1 1mL of the reaction solution is taken for high performance liquid chromatography measurement and analysis.

(2) Crude enzyme activity determination

Measuring the enzyme activity of the crude enzyme solution, reacting in a 2mL EP tube, 1mL of a reaction system with D-fructose content of 300g/L, reacting at 60 ℃, adding 900 mu L of NaH ₂ PO ₄ /Na ₂ HPO ₄ buffer，Co ²⁺ The crude enzyme solution with the final concentration of 140 mu mol/L and 100 mu L is reacted for 2min in a metal oscillator, immediately taken out and put into boiling water to be boiled for 5min to stop the reaction, the protein precipitate is separated from the D-fructose and D-psicose solution by centrifugation at 1200rpm, and the supernatant is put into a refrigerator at the temperature of minus 40 ℃ to be stored.

The product analysis method is consistent with the pure enzyme.

(3) Whole cell enzyme Activity determination

Measuring whole cell enzyme activity, centrifuging 1mL of bacterial liquid, and using NaH with pH of 7.0 ₂ PO ₄ /Na ₂ HPO ₄ buffer wash 3 times, suspend in 100. Mu.L NaH ₂ PO ₄ /Na ₂ HPO ₄ To buffer, 900. Mu.L of 300g/L D-fructose solution was added. And (3) reacting for 5min in the metal oscillation period, immediately taking out the mixture to a refrigerated centrifuge at 4 ℃, centrifuging at 12000rpm for 2min, taking supernatant, and placing the supernatant in a refrigerator at-40 ℃ for preservation, wherein the enzyme activity definition is consistent with that of the crude enzyme liquid. The product analysis method is consistent with the pure enzyme.

50mM Na ₂ HPO ₄ -NaH ₂ PO ₄ (pH 6.5, 7.0 or 8.5) buffer: 7.098g of Na is weighed ₂ HPO ₄ Dissolving in distilled water, and fixing the volume to 1L; 5.999g NaH was weighed out ₂ PO ₄ Dissolving in distilled water, and fixing the volume to 1L; the two buffers were mixed to adjust the pH.

Reaction substrate solution (100 g/L D-fructose): 10g D-fructose was weighed and dissolved in 50mM Na, pH 8.5 ₂ HPO ₄ -NaH ₂ PO ₄ In the buffer solution, the volume is fixed to 100mL, and the buffer solution is preserved in a refrigerator at 4 ℃. HPLC detection conditions: RID detector, mobile phase ultrapure water, chromatographic column Hi-Plex Ca 30X 7.7mm, column temperature 80 ℃, detection temperature 55 ℃ and flow rate 0.4mL/min.

D-psicose 3-epimerase enzyme activity unit: the amount of enzyme required to catalyze the production of 1. Mu. Mol of D-psicose within 1min is defined as one unit of enzyme activity.

Specific enzyme activity of D-psicose 3-epimerase: one unit of enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1. Mu. Mol of D-psicose per minute under assay conditions.

Equilibrium conversion: refers to the fact that when the reversible chemical reaction of converting D-fructose into D-psicose reaches a chemical equilibrium state, the amount of D-fructose converted into D-psicose accounts for a percentage of the initial amount of D-fructose.

Example 1: obtaining recombinant plasmid

(1) Chemically synthesizing a gene DSDPease of encoding the D-psicose 3-epimerase, the nucleotide sequence of which is shown as SEQ ID NO 7, and a gene RCDPease of encoding the D-psicose 3-epimerase, the nucleotide sequence of which is shown as SEQ ID NO 8.

(2) Primers required for fusion PCR of a promoter HpaII (with a nucleotide sequence shown as SEQ ID NO 3), a P43 (with a nucleotide sequence shown as SEQ ID NO 4), an srfA (with a nucleotide sequence shown as SEQ ID NO 5), an spovG (with a nucleotide sequence shown as SEQ ID NO 6) fragment and a DSDPEase, RCDPEase gene fragment are designed, and the used primer sequences are shown as table 2 and different promoter fragments are cloned by taking a bacillus subtilis genome as a gene template.

