CN112695006A

CN112695006A - Recombinant bacillus subtilis for expressing D-psicose-3-epimerase

Info

Publication number: CN112695006A
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: 2021-04-23
Anticipated expiration: 2041-02-05
Also published as: CN112695006B

Abstract

The invention discloses a recombinant bacillus subtilis for expressing D-psicose-3-epimerase, belonging to the technical field of bioengineering. The D-psicose 3-epimerase is obtained from Dorea sp.CAG317 and Clostridium cellulolyticum H10, and the recombinant Bacillus subtilis is constructed by taking Bacillus subtilis 168 as a chassis cell and pMA5 as an expression vector, so that the expression of the D-psicose 3-epimerase is realized. Through screening of a strong constitutive promoter, the catalytic efficiency of the recombinant strain is successfully improved to 12-13 times that of a single-enzyme recombinant strain under a weak acid condition, 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 recombinant bacillus subtilis for expressing D-psicose-3-epimerase, and belongs to the technical field of bioengineering.

Background

D-psicose (D-allulose) is an epimer of D-fructose at the C3 position, has a melting point of 96 ℃, and has high solubility in water: 291g of D-psicose can be dissolved in 100g of water at 25 ℃, the mass percent is 74% (w/w), and the solution density reaches 1.35 kg/L; the solubility is further increased at 50 ℃, 489g of D-psicose can be dissolved in each 100g of water, and the mass percent is 83% (w/w). D-psicose is a novel functional monosaccharide with special health care function discovered in recent years, the sweetness of the D-psicose is equal to 70% of that of cane sugar, the calorie of the D-psicose is equal to 0.3% of that of cane sugar, the D-psicose has similar taste and volume characteristics with the cane sugar, and the D-psicose can also generate Maillard reaction with amino acid or protein in food and can be used as an ideal substitute of the cane sugar in food. Toxicological experiments confirmed that D-psicose has a median Lethal Dose (LD)₅₀) 16.3g/kg, LD compared to fructose₅₀14.7g/kg, LD of erythritol₅₀At 15.3g/kg, D-psicose belongs to the "relatively harmless" category (lowest toxicity rating) according to toxicity rating. In 2014, D-psicose was officially approved by the U.S. Food and Drug Administration (FDA) as Generally Recognized As Safe (GRAS) allowing its application in food, dietary supplements, and pharmaceutical preparations.

D-psicose has various health-care effects, and can be used for resisting hyperglycemia, preventing or treating obesity and inflammation caused by obesity, advantageously regulating cholesterol metabolism, and exerting its biological effect by regulating intestinal microbiota. At a maximum dose of 2000mg/kg, D-psicose had no adverse effect on reproductive toxicity in rats. D-psicose belongs to a rare sugar in the natural world and can be synthesized by a chemical method and a microbiological method. Because the chemical method is not friendly to the environment, the biological method for synthesizing the D-psicose is widely concerned about the environmental protection; the biological method is generally used for preparing D-psicose under the catalysis of D-psicose 3-epimerase or D-tagatose 3-epimerase, but non-enzymatic browning is easy to occur in the preparation process, and is also called Maillard reaction.

Maillard reactions can affect the manufacture and storage of many food products and can occur in almost all food products, including the scorching effects of sugars and other carbohydrates, and maillard reactions that occur after carbohydrates, proteins, amino acids, etc. have been heat treated or stored, and both chemical reactions can produce a brownish color. Although some foods are intentionally prepared by utilizing the color produced by the Maillard reaction, the Maillard reaction in the case of D-psicose preparation by biotransformation consumes sugars to produce by-products and blackens the color of the product, thus burdening downstream purification processes.

In addition, in the prior art, the D-psicose 3-epimerase is an enzyme which can keep higher enzyme activity under an alkaline condition, and the enzyme activity is insufficient under a weak acid condition; in the previous studies, the inventors compared the activities of D-psicose 3-epimerase DPEase derived from Dorea sp.CAG317 and Clostridium cellulolyticum H10D-psicose 3-epimerase RCDPEase under different pH conditions, and the results are shown in Table 1:

table 1: enzyme activities of different D-psicose 3-epimerases under different pH conditions

pH	Relative enzyme activity of DSDPEase (%)	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
			6 disodium hydrogen phosphate/sodium dihydrogen phosphate	20.92	25.7
6.5 disodium hydrogen phosphate/sodium dihydrogen phosphate	31.51	34.24
			7 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

As can be seen, the enzyme activity of the D-psicose 3-epimerase is low at pH 6-7, while in many industrial reactions, pH 6-7 is a common reaction condition, and the enzyme activity of the D-psicose 3-epimerase is low at this time, so how to improve the enzyme activity of the D-psicose 3-epimerase under weak acid conditions becomes a hot point of research.

