CN113005132B - D-psicose-3-epimerase gene and application method thereof - Google Patents
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
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- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
Abstract
The invention discloses a D-psicose-3-epimerase gene and an application method thereof, belonging to the technical field of microbial engineering. The D-psicose-3-epimerase gene contains a nucleotide sequence of SEQ ID NO.1, the specific enzyme activity and the thermal stability of the enzyme coded by the gene are higher than those of most of D-psicose-3-epimerase reported at present, and the substrate conversion rate is also better. The gene is heterologously expressed in bacillus subtilis, the purified recombinant D-psicose-3-epimerase is used for catalyzing the conversion of D-fructose to generate D-psicose, and the obtained functional D-psicose can be directly used for the production of foods, medicines and the like.
Description
Technical Field
The invention relates to a D-psicose-3-epimerase gene and an application method thereof, belonging to the technical field of genetic engineering and enzyme engineering.
Background
Rare Sugar (Rare Sugar) is a monosaccharide and a derivative thereof naturally occurring in nature but with a very small content, has the taste similar to that of cane Sugar, has the advantages of low calorie, high stability, sweet coordination, no hygroscopicity, no cariogenic property, high tolerance and the like, can make up for the deficiency of the traditional sweetener, plays an important role in improving the diet of special people, and is widely concerned by people in recent years. At present, more than 50 rare sugars are reported, wherein D-psicose (a C-3 potential difference epimer of fructose) is taken as a typical representative, has the characteristic of low calorie, plays an important physiological activity role in various aspects such as inhibiting blood sugar rise and body fat accumulation, eliminating free radicals, protecting nerves, modifying medicines or active substances so as to optimize the functional activity of the medicines or the active substances, is a novel sweetener for people suffering from diabetes and obesity, and is applied to various fields such as food, health care, medicines and the like.
However, since D-psicose is produced in a small amount in nature and is difficult to purify, production of D-psicose is currently mainly achieved by chemical synthesis and biocatalysis. The chemical synthesis method comprises the conversion synthesis of D-fructose by using molybdic acid ions, the synthesis of 1,2,4, 5-di-O-isopropylidene-beta-D-fructopyranose, or the synthesis of ethanol and triethylamine by heating, and the like. However, the above chemical synthesis methods have various disadvantages of complicated purification process, low conversion rate, chemical waste pollution, by-product generation and the like, and are expensive in chemical synthesis cost, which is not favorable for large-scale production of D-psicose. Therefore, the synthesis of functional D-psicose using biotransformation technology has been a hot research topic internationally.
D-psicose 3-epimerase (D-psicose 3-epimerase, abbreviated as DPEase, EC5.1.3.30) is a typical hexulose 3-epimerase, which was first discovered in Agrobacterium tumefaciens in 2005 by Kim professor in Korean, and subsequently, more and more DPEases of different microbial origin were gradually discovered and clonally expressed in a heterologous host. The enzyme has higher affinity with a substrate D-psicose, can singly catalyze D-fructose to generate D-psicose, and is the most commonly used enzyme for synthesizing D-psicose by a biotransformation method. At present, the research on the DPEase mainly focuses on finding out the construction expression and exploring the enzymological properties and crystal structures of the DPEase, the research on a recombinant escherichia coli expression system is more, and few reports are made on the research on the production of D-psicose in the food industry by using the DPEase. As the DPEase expressed by cloning by taking escherichia coli as a host always has safety problems in industrial application, and the large-scale production of D-psicose is further expanded in order to promote the application of the DPEase in the food industry, a safer and more efficient food-grade bacillus subtilis recombinant expression system needs to be established and optimized to meet the requirement of industrial production.
Disclosure of Invention
In order to solve the above-mentioned problems, the present invention realizes the heterologous expression of D-psicose-3-epimerase by introducing a gene encoding D-psicose-3-epimerase derived from Clostridium isolateI INTA.CYC.091contig-100_981 into a genetically engineered bacterium.
The invention provides a D-psicose-3-epimerase gene capable of improving coding enzyme activity and thermal stability, and a nucleotide sequence of the gene is shown in SEQ ID NO. 1.
The invention also provides a recombinant plasmid containing the gene, and the recombinant plasmid can carry and express the D-psicose-3-epimerase gene.
In one embodiment, the plasmid is the escherichia coli-bacillus subtilis shuttle plasmid pHT 01.
The present invention also provides a microbial cell expressing the D-psicose 3-epimerase.
In one embodiment, the microbial cell is bacillus subtilis.