TABLE 2 primer sequences

(3) PCR reaction conditions: the two fragments are fused with PCR conditions, namely 58 ℃ for 30S,72 ℃ for 90S, and the cycle is 8 times; after the primer is added, continuing PCR for 3min at 95 ℃; cycling for 32 times at 95 ℃ for 30S,58 ℃ for 30S and 72 ℃ for 2 min; 72 ℃ for 10min; and (5) permanently preserving the temperature at 4 ℃.

Inverse PCR of pMA5 plasmid, designing primer pMA5-F ', pMA5-R', taking pMA5 empty as template, and PCR procedure at 95 deg.C for 3min; cycling for 32 times at 95 ℃ for 30S,58 ℃ for 30S and 72 ℃ for 7 min; 72 ℃ for 10min; and (5) permanently preserving the temperature at 4 ℃.

(4) PCR amplified product purification recovery

The PCR amplified products were spotted for viewing the band positions by agarose gel electrophoresis. The desired gel pieces were excised and purified for the target gene strictly according to the procedure of the nucleic acid gel recovery kit.

Through fusion PCR, the first step fuses the Promoter gene segment and the DPease gene segment together, and the fusion products obtained in the first step are added into the system for second fusion to obtain the Promoter1-DSDPease-Promoter2-RCDPease gene segment, and different Promoter combinations are fused by the method for PCR, so as to prepare for the next construction of recombinant plasmids.

(5) Construction of recombinant plasmids

Plasmid extraction was performed according to plasmid extraction kit GENEray biotechnology, then double digestion was performed on expression vector pMA5, the digestion sites were BamHI and MluI, and the digested product was subjected to nucleic acid gel validation and recovery purification, and the recovery step was performed according to gel recovery kit instructions.

And (3) connecting the target gene fragment with the linearized plasmid after enzyme digestion by using homologous recombinase to respectively obtain recombinant plasmids pMA5-DS, pMA5-RC, pMA5-DS-RC, pMA5-DS-HpaII-RC, pMA5-DS-srfA-RC, pMA5-DS-P43-RC, pMA5-DS-spovG-RC, pMA5-P43-DS-srfA-RC and pMA5-spovG-DS-srfA-RC.

Example 2: construction and expression of recombinant bacteria

The method comprises the following specific steps:

(1) Construction of amplified strains:

the recombinant plasmids pMA5-DS, pMA5-RC, pMA5-DS-RC, pMA5-DS-HpaII-RC, pMA5-DS-srfA-RC, pMA5-DS-P43-RC, pMA5-DS-spovG-RC, pMA5-P43-DS-srfA-RC, pMA5-spovG-DS-srfA-RC obtained in example 1 were each introduced into E.coli JM109 to carry out plasmid amplification, and after successful colony PCR, inoculated into 10mL LB solid medium at 37℃180 r.min ^-1 Culturing for 10h, and extracting plasmids according to the bacterial plasmid extraction kit.

(2) Recombinant strain construction:

respectively introducing the amplified plasmids obtained in the step (1) into bacillus subtilis B.subilis 168 by a chemical conversion mode, respectively inoculating the obtained recombinant strains into 10mL of LB solid medium after colony PCR verification, and culturing at 37 ℃ and 180 r.min ^-1 Culturing under conditions for 10 hours to obtain recombinant strains B.sub.168/pMA 5-DS, B.sub.168/pMA 5-RC, B.sub.168/pMA 5-DS-HpaII-RC, B.sub.168/pMA 5-DS-srfA-RC, B.sub.168/pMA 5-DS-P43-RC, B.sub.168/pMA 5-DS-spovG-RC, B.sub.168/pMA 5-P43-DS-srfA-RC, B.sub.168/pMA 5-SPOgG-DS-srfA-RC, B.sub.sub.168/pMA 5-srfA-RC, and preserving the strains at-80 ℃.