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

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

Disclosure of Invention

The technical problem is as follows:

the technical problem to be solved by the invention is to provide the recombinant bacillus subtilis of the D-psicose 3-epimerase which can successfully express and weaken the Maillard effect in the reaction process and can keep higher enzyme activity under the weak acid condition and the application thereof.

The technical scheme is as follows:

the D-psicose 3-epimerase genes (DSDPEase and RCDPEase) of a source Dorea sp.CAG317 and Clostridium cellulolyticum H10 are co-expressed in the bacillus subtilis 168; the promoter HpaII, the promoter P43, the promoter srfA and the promoter spovG are respectively inserted into the end of a gene segment DSDPEase5 'and the end of a gene segment RCDPEase 5' by constructing an expression system B.subtilis 168/pMA5-DS-RC, and the recombinant bacteria are expressed under corresponding culture conditions.

In order to solve the above problems, the present invention provides a recombinant Bacillus subtilis expressing D-psicose 3-epimerase derived from Dorea sp.CAG317 and D-psicose 3-epimerase derived from Clostridium cellulolyticum H10.

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

In one embodiment of the present invention, the recombinant bacillus subtilis is a pMA5 expression vector.

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

In one embodiment of the invention, the promoters HpaII, P43, srfA and spovG are inserted into the ends of the gene segment DSDPEase5 'and the gene segment RCDPEase 5', 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 H10 is shown in SEQ ID NO 2.

In one embodiment of the invention, the nucleotide sequence of the gene encoding D-psicose 3-epimerase derived from Dorea sp.CAG317 is shown in SEQ ID NO 7, and the nucleotide sequence of the gene encoding D-psicose 3-epimerase derived from Clostridium cellulolyticum H10 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 as SEQ ID NO 5; the nucleotide sequence of the promoter spovG is shown as 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: respectively carrying out enzyme digestion on D-psicose 3-epimerase DS derived from Dorea sp.CAG317 and D-psicose 3-epimerase RC derived from Clostridium cellulolyticum H10 and a pMA5 plasmid, and then connecting to obtain a recombinant plasmid pMA 5-DS-RC;

(2) constructing recombinant bacillus subtilis: and (2) transforming the recombinant plasmid pMA5-DS-RC obtained in the step (1) into the bacillus subtilis 168 to obtain the 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 present invention, OD of the recombinant Bacillus subtilis is added₆₀₀At least 15.

In one embodiment of the 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 application of the recombinant bacillus subtilis in weakening browning reaction in food or application in preparing high-sweetness low-calorie food.

The invention also provides the recombinant bacillus subtilis with improved enzyme activity under acidic conditions, and the Maillard reaction browning phenomenon in the D-psicose biotransformation process is weakened.

Advantageous effects

(1) The recombinant strains B.subtilis 168/pMA5-DS-RC, B.subtilis 168/pMA5-DS-srfA-RC, B.subtilis 168/pMA5-DS-HpaII-RC, B.subtilis 168/pMA5-DS-P43-RC, B.subtilis 168/pMA5-DS-srfA-RC, B.subtilis 168/pMA 567-DS-spovG-RC, B.subtilis 168/pMA5-P43-DS-srfA-RC, B.subtilis 168/pMA 5-DS-srfG-RC are constructed by successfully introducing D-psicose 3-epimerase genes (DSDPEase and RCDPEase) from Dorea sp 317 and Clostridium cellulolyticum H10 and co-expressing the recombinant strains.

(2) The recombinant strain B.subtilis 168/pMA5-spovG-DS-srfA-RC with the highest whole-cell enzyme activity obtained by the method is reacted under the condition of pH6.5 without browning, the enzyme activity is improved by 12-13 times compared with that of a single enzyme expression strain, the enzyme activity is still more than 50 percent by repeatedly utilizing batches of 13 times, and the problem of insufficient enzyme activity under the weak acid condition is solved.

