CN116286770A - D-psicose-3-epimerase from clostridium and application thereof - Google Patents
D-psicose-3-epimerase from clostridium and application thereof Download PDFInfo
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- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 30
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Abstract
The invention belongs to the technical field of genetic engineering and fermentation engineering, and provides D-psicose-3-epimerase from clostridium and application thereof, wherein a nucleotide sequence for encoding the D-psicose-3-epimerase is shown as SEQ ID No.1, and the nucleotide sequence is obtained through codon optimization.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and fermentation engineering, and particularly relates to a method for efficiently expressing D-psicose-3-epimerase in bacillus subtilis.
Background
Psicose (allose) is an epimer of D-fructose at the C-3 position, is white powder, is transparent and colorless in aqueous solution, is stable at normal temperature and normal pressure, exists in natural foods such as fruits, raisins, figs and the like, has sweetness of 70% of sucrose and calorie of 0.3% of sucrose, is a novel sweetener, and is a monosaccharide which naturally exists in nature but has a very small content, and is also called rare sugar. Psicose was approved by the FDA in 2015, and can be used in baked goods, candies, sweet pastes, dairy products, ice creams, desserts, beverages, and other products. The psicose has excellent processability, stability, high tolerance and strong capability of removing active oxygen, and is widely used in the fields of food, daily chemicals, health care products, medicines, beverages and the like.
D-psicose is present in a rare amount in nature, and is generally produced by enzymatic conversion at present. D-psicose-3-epimerase (i.e., DPE enzyme) is capable of converting cheaper D-fructose into D-psicose. Thus, DPE enzyme is a key factor in the biosynthesis of D-psicose. There are twenty types of DPE enzymes reported in the literature, namely, agrobacterium tumefaciens (Agrobacterium tumefaciens) from literature 1 (Samir R.D. et al Enzyme and Microbial Technology, 2020,140,109605), clostridium scintinus (Clostridium scindens) from Ruminococcus (Ruminococcus sp.) from literature 2 (Fu, G.et al Biotechnology and Applied Biochemistry,2019, 67 (5), 812-818), clostridium cellulolyticum (Clostridium cellulolyticum) from literature 3 (Su, L.et al Microbial Cell Factories, 2018, 17, 188), clostridium pallidum (Clostridium bolteae) from literature 4 (Jia. M. et al Applied Microbiology andBiotechnology,2014,98,717-725), clostridium scintinus (Clostridium scindens) from He, W.et al Journal of Agricultural and Food Chemistry, 2016, 64 (28), clostridium scintinus (3801-5707), clostridium (Mu, W.et al Biotechnology Letters, 2013, 35 (9), 1481), and the like. Wherein, the DPE enzyme from C.cellulolyticum in document 3 is heterologously expressed in bacillus subtilis, can reach 2246U/mL under the condition of a 3.6L fermentation tank, is the highest enzyme activity reported at present, but the optimal catalysis pH is alkaline condition, which limits the industrialized application to a certain extent.
The high temperature is advantageous in accelerating the reaction speed and suppressing the growth of harmful bacteria in industrial production. Most DPE enzymes found at present have poor thermal stability, are rapidly deactivated at temperatures above 50 ℃ and are not easy to store at normal temperature. And the known catalytic reaction conditions of the enzyme are slightly alkaline, and the high-temperature catalytic conditions of the alkalinity can lead the sugar solution to generate non-enzymatic browning, increase byproducts and reduce the yield. Therefore, it is important to provide DPE enzyme which has good thermal stability and can still maintain high catalytic activity under acidic or neutral conditions for the development of industrialization of D-psicose, and the present invention analyzes Protein crystals of the enzyme (method references Chan, H.et al Protein & Cell, 2012, 3, 123-131), and finds the root cause of the activity change.
Disclosure of Invention
In order to solve the problems in the prior art, the invention obtains the D-psicose-3-epimerase with high expression and good stability, namely DPE enzyme by a gene mining method. Aiming at the problem that DPE enzyme has low expression efficiency in bacillus subtilis, the nucleotide sequence for encoding DPE enzyme is subjected to codon optimization, and the protein expression level is improved under the condition of not changing the protein sequence; the provided D-psicose-3-epimerase can obtain higher enzyme activity under neutral and even acidic conditions and at higher temperature.
After the expression of DPE enzyme is optimized by the invention, the enzyme activity can reach 354U/mL at maximum under the condition of shaking culture, and can reach 2578U/mL under the condition of optimized fermentation in a 3.6L fermentation tank. Meanwhile, the invention also provides a method for obtaining DPE enzyme through mass fermentation of a 30L fermentation tank, wherein the enzyme activity is as high as 2660U/mL, and the D-psicose-3-epimerase with high expression and good stability is obtained.