(3) Expression of recombinant bacteria and enzyme activity assay:

respectively transferring the bacillus subtilis recombinant strain obtained in the step (2) to 10mL LB liquid mediumIn the middle, at 37 ℃ and 180 r.min ^-1 Culturing for 10h under the condition to obtain seed solution containing recombinant strain.

The seed solutions obtained above were transferred to 100mL of Super Rich medium at an inoculum size of 1% (v/v) and at 37℃180 r.min ^-1 Culturing for 24h under the condition, and expressing D-psicose 3-epimerase to obtain crude enzyme liquid containing the D-psicose 3-epimerase, namely obtaining the product: a DS-RC-containing crude enzyme solution, a DS-containing crude enzyme solution, a RC-containing crude enzyme solution, a DS-HpaII-RC-containing crude enzyme solution, a DS-P43-RC-containing crude enzyme solution, a DS-srfA-RC-containing crude enzyme solution, a pMA 5-DS-spovG-RC-containing crude enzyme solution, a P43-DS-srfA-RC-containing crude enzyme solution, and a spovG-DS-srfA-RC-containing crude enzyme solution.

The obtained crude enzyme solutions containing DS-RC, DS-HpaII-RC, DS-P43-RC, DS-srfA-RC, pMA5-DS-spovG-RC, P43-DS-srfA-RC and spovG-DS-srfA-RC were analyzed by SDS-PAGE gel electrophoresis, and the analysis results were shown in FIG. 1 and FIG. 2.

As can be seen from FIGS. 1 and 2, the recombinant strains B.sub.168/pMA 5-DS-RC, B.sub.168/pMA 5-DS-HpaII-RC, B.sub.168/pMA 5-DS-P43-RC, B.sub.168/pMA 5-DS-srfA-RC, B.sub.168/pMA 5-DS-spovG-RC, B.sub.168/pMA 5-P43-DS-srfA-RC, B.sub.168/pMA 5-spovG-DS-srfA-RC had a distinct band around 33kDa, indicating successful expression of DPEase in the recombinant strains.

Example 3: specific enzyme activity of recombinase under different pH conditions

The method comprises the following specific steps:

(1) Phosphate buffer solutions with pH of 6.5,pH 7.0,pH 8.5 are respectively prepared;

(2) D-fructose is respectively added into the phosphate buffer solution obtained in the step (1) to respectively obtain D-fructose solutions with the concentration of 300 g/L;

(3) To the D-fructose solution obtained in the step (2), 100. Mu.L of a DS-RC-containing crude enzyme solution, a DS-containing crude enzyme solution, a RC-containing crude enzyme solution, and a DS-HpaII-RC-containing crude enzyme solution were added, respectively. According to the method for measuring the enzyme activity of the crude enzyme liquid, the specific enzyme activities of different DPease crude enzyme liquids under different pH conditions are measured, the DSDPEase, RCDPEase single enzyme expression and the co-expression are compared in experiments, and the results are shown in Table 3. And (3) injection: the crude enzyme solution is prepared by crushing the same cell concentration.

Table 3: specific enzyme activity (U/mL) of single-enzyme and double-enzyme expression recombinant bacterium crude enzyme solution in phosphate buffer solutions with different pH values

pH	6.5	7.0	8.5
				B.subtilis 168/pMA5-DS	17.22	17.43	18.7
B.subtilis 168/pMA5-RC	16.71	16.6	17.23
				B.subtilis 168/pMA5-DS-RC	18.65	18.76	18.85

The specific enzyme activity of the recombinase was found to be 18.65U/mL at pH 6.5.

Definition of enzyme activity: the amount of enzyme required to convert 1. Mu. Mole of D-fructose in 1 minute was one activity unit U.