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

Drawings

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

FIG. 2: SDS-PAGE gel electrophoresis of the crude enzyme solution; subtilis 168/pMA5 empty control; lane 2: subtilis 168/pMA5-DS-srfA-RC supernatant; lane 3: sublis 168/pMA5-spovG-DS-srfA-RC supernatant; lane 4: sublis 168/pMA5-P43-DS-srfA-RC supernatant.

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

FIG. 4: the biomass and specific enzyme activity in the continuous fermentation process are changed.

Detailed Description

The media involved in the following examples are as follows:

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

LB solid medium: 10g/L of peptone, 10g/L of sodium chloride, 5g/L of yeast powder and 20g/L of 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 of sucrose and 20g/L, Na of yeast extract powder₂HPO₄·12H₂O 1g/L、Mn²⁺0.05mmol/L and 8g/L of 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 methods referred to in the following examples are as follows:

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

(1) determination of pure enzyme activity

Collecting cells, and treating the cells with pH 7.050 mmol. multidot.L^-1Na₂HPO₄-NaH₂PO₄Suspending with buffer solution, centrifuging, washing for 3 times, resuspending cells, adding lysozyme with final concentration of 1mg/mL, acting for a period of time, ultrasonically breaking cells, and centrifuging to obtain supernatant, i.e. crude enzyme solution. The DPEase pure enzyme is obtained by Ni column affinity chromatography purification and is used for detecting the activity of the pure enzyme. Enzyme activity assay reaction system (total system 1 mL): 900 μ L of pH 8.550 mmol. multidot.L^-1Na₂HPO₄-NaH₂PO₄Buffer-dissolved 100mg D-fructose and 2. mu.L 70mM Co²⁺Preheating the solution in 60 deg.C water bath for 5min, adding 100 μ L pure enzyme, and directly adding 100 μ L50 mmol/L for contrast reaction^-1Na₂HPO₄-NaH₂PO₄And (3) reacting the buffer solution in a constant-temperature water bath at 60 ℃ for 30min, and quickly terminating the reaction in a boiling water bath.Placing the enzyme reaction solution into a centrifuge at 10000 r.min^-1Centrifuging for 10min under the condition of (1), removing protein, precipitating, collecting supernatant, and diluting the product concentration of the reaction solution to 5 g.L^-1And taking 1mL of reaction solution to perform high performance liquid chromatography determination analysis.

(2) Crude enzyme activity assay

Measuring the activity of the crude enzyme liquid, reacting in a 2mL EP tube, wherein the reaction system is 1mL, the D-fructose content is 300g/L, the reaction temperature is 60 ℃, and 900 mu L of NaH is added₂PO₄/Na₂HPO₄ buffer，Co²⁺Reacting 100 μ L of crude enzyme solution with final concentration of 140 μmol/L in a metal oscillator for 2min, immediately taking out, putting into boiling water, boiling for 5min to terminate the reaction, centrifuging at 1200rpm to separate protein precipitate from D-fructose and D-psicose solution, putting the supernatant into a refrigerator at-40 deg.C, and storing.

The product analysis method was consistent with that of pure enzyme.

(3) Whole cell enzyme activity assay

Measuring the activity of the whole cell enzyme, centrifuging 1mL of bacterial liquid, and using NaH with pH 7.0₂PO₄/Na₂HPO₄buffer 3 times, suspend in 100. mu.L NaH₂PO₄/Na₂HPO₄To the buffer, 900. mu.L of 300g/L D-fructose solution was added. Reacting for 5min in a metal shaking period, immediately taking out to 4 ℃ refrigerated centrifuge, centrifuging at 12000rpm for 2min, taking supernatant, placing in a refrigerator at-40 ℃ and preserving, wherein the enzyme activity definition is consistent with that of the crude enzyme solution. The product analysis method was consistent with that of pure enzyme.

50mM Na₂HPO₄-NaH₂PO₄(pH 6.5, 7.0 or 8.5) buffer: 7.098g of Na were weighed₂HPO₄Dissolving in distilled water, and diluting to 1L; 5.999g NaH were weighed₂PO₄Dissolving in distilled water, and diluting to 1L; the two buffers were mixed to adjust the pH.