Specifically, according to the first aspect of the invention, a D-psicose-3-epimerase with improved expression level and good stability is provided, and a nucleotide sequence encoding the D-psicose-3-epimerase is shown as SEQ ID No.1 after codon optimization.
The invention provides a gene for encoding the D-psicose-3-epimerase of claim 1, wherein the nucleotide sequence of the gene is shown as SEQ ID No.1.
According to a second aspect of the present invention there is provided a host bacterium or recombinant vector expressing the D-psicose-3-epimerase of the first aspect of the present invention, the recombinant vector or host bacterium being a recombinant vector or host bacterium comprising a nucleotide sequence encoding the D-psicose-3-epimerase, the nucleotide sequence encoding the D-psicose-3-epimerase being as shown in SEQ ID No.1.
According to a preferred embodiment, the host bacterium is bacillus subtilis.
According to one embodiment, the host bacterium is bacillus subtilis b.
According to a preferred embodiment, the recombinant vector is pMA5.
According to a third aspect of the present invention there is provided a process for the preparation of D-psicose-3-epimerase, wherein B.subtilis is used for the fermentative preparation of D-psicose-3-epimerase.
According to a preferred embodiment, the medium formulation used in the method is as follows; 20. 20 g/L of soybean peptone, 5 g/L of corn steep liquor dry powder, 1 g/L of ammonium citrate, 50 g/L of glycerin, 2.31 g/L of monopotassium phosphate, 0.2 g/L of magnesium sulfate, 0.2 g/L of calcium chloride, 20 mug/mL of kanamycin final concentration and 20 mL of microelement liquid. The feed medium formulation is as follows: 200. 200 g/L of glycerol, 7.89 g/L of magnesium sulfate, 100. 100 g/L of soybean peptone, 3.5 g/L of corn steep liquor dry powder and 20 mL of trace element liquid. The fermentation culture conditions were as follows: 10 The feed was fed at a constant rate at a temperature of 37℃and a rotation speed of 300 rpm, pH=7.0, air flow rate of 1.5 VVM and cultured for 48 hours.
Further optimizing the conditions of the fermentation culture method, wherein the culture medium used in the method comprises the following formula:
20. 20 g/L casein hydrolysate, 15 g/L corn steep liquor extract, 1 g/L ammonium citrate, 40g/L glycerin, 2.31 g/L monopotassium phosphate, 0.2 g/L magnesium sulfate, 0.2 g/L calcium chloride, 20 mug/mL kanamycin final concentration and 20 mL trace element liquid. Feed medium: glycerin 100 g/L, magnesium sulfate 7.89 g/L, casein hydrolysate 100 g/L, corn steep liquor extract 10g/L and trace element liquid 20 mL, wherein the feeding mode is as follows: 10 mL/h constant-speed feeding. The fermentation conditions were 37℃at 400 rpm, pH=7.0 and air flow rate 2.0 VVM for 48 hours.
According to a fourth aspect of the present invention there is provided the use of a D-psicose-3-epimerase according to the first aspect of the present invention, a host bacterium or a recombinant vector according to the second aspect of the present invention for the preparation of D-psicose.
The excellent effects of the D-psicose-3-epimerase and the fermentation method disclosed by the invention mainly lie in the following aspects:
(1) A highly expressed D-psicose-3-epimerase having good thermostability and capable of reacting under neutral conditions was found. Has high activity in 55-65deg.C, and half-life of 9.3h at 60deg.C. There is a relatively good activity (greater than 80%) at pH 6.0-9.0, the best catalytic activity at ph=7.0, and still the higher catalytic activity at weak acid (ph=6.5).
(2) By utilizing a codon optimization method, the protein expression level is improved under the condition of not changing the protein sequence, the enzyme activity is improved from 126U/mL to 354U/mL, the enzyme activity is greatly improved, and the enzyme activity can reach 2578U/mL under the optimized fermentation condition of a 3.6L fermentation tank.
(3) Provides a large amount of fermentation method of the D-psicose-3-epimerase in a 30L fermentation tank, ensures that the DPE enzyme activity is as high as 2660U/mL, and provides a foundation for the industrial application of the DPE enzyme.
(4) The D-psicose-3-epimerase provided by the invention can obtain higher enzyme activity under neutral and even acidic conditions and at higher temperature.
