CN112921025B - Mutant of epimerase, coding gene, amino acid sequence and application thereof - Google Patents
Mutant of epimerase, coding gene, amino acid sequence and application thereof Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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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- C12Y—ENZYMES
- 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 an epimerase mutant, a coding gene and an amino acid thereof, and application in producing D-psicose, and particularly relates to an epimerase mutant obtained by a genetic engineering method; the method improves the thermal stability of the epimerase mutant, simultaneously improves the optimum temperature of the enzyme by 10-20 ℃, can exert good catalytic effect without metal ions, greatly reduces the production cost, has higher catalytic efficiency in a D-fructose-boric acid solution, and provides an effective way for the enzymatic industrial production of D-psicose.
Description
Technical Field
The invention relates to an enzyme mutant, in particular to an epimerase mutant, a coding gene and an amino acid thereof, and application of the epimerase mutant in production of D-psicose.
Background
D-psicose is a carbon-3 isomer of D-fructose, an unusual monosaccharide, found in natural products. In recent years, D-psicose has received widespread attention for its health benefits, including reducing body fat accumulation as an inhibitor of low calorie sweeteners, liver lipogenic enzymes, and intestinal enzyme glycosidases. In nature, the content of D-psicose is very small and is very difficult to obtain. Epimerase derived from a wild strain without modification is poor in thermostability, and industrial application has certain limitations, so that the use time of the enzyme is short, the application cost is high, and the large-scale industrial production of D-psicose is limited. Therefore, the epimerase with good thermal stability still has few resources, and is not beneficial to the industrial application and popularization of D-psicose.
There are several patent applications for mutants of epimerase to improve the efficiency or effect of catalyzing fructose to produce psicose. The patents CN201911384895.2, CN201911378513.5, CN201911204720.9 and the like obtain high-temperature resistant enzymes through point mutation, but the optimum temperature is too high, which is not beneficial to industrial production, the half-life period at the optimum temperature is short, and the recycling value of the enzymes is not large; the half-life of the enzyme is prolonged by point mutation, random mutation and the like in patents CN201810059404.6, CN201610816801.4, CN201911099450.X and the like, the thermal stability is improved, but the reaction balance is not obviously shifted to the right, and the conversion rate is still about 30%. The enzyme activity of the mutant obtained by CN201910655539.3 is obviously improved, but the optimum temperature is not improved, the solubility of the substrate and the product is limited at the temperature, the production of the substrate with high concentration in the industry is limited, and the industrial amplification is not facilitated.
Therefore, how to simultaneously improve the thermal stability of the epimerase and the efficiency of producing the D-psicose by the epimerase, so that the epimerase can perform catalytic reaction at a higher substrate concentration, and an effective way is provided for the enzymatic industrial production of the D-psicose, which is a technical problem to be solved at present.
Disclosure of Invention
Aiming at the defects in the prior art, the invention obtains the epimerase mutant by a genetic engineering method; the heat stability of the epimerase mutant is improved, the optimum temperature of the epimerase mutant is improved by 10-20 ℃, the mutant can play a good catalytic effect in water without other buffer solutions, and the method provides an effective way for the industrial production of D-psicose by an enzyme method.
The invention provides an epimerase mutant, wherein the amino acid sequence of the mutant is shown as SEQ ID NO.4 or SEQ ID NO. 5.
The second aspect of the invention provides an epimerase mutant, wherein the nucleotide sequence of the mutant is shown as SEQ ID NO.2 or SEQ ID NO. 3; or the gene sequence of the mutant is a nucleotide sequence encoding the amino acid sequence of the epimerase as described above.
The third aspect of the invention provides a method for constructing an epimerase mutant, which comprises the following steps; obtaining random mutants according to a random mutation kit, and screening mutants with excellent performance. The kit in the method is any commercially available QuickMutationTM gene random mutation kit.
The fourth aspect of the invention provides an application of the epimerase mutant in the production of D-psicose.
Further, the use comprises carrying out the reaction using the cell in a catalytic system for D-psicose production.
Further, the catalytic system for producing the D-psicose comprises 100-1000 g/L of fructose and 0-576 g/L of boric acid, and the whole cell concentration of the mutant with the amino acid sequence shown as SEQ ID NO.4 or SEQ ID NO.5 is 15-45 g/L; the preferable whole cell concentration is 25-35 g/L.
Furthermore, the temperature of a catalytic system for producing D-psicose is controlled within the range of 65-90 ℃, the reaction pH can fluctuate within the range of 6.5-8.5, the boric acid concentration can fluctuate within the range of 72-288 g/L, and the D-fructose can fluctuate within the range of 200-800 g/L;
in the technical scheme, the preferable system temperature is controlled within the range of 75-85 ℃.
In the technical scheme, the preferable reaction pH value can be in the range of 7.0-8.0.
For the technical scheme, the preferable concentration of the reaction boric acid can be in the range of 72-216 g/L.