Reaction substrate solution (100g/L D-fructose): 10g D-fructose was weighed out and dissolved in 50mM Na pH 8.5₂HPO₄-NaH₂PO₄The volume of the buffer solution is 100mL, and the solution is stored in a refrigerator at 4 ℃. HPLC detection conditions: RID detector, mobile phase ultrapure water, chromatographic column Hi-Plex Ca30X 7.7mm, column temperature 80 ℃, detection temperature 55 ℃ and flow rate 0.4 mL/min.

D-psicose 3-epimerase enzyme activity unit: the amount of enzyme required to catalyze the formation of 1. mu. mol of D-psicose within 1min was defined as one unit of enzyme activity.

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

Equilibrium conversion: means 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 is a percentage of the initial amount of D-fructose.

Example 1: obtaining of recombinant plasmid

(1) A gene DSDPEase which has a nucleotide sequence shown as SEQ ID NO 7 and codes D-psicose 3-epimerase and a gene RCDPEase which has a nucleotide sequence shown as SEQ ID NO 8 and codes D-psicose 3-epimerase are synthesized chemically.

(2) Designing primers required by fusion PCR of promoter HpaII (nucleotide sequence is shown as SEQ ID NO 3), P43 (nucleotide sequence is shown as SEQ ID NO 4), srfA (nucleotide sequence is shown as SEQ ID NO 5) and spovG (nucleotide sequence is shown as SEQ ID NO 6) fragments, DSDPEase and RCDPEase gene fragments, wherein the sequences of the used primers are shown as table 2, and cloning different promoter fragments by taking a bacillus subtilis genome as a gene template.

TABLE 2 primer sequences

(3) And (3) PCR reaction conditions: the two segments are fused under PCR conditions, 30S at 58 ℃ and 90S at 72 ℃ and are circulated for 8 times; after the primer is added, continuing PCR at 95 ℃ for 3 min; circulating for 32 times at 95 ℃ for 30S, 58 ℃ for 30S and 72 ℃ for 2 min; 10min at 72 ℃; keeping the temperature permanently at 4 ℃.

pMA5 plasmid reverse PCR, design primer pMA5-F ', pMA5-R', use pMA5 empty as template, PCR program is 95 degrees C3 min; circulating for 32 times at 95 ℃ for 30S, 58 ℃ for 30S and 72 ℃ for 7 min; 10min at 72 ℃; keeping the temperature permanently at 4 ℃.

(4) Purification and recovery of PCR amplified products

The PCR amplification products were spotted on a nucleic acid gel agarose gel electrophoresis to view the position of the bands. The desired gel pieces were excised and the target gene was purified strictly according to the instructions of the kit for recovery of nucleic acid gel.

Through fusion PCR, after a Promoter gene fragment and a DPEase gene fragment are fused together in the first step, fusion products obtained in the first step are added into a system for the second step of fusion to obtain a Promoter1-DSDPEase-Promoter2-RCDPEase gene fragment, and fusion PCR is carried out on different Promoter combinations by the method to prepare for constructing recombinant plasmids in the next step.

(5) Construction of recombinant plasmid

Carrying out plasmid extraction according to a plasmid extraction kit GENERAY biotechnology, carrying out double enzyme digestion on an expression vector pMA5, carrying out nucleic acid gel verification on a product obtained after enzyme digestion, recovering and purifying, and carrying out a recovery step according to the instructions of a gel recovery kit.

The target gene fragment is connected 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 pMA 5-spovG-DS-srfA-RC.

Example 2: construction and expression of recombinant bacteria

The method comprises the following specific steps:

(1) construction of the 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 introduced into E.coli JM109 for plasmid amplification by chemical transformation, and inoculated to 10mL of solid LBIn the culture medium, the temperature is 37 ℃, and the temperature is 180 r.min^-1Culturing for 10h, and extracting plasmids respectively according to the bacterial plasmid extraction kit.