(5) The D-psicose-3-epimerase provided by the invention has the advantages that the amino acid of a key site is changed with that of the existing DPE enzyme, so that the DPE enzyme can reach very high enzyme activity under neutral or even acidic conditions, and compared with the DPE enzyme in the same volume of a fermentation tank, the optimal activity pH=7.0 of the DPE enzyme provided by the invention has the highest enzyme activity of 2578U/mL. 95% of the highest enzyme activity at ph=6.5, 2449U/mL, and 90% of the highest enzyme activity at ph=6, 2320U/mL.
Drawings
Fig. 1: and (5) analyzing a DPE enzyme phylogenetic tree obtained by gene mining.
FIG. 2 expression of DPE enzymes of different branches in Bacillus subtilis.
FIG. 3 plasmid construction map of pMA5DPE enzyme.
Fig. 4: comparison of the expression level of DPE enzyme after codon optimization.
Fig. 5: DPE enzyme takes fructose as a substrate to catalyze and synthesize psicose. Wherein, FIG. 5A shows the analysis result of HPLC of psicose and ketose standard substance; FIG. 5B shows the results of HPLC analysis of fructose standards; FIG. 5C shows the results of HPLC analysis of the reaction product of DPE enzyme with fructose.
FIG. 6 stability test results of DPE enzyme. Wherein fig. 6A: test results of pH versus catalytic activity; fig. 6B: test results of temperature versus catalytic Activity FIG. 6C Co 2+ Test results for catalytic activity; fig. 6D: DPE enzyme activity changes over time at 60 ℃.
Detailed Description
The present invention will be described in detail with reference to examples and fig. 1 to 6.
Specifically, according to the first aspect of the invention, a D-psicose-3-epimerase with improved expression quantity and good stability is provided, and a sequence after codon optimization is shown as SEQ ID No.1.
According to a second aspect of the present invention there is provided a host bacterium or recombinant vector expressing the D-psicose-3-epimerase of the first aspect of the present invention, the recombinant vector or host bacterium being a vector or host bacterium comprising a nucleotide sequence encoding the D-psicose-3-epimerase, the nucleotide sequence encoding the D-psicose-3-epimerase being shown in SEQ ID No.1.
According to a preferred embodiment, the host bacterium is bacillus subtilis.
According to one embodiment, the host bacterium is bacillus subtilis b.
According to a preferred embodiment, the recombinant vector is pMA5.
According to a third aspect of the present invention there is provided a process for the preparation of D-psicose-3-epimerase, wherein B.subtilis is used for the fermentative preparation of D-psicose-3-epimerase.
According to a preferred embodiment, the medium formulation used in the method is as follows; 20. 20 g/L of soybean peptone, 5 g/L of corn steep liquor dry powder, 1 g/L of ammonium citrate, 50 g/L of glycerin, 2.31 g/L of monopotassium phosphate, 0.2 g/L of magnesium sulfate, 0.2 g/L of calcium chloride, 20 mug/mL of kanamycin final concentration and 20 mL of microelement liquid. The feed medium formulation is as follows: 200. 200 g/L of glycerol, 7.89 g/L of magnesium sulfate, 100. 100 g/L of soybean peptone, 3.5 g/L of corn steep liquor dry powder and 20 mL of trace element liquid. The fermentation culture conditions were as follows: 10 The feed was fed at a constant rate at a temperature of 37℃and a rotational speed of 300 rpm, at a pH=7.0, at an air flow rate of 1.5 VVM and cultured for 48 hours.
Further optimizing the conditions of the fermentation culture method, wherein the culture medium used in the method comprises the following formula:
20. 20 g/L casein hydrolysate, 15 g/L corn steep liquor extract, 1 g/L ammonium citrate, 40g/L glycerin, 2.31 g/L monopotassium phosphate, 0.2 g/L magnesium sulfate, 0.2 g/L calcium chloride, 20 mug/mL kanamycin final concentration and 20 mL trace element liquid. Feed medium: glycerin 100 g/L, magnesium sulfate 7.89 g/L, casein hydrolysate 100 g/L, corn steep liquor extract 10g/L and trace element liquid 20 mL, wherein the feeding mode is as follows: 10 mL/h constant-speed feeding. The fermentation conditions were 37℃at 400 rpm, pH=7.0 and air flow rate 2.0 VVM for 48 hours.
According to a fourth aspect of the present invention there is provided the use of a D-psicose-3-epimerase according to the first aspect of the present invention, a host bacterium or a recombinant vector according to the second aspect of the present invention for the preparation of D-psicose.
The invention also provides a gene for encoding the D-psicose-3-epimerase, and the nucleotide sequence of the gene is SEQ ID No.1.