In one embodiment, the Bacillus subtilis is a Bacillus subtilis WB600 host.
In one embodiment, the bacillus subtilis is constructed by the following steps: by utilizing a double enzyme digestion construction method, a D-psicose 3-epimerase gene with a nucleotide sequence shown as SEQ ID NO.1 is spliced to an expression vector pHT01 to construct a recombinant plasmid pHT01/dpe, and the recombinant plasmid pHT01/dpe is transformed into Bacillus subtilis WB 600.
The invention also provides a method for producing the D-psicose-3-epimerase, which uses the bacillus subtilis for producing the D-psicose-3-epimerase by fermentation.
In one embodiment, the fermentation is to ferment the bacillus subtilis in a TB culture medium at 25-37 ℃ for 20-60 h.
In one embodiment, the fermentation is to inoculate a certain amount of recombinant bacillus subtilis into LB culture medium containing ampicillin, culture the recombinant bacillus subtilis at 37 ℃ and 200r/min to logarithmic growth phase, inoculate the cultured seed culture solution into TB culture medium containing ampicillin according to the inoculation amount of 4% (v/v), and perform fermentation culture in a shaker at 25-37 ℃ for 20-60 h to produce the recombinant D-psicose-3-epimerase.
The invention also provides an application of the D-psicose-3-epimerase gene or Bacillus subtilis for expressing the D-psicose-3-epimerase in production of functional D-psicose, wherein the application takes the D-psicose-3-epimerase or an enzyme preparation containing the D-psicose-3-epimerase as a catalyst and takes D-fructose as a substrate to promote the conversion of the D-fructose into the functional rare sugar D-psicose, and the obtained functional D-psicose can be directly used for production of foods, medicines and the like.
In one embodiment, the enzyme preparation is a crude enzyme solution obtained by fermentation production of genetically engineered bacteria or a pure enzyme obtained by separation and purification, and the crude enzyme solution or the pure enzyme is added to a substrate in the form of the enzyme preparation.
The invention has the beneficial effects that:
1) heterologous expression of D-psicose-3-epimerase from Clostridium difficile in Bacillus subtilis is realized, the method is safe and efficient, the D-psicose-3-epimerase crude enzyme solution can be applied to production of foods and medicines, and the catalytic activity of the expressed D-psicose-3-epimerase crude enzyme solution can reach 188.4U/mg;
2) compared with other reported similar enzymes, the D-psicose-3-epimerase has the advantages that the D-psicose-3-epimerase is a new source of D-psicose-3-epimerase, the source is not reported before, the specific enzyme activity and the thermal stability of the D-psicose-3-epimerase are higher than those of most D-psicose-3-epimerase reported before, and the substrate conversion rate is also better.
3) The D-psicose-3-epimerase has high substrate conversion rate and product purity, and can effectively improve the yield of D-psicose and reduce the industrial production cost, thereby having industrial application value.
Drawings
FIG. 1 shows the construction of a recombinant plasmid of D-psicose-3-epimerase
FIG. 2 SDS-PAGE analysis of purified recombinant D-psicose-3-epimerase.
FIG. 3 HPLC analysis for the production of a product using recombinant D-psicose-3-epimerase.
Detailed Description
The method for measuring the enzyme activity of the D-psicose-3-epimerase comprises the following steps: 200. mu.L of the enzyme solution was added to 800. mu.L of Tris-HCl buffer (50mM, pH 7.5, 0.1mM Co) containing fructose2+) To give a final fructose concentration of 80g/L, the total reaction volume was 1 mL. Oscillating, mixing, placing in 55 deg.C water bath, reacting for 10min, transferring into boiling water, inactivating enzyme for 10min to terminate reaction; in this case, the enzyme solution was replaced with an equal amount of Tris-HCl buffer as a blank. After enzyme deactivation, the reaction solution is centrifuged for 10min at 10000rpm, the supernatant is taken and passed through a 0.22 mu m water system membrane to remove impurities, and then the concentration of D-fructose and D-psicose in the reaction solution can be analyzed by high performance liquid chromatography.
Definition of enzyme activity unit: under the standard enzyme reaction condition, the enzyme amount required by the enzyme to catalyze D-fructose to generate 1 mu mol of D-psicose in unit time is one enzyme activity unit.