(2) Constructing a recombinant strain:

respectively introducing the amplified plasmids obtained in the step (1) into bacillus subtilis 168 in a chemical conversion mode, respectively inoculating the obtained recombinant strains into 10mL of LB solid culture medium after colony PCR verification, and respectively inoculating at 37 ℃ for 180 r.min^-1Culturing for 10h under the condition to obtain recombinant strains B.subtilis 168/pMA5-DS, B.subtilis 168/pMA5-RC, B.subtilis 168/pMA5-DS-RC, B.subtilis 168/pMA5-DS-HpaII-RC, B.subtilis 168/pMA5-DS-srfA-RC, B.subtilis 168/pMA5-DS-P43-RC, B.subtilis 168/pMA5-DS-spovG-RC, B.subtilis 168/pMA5-P43-DS-srfA-RC, B.subtilis 168/pMA5-spovG-DS-srfA-RC and strain preservation at-80 ℃.

(3) Expression and enzyme activity determination of the recombinant bacteria:

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

The seed solutions obtained above were each transferred to 100mL of Super Rich medium at an inoculum size of 1% (v/v), and the mixture was incubated at 37 ℃ for 180 r.min^-1Culturing for 24h under the condition, expressing the D-psicose 3-epimerase to respectively obtain crude enzyme liquid containing the D-psicose 3-epimerase, and obtaining the following products: a DS-RC-containing crude enzyme solution, a DS-containing crude enzyme solution, an 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 DS-RC-containing crude enzyme solution, DS-RC-containing crude enzyme solution, DS-HpaII-RC-containing crude enzyme solution, DS-P43-RC-containing crude enzyme solution, DS-srfA-RC-containing crude enzyme solution, pMA 5-DS-spovG-RC-containing crude enzyme solution, P43-DS-srfA-RC-containing crude enzyme solution, and spovG-DS-srfA-RC-containing crude enzyme solution were subjected to SDS-PAGE gel electrophoresis analysis, and the results of the analysis are shown in FIGS. 1 and 2.

As can be seen from FIGS. 1 and 2, the recombinant strains B.subtilis 168/pMA5-DS-RC, B.subtilis 168/pMA5-DS-HpaII-RC, B.subtilis 168/pMA5-DS-P43-RC, B.subtilis 168/pMA5-DS-srfA-RC, B.subtilis 168/pMA5-DS-spovG-RC, B.subtilis 168/pMA5-P43-DS-srfA-RC, and B.subtilis 168/pMA5-spovG-DS-srfA-RC all have obvious bands near 33, indicating that DPEase in the recombinant strains can be successfully expressed.

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

The method comprises the following specific steps:

(1) respectively preparing phosphate buffer solutions with pH of 6.5, pH of 7.0 and pH of 8.5;

(2) respectively adding D-fructose 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 step (2), 100. mu.L of a DS-RC-containing crude enzyme solution, a DS-containing crude enzyme solution, an 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 solution, the specific enzyme activities of different DPEase crude enzyme solutions under different pH conditions are measured, the single enzyme expression and the coexpression of the DSDPEase and the RCDPEase are compared in the experiment, and the results are shown in Table 3. Note: the crude enzyme solution is prepared by crushing the same cells at the same concentration.

Table 3: specific enzyme activity (U/mL) of crude enzyme liquid of recombinant bacteria expressed by single enzyme and double enzymes 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 is 18.65U/mL under the condition of pH 6.5.

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

Specific enzyme activity definition: crude enzyme solution per ml of bacterial solution/enzyme activity per ml of bacterial solution.

(3) Adding D-fructose 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-spovG-RC, a crude enzyme solution containing P43-DS-srfA-RC, and a crude enzyme solution containing spovG-DS-srfA-RC were added, and the specific enzyme activities of the different DPEase crude enzyme solutions were measured at pH6.5 according to the crude enzyme solution enzyme activity measurement method, and the results are shown in Table 4. Note: the crude enzyme solution is prepared by crushing the same cells at the same concentration.

Table 4: enzyme activity of different recombinant bacteria crude enzyme liquid in phosphate buffer solution with pH of 6.5

Through promoter screening, the specific enzyme activity of the recombinase prepared by 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 strain B.subtilis 168/pMA5-DS and B.subtilis 168/pMA 5-RC.