Example 1: mining DPE enzyme from Clostridium genome
1954 transcriptomes SRA (Sequence Read Archive) data of Clostridium species in NCBI (National Center for Biotechnology Information ) were downloaded in full and spliced to build a local Clostridium database. Taking DPE enzyme from Ruminococcus sp as query sequence, 146 similarity over 8e is obtained after local BLAST to local database -18 Is a protein sequence of (a). The obtained protein sequence was subjected to weight removal to obtain 95 nucleotide sequences. The amino acid sequence encoded by the 95 nucleotide sequences is treed by the method of Jukes-Cantor and Neighbor-Joing. From the resulting developmental tree, it can be seen that all DPE enzymes can be divided into 5 branches, the specific branches are shown in FIG. 1, and branch I, branch II, branch III, branch IV and branch V respectively. The DPE enzyme from the branch I source (hereinafter referred to as DPE enzyme) was found to have the highest expression efficiency under the same conditions by taking one sequence from 5 branches, respectively, and the DPE enzyme from the branch I source was found to have the best expression efficiency as compared with the case shown in FIG. 2, and further studies found to have stability and catalytic activity superior to those of the known DPE enzyme.
Example 2: construction and expression of original DPE enzyme expression plasmid
1) PCR of DPE enzyme sequence
Primers containing homology arms were designed as follows:
DPE-F：GTGCCACCTAAAAAGGAGCGATTTACATATGAAACATGGTATATAC
DPE-R：GAGGTGAATTTCGACCTCTAGATCAGGAGTGTTTATGACATTCTAATAC
PCR is performed by using the synthesized nucleotide sequence of DPE enzyme as a template to obtain a gene fragment containing a homology arm with the expression plasmid.
2) pMA5 is subjected to double digestion by NedI and MulI, then is subjected to gel recovery, and is recombined with a DPE enzyme nucleotide sequence obtained by PCR by using Gibson and then is transferred into eco.li DH5 alpha competent cells. Plasmid extraction was performed on positive clones to obtain pMA5DPEO, and FIG. 3 shows a plasmid map of pMA5 DPEO.
3) Transferring the plasmid obtained in the step 2) into a competent cell of the bacillus subtilis B.subtilis 168 to obtain recombinant bacillus subtilis B.subtilis 168-DEPaseO.
4) Picking up the recombinant bacillus subtilis 168-DEPaseO obtained in the step 3), inoculating positive transformants on LB medium plates containing kanamycin (with a final concentration of 50 mug/mL), inoculating the positive transformants in 30 mL LB medium containing kanamycin (with a final concentration of 50 mug/mL), and culturing at 30 ℃ and 200 rpm overnight; the culture was carried out in LB medium of 50 mL or more at 30℃and 200 rpm for 48 hours according to an inoculum size of 2%. The cells were collected from the fermentation broth 5000 xg, washed with PBS buffer, resuspended and diluted 5-fold. After the cells are broken by using an ultrasonic breaker, centrifuging at 12000 rpm for 15min, collecting supernatant, and obtaining the supernatant as crude enzyme liquid.
And (3) performing enzyme activity measurement on the crude enzyme solution in the step (4). The enzyme reaction conditions are as follows: 100 g/L D-fructose prepared by HEPS (pH 7.0) is used as a substrate, 800 mu L of the substrate and 200 mu L of diluted enzyme solution are precisely reacted for 10 min, the reaction is quenched by boiling, and the reaction is filtered by a microporous filter membrane with the thickness of 0.45 mu m, and the filtrate is subjected to high performance liquid analysis.
The high performance liquid chromatography analysis was performed as follows: the instrument was an Agilent1260 high performance liquid chromatograph equipped with a differential detector, the column was a Carbomix Pb-NP10 column (8%, 7.8X300 mm,10 μm, sirocco technology), the mobile phase was water, the flow rate was 0.5 mL/min, and the column temperature was 70 ℃.
The DPE enzyme activity expressed by recombinant Bacillus subtilis B.subtilis 168-DEPaseO was determined by high performance liquid chromatography to be 126U/mL.