Specific enzyme activity definition: crude enzyme obtained per milliliter of bacterial liquid/enzyme activity possessed by each milliliter of bacterial liquid.

(3) D-fructose is added into the phosphate buffer solution with the pH of 6.5 obtained in the step (1) to obtain a D-fructose solution with the concentration of 300 g/L; to the obtained D-fructose solution, 100. Mu.L of a crude enzyme solution containing DS-P43-RC, a crude enzyme solution containing DS-srfA-RC, a crude enzyme solution containing pMA5-DS-srfA-RC, a crude enzyme solution containing P43-DS-srfA-RC, and a crude enzyme solution containing spofG-DS-srfA-RC were added, and the specific enzyme activities of the respective DPease crude enzyme solutions at pH of 6.5 were measured by the crude enzyme activity measurement method, and the results are shown in Table 4. And (3) injection: the crude enzyme solution is prepared by crushing the same cell concentration.

Table 4: enzyme activity of different recombinant bacterium crude enzyme solutions in phosphate buffer solution with pH value of 6.5

Through promoter screening, the specific enzyme activity of the recombinant enzyme prepared by adopting the recombinant strain B.subtilis 168/pMA5-spovG-DS-srfA-RC under the condition of pH6.5 is 228.5U/mL, and the catalytic efficiency is improved by 12-13 times compared with that of the recombinant strains B.subtilis 168/pMA5-DS and B.subtilis 168/pMA 5-RC.

Further, experiments with recombinant strain B.subilis 168/pMA5-spovG-DS-srfA-RC of example 3 were repeated 13 times, and the results are shown in Table 5; the enzyme activity after repeated 13 times is still more than 50%, and the problem of insufficient enzyme activity under weak acid condition is solved.

Table 5: balanced conversion rate and residual enzyme activity of different batches

Example 4: preparation of D-psicose by recombinant bacteria

D-psicose was synthesized by transforming different concentrations of D-fructose with B.subtilis 168/pMA5-spovG-DS-srfA-RC recombinant strain.

The method comprises the following specific steps:

(1) The B.subtilis 168/pMA5-spovG-DS-srfA-RC recombinant strain prepared in example 2 was transferred to LB solid medium containing kanamycin for activation, and placed in a 37 ℃ biochemical incubator for culturing for 12 hours, to obtain single colonies.

(2) The single colony after activation was picked and cultured in 10mL of LB liquid medium containing 50. Mu.g/mL of kanamycin for 12 hours, transferred to 500mL of Soper Rich medium containing 50. Mu.g/mL of kanamycin at an inoculum size of 2% (v/v), cultured at 220rpm and 37℃for 24 hours, and cells were collected separately using a high-speed refrigerated centrifuge.

(3) Preparing 300g/L,500g/L and 750g/L of D-fructose solution respectively by adopting phosphate buffer solution with pH of 6.5, wherein the pH of the solution is 6.5, and placing the solution in a 250mL triangular flask; heating D-fructose solutions with different concentrations on a magnetic stirrer, adding the cells collected in the step (2) when the temperature is constant, wherein the concentration of the cells in the reaction system of each concentration gradient is OD ₆₀₀ =15, temperature 60 ℃, rotation speed 200 rpm;

1mL of the reaction solution was taken from the different reaction systems every 10 minutes, immediately centrifuged at a high speed in a refrigerated centrifuge at 4℃for 14000r/min and centrifuged for 3min, and the cells were isolated to terminate the reaction. The supernatant was obtained and analyzed, and the results are shown in Table 6, and the detection method is as follows: agilent 1260 high performance liquid chromatograph, differential detector, detection temperature 55 ℃; hi Plex Ca ion exchange column, mobile phase ultrapure water, flow rate 0.4mL/min; single sample run time 40min.