Also, the experiment of the recombinant strain B.subtilis 168/pMA5-spovG-DS-srfA-RC in example 3 was repeated for 13 times, and the results are shown in Table 5; the enzyme activity after repeating the batch for 13 times is still more than 50 percent, and the problem of insufficient enzyme activity under the weak acid condition is solved.

Table 5: different batches balance conversion rate and residual enzyme activity

Example 4: preparation of D-psicose by recombinant bacteria

And D-psicose is synthesized by transforming D-fructose with different concentrations by using 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 cultured in a biochemical incubator at 37 ℃ for 12 hours to obtain a single colony.

(2) The activated single colonies were picked up and cultured in 10mL of LB liquid medium containing 50. mu.g/mL of kanamycin for 12 hours, transferred to 500mL of Ulper 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 the cells were collected using a high-speed refrigerated centrifuge.

(3) Respectively preparing 300g/L, 500g/L and 750g/L D-fructose solutions by adopting a phosphate buffer solution with the pH of 6.5, and dissolvingThe pH of the solution is 6.5, and the solution is placed in a 250mL triangular flask; d-fructose solutions with different concentrations are placed on a magnetic stirrer to be heated, the cells collected in the step (2) are added when the temperature is constant, and the concentration of the cells in each reaction system with concentration gradient is OD₆₀₀15 ℃ at 60 ℃ and at a rotation speed of 200 rpm;

taking 1mL of reaction solution from different reaction systems every 10 minutes, immediately carrying out high-speed centrifugation at 14000r/min by a refrigerated centrifuge at 4 ℃, centrifuging for 3min, and separating cells to stop the reaction. The obtained supernatant was analyzed and the results are shown in table 6, the detection method is as follows: an Agilent 1260 high performance liquid chromatograph and a differential detector, wherein the detection temperature is 55 ℃; hi Plex Ca ion exchange column, mobile phase ultrapure water, flow rate of 0.4 mL/min; individual samples run for 40 min.

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

As previously described herein, under mildly acidic conditions, the release of melanin by the maillard reaction is prevented. From the data in table 6, it is clear that the equilibrium conversion using a fructose solution with a concentration of 500g/L and 750g/L is almost 33% under weak acid (pH 6.5). At a fructose concentration of 750g/L, the recombinant strain B.subtilis 168/pMA5-spovG-DS-srfA-RC produced 244.3g/L of D-psicose in 30 minutes with a conversion of 32.6%.

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

(1) preparing a seed solution: 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. A single colony is picked up and transferred into 10mL LB liquid culture medium, and is cultured for 12H at 37 ℃ in a shaking table at 180rpm, then is transferred into 100mL seed culture medium (15 g/L of sucrose, 20g/L, Na2HPO 4.12H 2O 1g/L, Mn2+0.05mmol/L of yeast extract powder and 8g/L of sodium chloride) according to the inoculation amount of 4% (v/v), and is continuously placed into the shaking table at 37 ℃ and is cultured for 16H at 180rpm, and seed liquid is obtained.

(2) Inoculating the seed liquid obtained in the step (1) into a 5L bioreactor containing 2L of fermentation medium (the formula is the same as the seed liquid) at an inoculation amount 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, and the DO is controlled to be 30%; when the pH value is 7.0, the supplementary materials (sucrose 300g/L and yeast powder 70g/L) are acidic, and replace acid to be connected with an acid pump, namely when the pH value in the fermentation tank is more than 7.0, the acid pump automatically adds a proper amount of supplementary materials to restore the pH value to 7.0. Fermenting continuously for 60h, collecting samples with different fermentation time, and freezing for storage.

Results as shown in fig. 4, biomass increased rapidly within the first 6 h; the pH value is increased due to the exhaustion of the sucrose, the supplementary material (matched with an acid pump) starts to flow, and the thallus density is 2-3 OD₆₀₀The speed of/h is increased, the density of the thalli is increased to 98.8U/L in 30h, the enzyme activity is 387782U/L, after 36h enters a stable period, the bacterial count is not increased, the nutrient substance is used for protein expression, and the enzyme activity is improved.

OD₆₀₀The cell density of (A) was increased to 108.4 in 42 hours, and the enzyme activity was 480098U/L, as shown in Table 7.