Example 3: DPE enzyme codon optimization for improving expression efficiency
1) In order to increase the expression efficiency of DPE enzyme in Bacillus subtilis. The DPE enzyme nucleotide sequence is optimally screened against codons of bacillus subtilis, and the finally optimized sequence is determined by the expression quantity as follows (SEQ ID No. 1):
ATGAAACATGGCATTTATTATGCATATTGGGAACAAGAATGGGAAGCAGATTATAAATATTATATTGAAAAAGTTGCAAAACTGGGCTTTGATATTCTGGAAATTGCAGCATCACCGCTGCCGTTTTATTCAGATAACCAAATTAATGAACTGAAAGCATGCGCAAGAGGCAATGGCATTACACTGACAGTTGGCCATGGCCCGTCAGCAGAACAAAATCTGTCATCACCGGATCCGGATATTAGAAAAAATGCAAAAGCATTTTATACAGATCTGCTGAAAAGACTGTATAAACTGGATGTTCATCTGATCGGCGGCGCACTGTATTCATATTGGCCGATTGATTATACAAAAACAATTGATAAAAAAGGCGATTGGGAAAGATCAGTTGAATCAGTTAGAGAAGTTGCAAAAGTTGCAGAAGCATGCGGCGTTGATTTCTGCCTTGAAGTTCTGAATAGATTTGAAAATTATCTGATTAATACAGCACAAGAAGGCGTTGATTTTGTTAAACAAGTTGATCATAATAACGTTAAAGTTATGCTGGACACATTTCACATGAATATTGAAGAAGATTCAATTGGCGGCGCAATTAGAACAGCGGGCTCATATCTGGGCCATCTGCATACGGGCGAATGCAATAGAAAAGTTCCGGGCAGAGGCAGAATTCCGTGGGTTGAAATTGGCGAAGCACTGGCAGATATTGGCTATAATGGCTCAGTTGTTATGGAACCGTTTGTTAGAATGGGCGGCACAGTTGGCTCAAATATTAAAGTTTGGAGAGATATTTCAAATGGCGCAGATGAAAAAATGCTGGATAGAGAAGCACAAGCAGCACTGGATTTTTCAAGATATGTTCTGGAATGCCATAAACATTCATAA
the nucleotide sequence before optimization is shown as SEQ ID No.2, and the amino acid sequence after optimization is shown as SEQ ID No. 3.
SEQ ID No.2 is shown below:
SEQ ID No.2
ATGAAACATGGTATATACTACGCATATTGGGAACAAGAATGGGAAGCTGATTACAAATACTATATTGAGAAGGTTGCAAAGCTTGGTTTTGATATTCTAGAGATTGCAGCTTCACCGCTACCTTTTTACAGTGACAAACAGATTAATGAGCTCAAGGCATGTGCCAGAGGCAATGGAATTACACTTACGGTAGGCCATGGGCCTAGTGCAGAACAAAACCTGTCTTCTCCCGACCCCGATATTCGCAAAAATGCTAAAGCTTTTTATACCGATTTACTCAAACGACTTTACAAGCTGGATGTACATTTGATAGGTGGGGCTTTATATTCTTATTGGCCGATAGATTACACAAAGACAATTGATAAAAAAGGCGATTGGGAACGCAGCGTTGAAAGTGTTCGAGAAGTTGCTAAGGTGGCCGAAGCCTGTGGAGTGGATTTCTGCCTAGAGGTTCTTAATAGATTTGAGAATTATTTAATTAACACAGCACAAGAGGGTGTAGATTTTGTAAAACAGGTTGACCATAACAATGTAAAGGTAATGCTTGATACCTTCCATATGAATATTGAGGAAGATAGTATCGGAGGTGCAATCAGGACTGCGGGCTCTTACTTGGGACATTTACACACTGGCGAATGTAATCGTAAAGTTCCCGGCAGAGGAAGAATTCCATGGGTAGAAATTGGTGAGGCTCTTGCTGACATAGGTTATAACGGTAGTGTTGTTATGGAACCTTTTGTTAGAATGGGCGGAACTGTCGGATCTAATATTAAGGTTTGGCGTGACATTAGTAACGGTGCAGATGAGAAAATGCTGGATAGAGAAGCACAGGCCGCACTTGATTTCTCCAGATATGTATTAGAATGTCATAAACACTCCTGA
SEQ ID No.3 is shown below:
SEQ ID No.3
MKHGIYYAYWEQEWEADYKYYIEKVAKLGFDILEIAASPLPFYSDNQINELKACARGNGITLTVGHGPSAEQNLSSPDPDIRKNAKAFYTDLLKRLYKLDVHLIGGALYSYWPIDYTKTIDKKGDWERSVESVREVAKVAEACGVDFCLEVLNRFENYLINTAQEGVDFVKQVDHNNVKVMLDTFHMNIEEDSIGGAIRTAGSYLGHLHTGECNRKVPGRGRIPWVEIGEALADIGYNGSVVMEPFVRMGGTVGSNIKVWRDISNGADEKMLDREAQAALDFSRYVLECHKHS
2) And (3) transforming the optimized DPE enzyme expression plasmid into B.subtilis 168 competent cells to obtain recombinant bacillus subtilis B.subtilis 168-DEPaseY.