Example 1 construction of Bacillus subtilis secretion expression System
Designing a corresponding amplification primer according to the gene of the D-psicose-3-epimerase:
an upstream primer: 5'-TACATAGGATCCATGAAACACGGAATCTACTACGCCTATTGGGAAAAGCAAT-3', respectively;
a downstream primer:
5’-CACGCCTCTAGATTAATCCAGCATGTAGCGCTGGAATGTGACAGCGTTTTTGGCGTCGAGGTCG-3’。
using the above primers and using synthetic plasmid containing Clostridium isolate INTA. CYC.091contig-100_981DPEase gene as template, cloning the target gene SEQ ID NO.1 containing BamH I and Xba I restriction sites at both ends.
The PCR system of the D-psicose-3-epimerase gene is as follows: taq Buffer(Mg2+Plus) 10. mu.L, dNTP mix (2.5 mM each) 4. mu.L, forward primer (10. mu.M) 1. mu.L, reverse primer (10. mu.M) 1. mu.L, template DNA 1. mu.L, Taq DNA Polymerase (1.25U/. mu.L) 1. mu.L, and double distilled water was added to 50. mu.L. The PCR amplification conditions were: performing pre-denaturation at 98 ℃ for 3 min; then 30 cycles (98 ℃ for 10s, 60 ℃ for 15s, 68 ℃ for 2min) are carried out; finally, the temperature is kept for 10min at 68 ℃.
Inserting the D-psicose-3-epimerase gene amplified by PCR into a pMD 18-T simple plasmid to obtain a cloning vector pMD 18-T simple/dpe, carrying out double enzyme digestion on the vector, recovering a target gene fragment containing a cohesive end, and inserting the target gene fragment into a pHT01 plasmid treated by the same endonuclease to obtain an expression vector pHT 01/dpe. And then the plasmid is transformed into Escherichia coli E.coli JM109, an LB plate with ampicillin is coated, a transformant is selected for sequencing and colony PCR verification, and a recombinant plasmid containing the D-psicose-3-epimerase gene is extracted. And (3) transforming the recombinant plasmid into B.subtilis WB600 to obtain the recombinant gene engineering bacterium.
EXAMPLE 2 fermentative production of recombinant D-psicose-3-epimerase
Selecting a single bacillus subtilis colony containing the recombinant plasmid from a preservation plate, inoculating the single bacillus subtilis colony into 50mL of LB culture medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0) containing ampicillin, and performing shake-flask culture at 37 ℃ and 200rpm for overnight; was inoculated into 50mL of TB medium (1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17mM KH) containing ampicillin at a dose of 4%2PO4，72mM K2HPO4And the pH value is 6.0), performing shake flask culture at 25-37 ℃ and 200rpm for 20-60 h; centrifuging 30mL of fermentation liquid with OD of 6 at 4 ℃ and 10000rpm for 10min, discarding the supernatant, washing the surface of a thallus precipitate with 50mM Tris-HCl with pH 7.5 for 3 times, then adopting Binding Buffer (50mM Tris-HCl,500mM NaCl, pH 7.5) to resuspend the thallus, then adding 1mg/mL lysozyme, carrying out water bath heat preservation at 37 ℃ for 30min, carrying out ultrasonic crushing on ice for 15min under the procedures of 200W, opening for 1s and closing for 2s, finally centrifuging at 4 ℃ and 10000rpm for 20min, and taking the supernatant, namely an intracellular enzyme solution with crude enzyme activity of 102.12U/mL.
And (3) fermenting the bacillus subtilis for producing the recombinant D-psicose-3-epimerase in a TB culture medium at 30 ℃ for different time according to the same method, taking an enzyme solution to measure the activity and the protein concentration of the bacillus subtilis, and calculating to obtain the specific enzyme activity of 188.4U/mg.
EXAMPLE 3 isolation and purification of recombinant D-psicose-3-epimerase
After the crude enzyme solution was subjected to membrane treatment with a 0.45 μm aqueous membrane, the nickel column was equilibrated with 5 column volumes of solution A (500mM NaCl,50mM Tris-HCl, pH 7.5); loading at a flow rate of 1mL/min, re-equilibrating with solution A, washing off a part of the contaminating proteins with eluent 1 containing low concentration of imidazole (500mM NaCl,50mM Tris-HCl,20mM imidazole, pH 7.5), washing off the target proteins with 60% eluent 2(500mM NaCl,50mM Tris-HCl,500mM imidazole, pH 7.5), collecting the corresponding eluents according to the elution peaks, placing them in a dialysis bag, dialyzing overnight at 4 ℃ with 50mM Tris-HCl, pH 7.5, and then identifying them by SDS-PAGE protein electrophoresis (FIG. 2) and enzyme activity determination. The calculated molecular weight of the recombinant D-psicose-3-epimerase was reported to be 32.3kDa, which is consistent with the molecular weight estimated by SDS-PAGE.