Table 6: different substrate concentrations at different times produce D-psicose concentration (g/L)

As previously described herein, reaction under weakly acidic conditions prevents the Maillard reaction from liberating melanin. From the data in Table 6, it is seen that the equilibrium conversion was almost 33% under weak acid (pH=6.5) conditions using a fructose solution at a concentration of 500g/L and 750 g/L. At a fructose concentration of 750g/L, recombinant strain B.subtilis 168/pMA5-spovG-DS-srfA-RC produced 244.3g/L of D-psicose within 30 minutes with a conversion of 32.6%.

Example 5: the specific enzyme activity of recombinant bacteria B.subtilis 168/pMA5-spovG-DS-srfA-RC at the level of a fermentation tank comprises the following specific steps:

(1) Preparing seed liquid: the B.subtilis 168/pMA5-spovG-DS-srfA-RC strain prepared in example 2 was streaked on LB solid plate medium and cultured in a biochemical incubator at 37℃for 12 hours. Single colonies are picked and transferred to 10mL of LB liquid medium, placed on a shaking table at 37 ℃ for culturing at 180rpm for 12 hours, then transferred to 100mL of seed medium (15 g/L of sucrose, 20g/L, na2 HPO4.12H2O 1g/L, mn2+0.05mmol/L and 8g/L of sodium chloride) according to the inoculum size of 4% (v/v), and continuously placed on the shaking table at 37 ℃ for culturing at 180rpm for 16 hours to obtain seed liquid.

(2) Inoculating the seed solution obtained in the step (1) into a 5L bioreactor containing 2L fermentation medium (the same formula as the seed solution) with an inoculum size of 4% (v/v) for fed-batch fermentation. The culture conditions of the fermentation tank are as follows: the temperature is 37 ℃; the rotating speed is coupled with DO to ensure that DO is controlled at 30%; and the pH value is 7.0, and the feed (sucrose 300g/L and yeast powder 70 g/L) is slightly acidic, so that the acid pump is connected with the acid pump instead of acid, namely when the pH value in the fermentation tank is more than 7.0, the acid pump automatically adds a proper amount of feed to restore the pH value to 7.0. And (5) continuously fermenting for 60 hours, collecting samples with different fermentation times, and freezing and preserving.

As a result, as shown in FIG. 4, the biomass increased rapidly in the first 6 hours; the pH value is increased due to the sucrose depletion, the feeding material (matched with an acid pump) starts to flow, and the cell density is 2-3 OD ₆₀₀ The speed per hour is increased, the cell density is increased to 98.8U/L for 30 hours, the enzyme activity is 387782U/L, the bacterial count is not increased after 36 hours enter a stable period, the nutrient substances are used for protein expression, and the enzyme activity starts to be improved.

OD ₆₀₀ The cell density of (C) was increased to 108.4 at 42 hours, and the enzyme activity was 480098U/L, and the results are shown in Table 7.

This result is about an 11-fold increase in the enzyme activity over the previous recombinant Gluconobacter expression of D-psicose 3-epimerase (see in particular Yang J, tian C, zhang T, ren C, zhu Y, et al (2019) Development of food-grade expression system for D-allose 3-epimerase preparation with tandem isoenzyme genes in Corynebacterium glutamicum and its application in conversion of cane molasses to D-allose. Biotechnol Bioeng 116:745-756). The specific activities were all determined by catalyzing 300g/L D-fructose to synthesize D-psicose in phosphate buffer with pH of 6.5.

In addition, strain B.subtilis 168/pMA5-spovG-DS-srfA-RC, the promoter contained by strain B.subtilis 168/pMA5-spovG-DS-srfA-RC was constitutive, and no inducer was required during fermentation.

Table 7: subtilis 168/pMA5-spovG-DS-srfA-RC fermentation culture

Example 6: recombinant strains attenuate browning reactions

The invention researches the influence on the browning of the reaction liquid under the condition of different pH values (6.5-8.5) when the crude enzyme liquid and the whole cell catalyzed D-fructose are reacted at 60 ℃ to generate the D-psicose reaction liquid.