3) Recombinant Bacillus subtilis B.subulis 168-DEPaseO and B.subulis 168-DEPaseY were subjected to a small amount of fermentation under the same conditions. After obtaining the cells, after the cells with the same weight are lysed, SDS-PAGE analysis is performed on the supernatant, and the two are compared, and specific comparison results are shown in the accompanying figure 4, so that the concentration of the DPE enzyme after optimization is larger, and the expression quantity is greatly improved.
The enzyme activity of B.subtilis 168-DEPasey was determined by the method of example 2 and reached 354U/mL. The enzyme activity was increased by a factor of 2 compared to recombinant bacillus subtilis without codon optimization, which corresponds to the comparison in fig. 4.
The enzyme reaction solution of the above example was analyzed using D-fructose (5.49, mg/mL) and D-psicose (5.25, mg/mL) produced by Sigma as standard substances, and the sample amount was 10. Mu.L. The analysis results of the standard substances are shown in fig. 5A and fig. 5B. The experimental analysis results in example 3 are shown in fig. 5C. According to the high performance liquid analysis result, the DPE enzyme expressed by the recombinant bacillus subtilis can catalyze epimerization reaction between D-fructose and D-psicose. The novel DPE enzyme discovered in the invention can be used for realizing the bioconversion production of D-psicose, and the enzyme catalytic reaction uses cheaper D-fructose as a substrate, so that the DPE enzyme has the advantages of low cost and great advantages.
To further investigate the performance of DPE enzymes, the acid and alkali resistance, the high temperature resistance, and the durability of DPE enzymes under different experimental conditions were investigated, respectively, see example 4.
Example 4 optimization of the reaction conditions and stability test of DPE enzyme
1) The DPE enzyme obtained by the invention reacts with 100 g/L D-fructose at different pH values respectively, the obtained result is shown in figure 6, the DPE enzyme provided by the invention has relatively good activity (more than 80%) at the pH value of 6.0-9.0, has the best catalytic activity at the pH value of 7.0, and still has higher catalytic activity under the condition of weak acid (pH=6.5). The DPE enzyme has better pH stability.
2) The resulting DPE enzyme was reacted with 100 g/L D-fructose at different temperatures in a buffer at pH 7.0, respectively. The DPE enzyme has better activity at 55-65 ℃ and the optimal reaction temperature is 60 ℃.
3) Co is added into the reaction system 2+ The reaction rate of DPE enzyme can be improved. The obtained DPE enzyme is respectively mixed with Co with different concentrations at 60 DEG C 2+ The reaction was carried out at 2. Mu.M to 32. Mu.M Co 2+ Within the range, the reactivity increases linearly; when Co is 2+ The concentration is more than 50 mu M, and the reactivity is relatively stable. Finally determining Co in the reaction system 2+ A concentration of 0.05. 0.05mM indicates that the DPE enzyme obtained according to the invention requires a lower concentration of Co 2+ An efficient reaction can be achieved.
4) At 0.05mM Co 2+ The half-life of the DPE enzyme at 60℃was determined with participation in the reaction. The enzyme activity was 100% based on the initial enzyme reaction. The DPE enzyme activity decreased slowly with time, and was still 80% active after 12 hours, with a half-life of 9.3 hours at 60 ℃When (1). The enzyme has good heat-resistant advantage and stability advantage, and can be reused. Wherein the relevant properties of the DPE enzyme in respect of thermostability and stability are shown in FIG. 6.
Through the optimization, the optimal reaction conditions of the DPE enzyme in the invention are finally determined as follows: pH=7.0 buffer, 100 g/L D-fructose, 0.05mM Co 2+ ，60℃。
EXAMPLE 5 fermentation of recombinant Bacillus subtilis B.subtilis 168-DEPaseY in a 3.6L fermenter
1) Positive transformants of the recombinant B.subtilis 168-DEPasey obtained in example 3 were picked up on LB medium plates containing kanamycin (final concentration: 50. Mu.g/mL), inoculated in 500mL of LB liquid medium containing kanamycin (final concentration: 50. Mu.g/mL), and cultured at 30℃for 12-14 hours at 200 rpm. The culture was inoculated into a 3.6L fermenter containing 1L fermentation medium (soybean peptone 20 g/L, corn steep liquor dry powder 5 g/L, ammonium citrate 1 g/L, glycerol 50 g/L, potassium dihydrogen phosphate 2.31 g/L, magnesium sulfate 0.2 g/L, calcium chloride 0.2 g/L, kanamycin final concentration 20. Mu.g/mL, trace element liquid 20 mL) at 37℃at 250 rpm, pH=7.0, air flow rate 1.0 VVM for 48 hours according to an inoculum size of 10%. Feed medium: 200. 200 g/L of glycerol, 7.89 g/L of magnesium sulfate, 100. 100 g/L of soybean peptone, 3.5 g/L of corn steep liquor dry powder and 20 mL of trace element liquid.