This result is about 11-fold higher than the previous results regarding the activity of recombinant Gluconobacter expressing D-psicose 3-epimerase (see, specifically, Yang J, Tian C, Zhang T, Ren C, Zhu Y, et al (2019) Development of food-grade expression system for D-allose 3-expression with the aid of the enzyme expression of microorganism genes in Corynebacterium glutamicum and enzyme application in conversion of can monomers to D-allose. The specific enzyme activities are measured by catalyzing 300g/L D-fructose to synthesize D-psicose in phosphate buffer solution with the pH value of 6.5.

In addition, the promoter contained by the strain B.subtilis 168/pMA5-spovG-DS-srfA-RC and the strain B.subtilis 168/pMA5-spovG-DS-srfA-RC is constitutive, and an inducer is not needed in the fermentation process.

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

Example 6: the browning reaction is weakened by the recombinant strain

The invention researches the influence of different pH (6.5-8.5) conditions on browning of a reaction solution when the reaction solution is reacted at 60 ℃ by crude enzyme solution and a whole-cell catalytic D-fructose to generate D-psicose.

The method comprises the following specific steps:

(2) respectively adding D-fructose 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 is subpackaged into 10 tubes, each tube is 900 mu L, and empty controls, 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 and 8.5-DS-RC are respectively marked on the tube body.

(3) Crude enzyme solutions (OD, each prepared from B.subtilis 168/pMA5-DS, B.subtilis 168/pMA5-RC and B.subtilis 168/pMA5-DS-RC strains, respectively₆₀₀60 whole cell disruption) was added to each tube of fructose solution dispensed in step (2), and the mixture was stored at 60 ℃ for 30 min.

The sugar solution reacts with amino groups in the protein, and the Maillard reaction releases melanin. The results of the studies reported that the absorbance of the sample solution at 420nm was proportional to the melanin concentration, as shown in Table 8.

Table 8: browning degree OD of different crude enzyme solutions under the same pH₄₂₀

Note: OD₄₂₀The magnitude of the value is influenced by a number of factors, with pH and temperature being the primary factors.

From example 3, the co-expressing strainsSubtilis 168/pMA5-DS-RC, the enzyme activity is higher, and the color change and OD of the reaction solution are changed along with the change of pH₄₂₀The comparison result is shown in fig. 3, and the browning degree and the pH magnitude show positive correlation.

The key to solve the browning problem is to reduce the pH of the reaction solution, and it can be understood from example 4 that the enzymatic properties of the single-enzyme-expressed DPEase cannot meet the requirement. Therefore, the invention improves the enzyme activity of the whole cell under the weak acid condition and can effectively solve the problem.

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

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Claims

1. A recombinant Bacillus subtilis which expresses a D-psicose 3-epimerase derived from Dorea sp.CAG317 and a D-psicose 3-epimerase derived from Clostridium cellulolyticum H10.

2. The recombinant Bacillus subtilis of claim 1 wherein Bacillus subtilis 168 is the expression host.

3. The recombinant Bacillus subtilis of claim 1 or claim 2 wherein the expression vector is pMA 5.

4. The recombinant Bacillus subtilis of any one of claims 1 to 3, wherein expression of D-psicose 3-epimerase is enhanced by one or more of the promoters HpaII, P43, srfA, spovG.

5. The recombinant Bacillus subtilis of any one of claims 1 to 4, wherein the amino acid sequence of D-psicose 3-epimerase derived from Dorea sp.CAG317 is shown in SEQ ID NO 1, and the amino acid sequence of D-psicose 3-epimerase derived from Clostridium cellulolyticum H10 is shown in SEQ ID NO 2.

6. A method for constructing the recombinant Bacillus subtilis of any one of claims 1 to 5, comprising the steps of:

7. A method for reducing browning reaction, characterized in that the recombinant Bacillus subtilis according to any one of claims 1 to 5 is added to a reaction system containing D-fructose to carry out the reaction.

8. The method of claim 7, wherein the OD of the recombinant Bacillus subtilis is added₆₀₀At least 15.

9. The method of claim 7 or 8, wherein the reaction conditions are: the pH value is 6.0-7.0, and the temperature is 55-65 ℃.

10. Use of the recombinant bacillus subtilis of any one of claims 1 to 5 for reducing browning reactions during food processing, or for preparing a high-sweetness, low-calorie food.