The method comprises the following specific steps:

(2) D-fructose is respectively added into the phosphate buffer solution obtained in the step (1) to respectively obtain D-fructose solutions with the concentration of 300 g/L; the D-fructose solution was packed into 10 tubes of 900. Mu.L each, and the tube body was marked with empty control, 6.5-DS,6.5-RC,6.5-DS-RC,7.0-DS,7.0-RC,7.0-DS-RC,8.5-DS,8.5-RC,8.5-DS-RC, respectively.

(3) Crude enzyme solutions (OD in each case) prepared from B.sub.168/pMA 5-DS, B.sub.168/pMA 5-RC and B.sub.168/pMA 5-DS-RC strains were prepared, respectively ₆₀₀ Whole cell disruption of =60), 100 μl of each was added to each tube of fructose solution dispensed in step (2), and stored at 60 ℃ for 30min.

The sugar solution reacts with amino groups in the protein, and the Maillard reaction releases melanin. Studies have reported that at 420nm, the absorbance of the sample solution is proportional to the melanin concentration, and the results are shown in table 8.

Table 8: browning degree OD of different crude enzyme solutions at the same pH ₄₂₀

Note that: OD (optical density) ₄₂₀ The magnitude of the value is influenced by a number of factors, of which pH and temperature are the main factors.

As is clear from example 3, the co-expression strain B.subilis 168/pMA5-DS-RC has high enzyme activity, and the color of the reaction solution changes with the change of pH and OD ₄₂₀ The comparison is shown in FIG. 3, where the degree of browning is positively correlated with pH.

The key to solving the browning problem is to lower the pH of the reaction solution, and it is understood from example 4 that the expression of DPease enzyme by the single enzyme cannot meet this requirement. Therefore, the invention improves the enzyme activity of whole cells under weak acid condition and can effectively solve the problem.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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

<110> university of Jiangnan

<120> recombinant Bacillus subtilis expressing D-psicose-3-epimerase

<130> BAA201691A

<160> 8

<170> PatentIn version 3.3

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Claims

1. A recombinant bacillus subtilis is characterized by comprising the following componentsBacillus 168 is used as expression host, pMA5 is used as expression vector to express the gene with amino acid sequence shown as SEQ ID NO. 1Dorea sp.The CAG 317D-psicose 3-epimerase and the amino acid sequence of which are shown in SEQ ID NO. 2 are derived fromClostridium cellulolyticumDpsicose 3-epimerase of H10; meanwhile, the promoter spovG is adopted to strengthen expressionDorea sp.D-psicose 3-epimerase of CAG317 and enhanced expression using srfA promoterClostridium cellulolyticumDpsicose 3-epimerase of H10; the nucleotide sequence of the promoter srfA is shown as SEQ ID NO. 5; the nucleotide sequence of the promoter spovG is shown in SEQ ID No. 6.

2. A method of constructing the recombinant bacillus subtilis of claim 1, comprising the steps of:

(1) Constructing a recombinant plasmid: will comprise the promoter spovG and be derived fromDorea sp.Fragments of the D-psicose 3-epimerase DS of CAG317, comprising the promoter srfA and being derived fromClostridium cellulolyticumThe fragment of the D-psicose 3-epimerase RC of H10 is connected with the pMA5 plasmid after enzyme digestion to obtain a recombinant plasmid pMA5-DS-RC;

3. A method for reducing browning reaction, comprising adding the recombinant Bacillus subtilis according to claim 1 to a reaction system containing D-fructose for reaction.

4. The method of claim 3, wherein the OD of the recombinant bacillus subtilis is added ₆₀₀ At least 15.

5. The method of claim 3 or 4, wherein the reaction conditions are: the pH is 6.0-7.0, and the temperature is 55-65 ℃.

6. The use of the recombinant bacillus subtilis of claim 1 for reducing browning reactions during food processing or for preparing D-psicose.