2) The fermentation broth in step 1) was centrifuged to obtain a cell, and the enzyme activity was measured by the method of example 2, resulting in 1896/U/mL.
Example 6:3.6 Optimization of culture conditions in an L fermenter
1) Optimization of the culture medium. The initial medium in example 5 was replaced with: 20. 20 g/L casein hydrolysate, 15 g/L corn steep liquor extract, 1 g/L ammonium citrate, 40g/L glycerin, 2.31 g/L monopotassium phosphate, 0.2 g/L magnesium sulfate, 0.2 g/L calcium chloride, 20 mug/mL kanamycin final concentration and 20 mL trace element liquid. Feed medium: 100 g/L of glycerin g/L, 7.89 g/L of magnesium sulfate, 100 g/L of casein hydrolysate, 10g/L of corn steep liquor extract and 20. 20 mL of trace element liquid. The fermentation conditions were unchanged, and the enzyme activity was determined to be 2285U/mL.
2) Fermentation conditions are optimized together with the culture medium. The fermentation medium and the feed medium of step 1) were used, and the fermentation conditions were optimized to 37℃at 400 rpm, pH=7.0, and air flow rate of 2.0 VVM for 48 hours. The enzyme activity under this condition was determined to be 2578U/mL.
Example 7: fermenting recombinant Bacillus subtilis B.subtilis 168-DEPaseY in 30L fermenter
1) Seed culture was performed according to the method in example 5. The culture was inoculated at an inoculum size of 2% -5% into a 30L fermenter containing the fermentation medium of example 5 of 15L at 37℃and 300 rpm, pH=7.0, air flow rate of 1.5 VVM for 48 hours. The fermentation was fed at a constant rate (10 mL/h) and the feed medium was the same as in example 5.
2) The fermentation broth in step 1) was centrifuged to obtain a cell, and the enzyme activity was measured by the method of example 2, resulting in 2460U/mL.
Example 8: fermenting recombinant Bacillus subtilis B.subtilis 168-DEPaseY in 30L fermenter under optimized conditions
1) Seed culture was performed according to the method in example 5. 30L fermenters containing 15L of the fermentation medium of example 6 were inoculated at 2% -5% inoculum size, and incubated at 37℃at 400 rpm, pH=7.0, air flow rate 2.0 VVM for 48 hours. The fermentation was fed at a constant rate (10 mL/h) and the feed medium was the same as in example 6.
2) The fermentation broth in step 1) was centrifuged to obtain a cell, and the enzyme activity was measured by the method of example 2, resulting in 2660U/mL. The enzyme activity under the condition of 30L fermentation tank is equivalent to that of 3.6L fermentation tank, and the unit OD is measured simultaneously 600 The enzyme activities of (2) are equivalent in value. The enzyme activity level equivalent to that of a small-volume fermentation tank can be maintained after the volume is enlarged, so the establishment of the method is beneficial to promoting the industrialized development of the enzyme.
Example 9: comparison of DPE enzyme Performance from different sources
The DPE enzyme provided by the invention is compared with DPE enzyme activity obtained in the prior art document, acid and alkali resistance, stability, half life and the like are comprehensively compared, and specific data are shown in table 1:
TABLE 1 comparison of DPE enzymes of different origins
The DPE enzyme activities of different sources in Table 1 are all the best performances, and because of different sources and different characteristics, the best activities cannot be compared under certain same conditions, so that the best performances of the DPE enzyme activities can only be compared by adopting the comprehensive best performances, and as can be seen from Table 1, the optimal pH of the DPE enzyme in the invention is 7.0, the optimal temperature is 60 ℃, and the high enzyme activity 2660U/mL can be obtained by the fermentation method provided by the invention. The acidic reaction condition can reduce the occurrence of side reaction, the reaction can be kept at a higher reaction rate at 60 ℃, and the longer half-life period can realize the recycling of the enzyme.
Compared to the DPE enzyme activity of c.cellulolyticum H10 from document 3 (Su, l.et al Microbial Cell Factories, 2018, 17, 188), the DPE enzyme of c.cellulolyticum H10 has a maximum enzyme activity of 2246U/mL, but its optimal activity is at ph=8, according to document 3 it is reported that the DPE enzyme has an enzyme activity of about 90% of the maximum enzyme activity at ph=7, about 2021U/mL; at ph=6.5, 85% of the highest enzyme activity, approximately 1900U/mL; the pH=6 was 80% of the highest enzyme activity, about 1700U/mL.