EXAMPLE 4 production of recombinant D-psicose-3-epimerase
The recombinant D-psicose-3-epimerase is used for producing and preparing D-psicose, and the reaction process is as follows: first, a substrate solution (50mM Tris-HCl, pH 7.5, 0.1mM Co) containing 80g/L fructose2+) Preheating in 55 deg.C water bath shaker for 10min, adding 10U/g enzyme solution, reacting in 55 deg.C water bath shaker for 2 hr, transferring to boiling water, inactivating enzyme for 20min to terminate reaction; in this, the enzyme solution was replaced with an equal amount of Tris-HCl buffer as a blank. After enzyme deactivation, the reaction solution is centrifuged for 10min at 10000rpm, the supernatant is taken and passed through a 0.22 mu m water system membrane to remove impurities, and then the concentration of D-fructose and D-psicose in the reaction solution can be analyzed by high performance liquid chromatography. The chromatographic analysis results are shown in fig. 3, and the conversion rate calculated can reach 31%.
Comparative example:
the specific implementation manner is the same as that of examples 1-4, except that the gene derived from clostridium isolate inta. cyc.091contig-100_981 is used for replacing the D-psicose-3-epimerase gene from other sources reported in the prior art, a recombinant gene engineering bacterium is constructed according to the same strategy as that of examples 1-4, the bacterium is cultured under the same conditions to obtain an enzyme solution, the activity and the enzymological properties of the enzyme solution are measured, and the comparison result is shown in table 1.
TABLE 1D-psicose-3-epimerase from different sources
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 one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> a new source of D-psicose-3-epimerase gene and method of use thereof
<130> BAA210183A
<160> 3
<170> PatentIn version 3.3
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cccttctact ctgagcagca aatgaaagat atcaaggctt gtgccgaggc caatggcatc 180
acattgacgt gtggccacgg ccccagtcct gaccagaacc tggcctcatc agaccctgcc 240
gtccgagccc atgccaagtc atttttcacc gacctgctgt cacgcctcga aaaaatggac 300
atccatgtca tcggcggcgg gatctactcc tactggccag ttgattattc catgccgatc 360
gacaaacctg gcgattgggc ccgcagcgtc gaaggcgtgg ccgaaatggc caaagtcgcc 420
gaatcctgcg gtgtggacta ttgcctggag gtcctcaacc gttttgaagg ctacctcttg 480
aacactgccg aagaagctgt ccagtttgtc caggaggtca accaccctcg agtcaaaatc 540
atgctcgata ccttccatat gaacatcgaa gaagacagca tcggcggcgc catccgtcgc 600
gccggccagc atctgggcca tttccatact ggtgaatgca accgccgcgt acccggccga 660
ggccgcaccc cctggcgcga aatcggcgaa gccctgaggg acatcggcta cacaggcgcc 720
gtcgtcatgg agcccttcgt ccgcatgggc ggccaagtcg gctccgatat caagatctgg 780
cgcgaaatga atcccggtgc cgatgacgcc cagctcgacc tcgacgccaa aaacgctgtc 840
acattccagc gctacatgct ggattga 867
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Claims (7)
1. A method for producing D-psicose-3-epimerase, characterized in that recombinant Bacillus subtilis expressing D-psicose-3-epimerase is used for the fermentative production of D-psicose-3-epimerase; the nucleotide sequence of the coding gene of the D-psicose-3-epimerase is shown in SEQ ID NO. 1.
2. The method according to claim 1, wherein the recombinant Bacillus subtilis expresses D-psicose-3-epimerase represented by SEQ ID No.1 using an Escherichia coli-Bacillus subtilis shuttle plasmid pHT01 as a vector.
3. The method of claim 1, wherein the recombinant Bacillus subtilis is a Bacillus subtilis WB600 host.
4. The method according to claim 1, wherein the fermentation is carried out by fermenting the bacillus subtilis in a TB medium at 25-37 ℃ for 20-60 h.
5. Use of the method according to any one of claims 1 to 4 for the preparation of an enzyme preparation comprising D-psicose-3-epimerase.
6. The use according to claim 5, wherein the enzyme preparation is prepared by separating and purifying a crude enzyme solution obtained from fermentation production.
7. The application of the D-psicose-3-epimerase gene shown in SEQ ID NO.1 or the recombinant bacillus subtilis expressing the D-psicose-3-epimerase shown in SEQ ID NO.1 in the production of D-psicose.
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