The optimal activity of the DPE enzyme provided by the invention is pH=7.0, and the highest enzyme activity is 2660U/mL. 95% of the highest enzyme activity at ph=6.5, 2527U/mL, and 90% of the highest enzyme activity at ph=6, 2394U/mL. And the half-life of the DPE enzyme from C.cellulolyticum H10 is 6.3H, the optimal activity half-life of the DPE enzyme provided by the invention is 9.3H, and the high-temperature stability is better. Compared with the same volume of 3.6L of the fermentation tank, the DPE enzyme provided by the invention has optimal activity pH=7.0 and the highest enzyme activity is 2578U/mL. 95% of the highest enzyme activity at ph=6.5, 2449U/mL, 90% of the highest enzyme activity at ph=6, 2320U/mL; whereas the highest enzyme activity of DPE enzyme of C.cellulolyticum H10 is 2246U/mL, the DPE enzyme obtained by the invention has higher activity under neutral or acidic condition compared with the existing DPE enzyme.
Comparing the sequences of the two, the DPE enzyme provided by the invention has high similarity with DPE enzyme in Clostridium cellulolyticum H. But in combination with analysis of the Protein crystals of both (method references Chan, H. Et al, protein & Cell, 2012, 3, 123-131), it was found that the DPE enzyme of the present invention was altered from the amino acids near the key active site of the DPE enzyme in Clostridium cellulolyticum H, which directly resulted in a difference in performance between the two, such that the DPE enzyme activity of the present invention had an optimal pH of 7 and the highest enzyme activity under neutral conditions 2578U/mL was much higher than that of c.cellulolyticum H10; the stability of the DPE enzyme provided by the invention under the optimal temperature condition is far better than Clostridium cellulolyticum H, and the DPE enzyme provided by the invention is more suitable for industrial requirements.
The high-expression DPE enzyme discovered in the invention can be used for realizing the bioconversion production of D-psicose, and the enzyme catalytic reaction uses cheaper D-fructose as a substrate, so that the DPE enzyme has low cost and great advantages and industrial values.
It should be understood that while the present invention has been described by way of example in terms of its preferred embodiments, it is not limited to the above embodiments, but is capable of numerous modifications and variations by those skilled in the art. The fermentation process of the DPE enzyme can be adjusted and changed accordingly according to specific needs. It will thus be appreciated that those skilled in the art will be able to devise numerous alternative arrangements which, although not explicitly described herein, embody the principles of the invention and are included within its spirit and scope.
Claims (9)
1. The high-expression D-psicose-3-epimerase is characterized in that a nucleotide sequence for encoding the D-psicose-3-epimerase is shown as SEQ ID No.1 after codon optimization.
2. A recombinant vector or host bacterium for expressing the D-psicose-3-epimerase according to claim 1, wherein the recombinant vector or host bacterium is a recombinant vector or host bacterium comprising a nucleotide sequence encoding the D-psicose-3-epimerase, and the nucleotide sequence encoding the D-psicose-3-epimerase is shown in SEQ ID No.1.
3. The host bacterium of claim 2, wherein the host bacterium is bacillus subtilis.
4. The recombinant vector of claim 2, wherein the recombinant vector is pMA5.
5. A method for producing D-psicose-3-epimerase, wherein the method is characterized in that the D-psicose-3-epimerase is produced by fermentation using the host bacterium according to claim 2.
6. The method of claim 5, wherein the method comprises an inoculum size of 2% to 5% and the medium is formulated as follows: 20. 20 g/L casein hydrolysate, 15 g/L corn steep liquor extract, 1 g/L ammonium citrate, 40g/L glycerin, 2.31 g/L monopotassium phosphate, 0.2 g/L magnesium sulfate, 0.2 g/L calcium chloride, 20 mug/mL kanamycin final concentration and 20 mL trace element liquid.
7. The method of any one of claims 5-6, wherein the feed medium used in the method is formulated as follows: feed medium: glycerin 100 g/L, magnesium sulfate 7.89 g/L, casein hydrolysate 100 g/L, corn steep liquor extract 10g/L and trace element liquid 20 mL, wherein the feeding mode is as follows: 10 mL/h constant-speed feeding.
8. The method according to any one of claims 5 to 6, wherein the fermentation conditions in the method are temperature 37 ℃, rotation speed 400 rpm, ph=7.0, air flow 2.0 VVM for 48 hours.
9. Use of a D-psicose-3-epimerase as claimed in claim 1, a host bacterium as claimed in any one of claims 2 to 4 or a recombinant vector for the preparation of D-psicose.
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