Compared with the prior art, the invention has the following beneficial effects:
the invention obtains epimerase mutants E.coli BL21-pET29(a) -DTEase-153-211 and E.coli BL21-pET29(a) -DTEase-203-237 by a genetic engineering method; compared with wild plants, the property of the strain is obviously improved, and is embodied in the following aspects: (1) the optimum temperature of the mutant is 10-20 ℃ higher than that of the original strain; (2) the production intensity at the optimum temperature is 1.54 times and 1.17 times of that of the original bacteria; (3) the thermal stability of the enzyme is obviously improved; (4) the mutant can play a good catalytic effect by using pure water as a reaction solution without adding metal ions, so that the reaction cost is reduced, the subsequent purification process is simplified, the production period is shortened, and the method has important significance for large-scale production; (5) in the mutant, the conversion rate of the psicose can be further improved to 73.2% by adding borate under the condition that the high-concentration fructose is 700g/L as a substrate. (6) The whole cells are used as the catalyst, so that the steps of cell crushing and enzyme purification can be eliminated, and the enzyme with better property is catalyzed by the whole cells, so that the step of immobilization is omitted, and the method has industrial feasibility.
Drawings
FIG. 1 SDS-PAGE analysis of epimerase in recombinant E.coli. Wherein Lane M is a molecular weight marker; 1, negative control 2, recombinant bacteria; 3, purifying the epimerase;
FIG. 2 temperature optima of mutants;
FIG. 3 thermal stability data;
FIG. 4 the effect of different molar ratios of boric acid addition on the conversion of psicose.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
In the present invention, percentages and percentages are by mass unless otherwise specifically indicated. Unless otherwise specified, the experimental methods used are conventional methods, and the materials, reagents and the like used are commercially available.
The enzymatic properties, thermostability and half-life detection methods referred to in the examples of the present application are as follows:
1. determination of enzymatic Properties
Definition of enzyme activity: 1U enzyme activity is defined as the amount of enzyme required to catalyze the production of 1moL of D-psicose per minute at 60 ℃ as one enzyme activity unit (U), and the reaction system contains 100g/L fructose and 30g/L whole cells and is reacted in 50mM Tris-HCl (pH 7.5). The D-psicose production was checked by HPLC to determine the enzyme activity.
The enzymatic properties after mutation can be changed, the optimal enzyme activity conditions are explored, and the enzymatic properties of the non-mutated WT (original strain) and the mutant under the same conditions are analyzed and compared respectively.
2. Thermal stability and half-life assays
Determination of temperature stability: the enzyme solution was diluted to a concentration of 30g/L with PBS, incubated at 35 ℃ and 45 ℃ and the residual enzyme activity was measured at different times of incubation.
t1/2The value refers to the corresponding time when the residual enzyme activity is 50% after the enzyme is treated at a specific temperature for a period of time. The specific determination method is as follows: the residual enzyme activities of the enzymes treated at 35 ℃ and 45 ℃ for different periods of time were measured and calculated, respectively, using the enzyme activities without heat treatment as 100%. The treatment time is used as the abscissa and Ln (% residual enzyme activity) is used as the ordinate, a curve of time-Ln (% residual enzyme activity) is drawn, and t is calculated according to the graph1/2＝Ln2/Kd，KdIs the slope of the graph.
EXAMPLE 1 construction of mutants
The epimerase Gene DTEase (Gene ID:56822880) from the bacterium Flavonifractalutii, the codon-optimized sequence of which is shown in SEQ ID NO: 1.
Obtaining of mutants
The mutant is a method for obtaining the mutant by mutation on the basis of the epimerase amino acid sequence translated by SEQ ID NO. 1, comprising the following steps:
1. according to QuickMutionTMDesigning a primer and random mutation for the gene random mutation kit, wherein the primer sequence is as follows:
random-F：gatataCATATGAACCCGATCGGTATGC；SEQ ID NO.6；
random-R：ggtgCTCGAGCGCGGTCAGTTC；SEQ ID NO.7；
2. random mutation PCR reaction
Random mutation PCR reaction the random mutation PCR reaction system was set with reference to the following table:
TABLE 1
| Reagent | Final concentration | Volume of |
| Double distilled water or MilliQ water | - | 12.2μL |
| RandomMut buffer(10×) | 1× | 2μL |
| Mutation enhancer(10×) | 1× | 2uL |
| dNTP(2.5mM) | 0.25mM | 2μL |
| Template DNA | 0.2pg-5ng/μL | 1μL |
| Primer mixtures (10. mu.M each) | 0.2. mu.M each | 0.4μL |
| RandomMut DNA polymerase | - | 0.4μL |
| Total volume | - | 20μL |
The PCR instrument was set up according to the following parameters:
TABLE 2
3. Transformation of competent cells
10 μ L of PCR product was taken and detected in 1% agarose, and after observing the target band, 1 μ L of DMT enzyme was added to the remaining PCR product, and incubated for 1 h. Adding 2-5 μ L of the digested product into 50 μ L of LDMT competent cells, mixing, ice-cooling for 30min, heating in 42 deg.C water bath for 45s, and immediately placing on ice for 2-5 min. Then 250. mu.L of LB medium was added, cultured at 200rpm and 37 ℃ for 1h, and 100. mu.L of the bacterial solution was applied to the kanamycin-resistant plate for overnight culture. A plurality of mutants are obtained, wherein the mutants with outstanding properties are named as E.coli BL21-pET29(a) -DTEase-153-211 and E.coli BL21-pET29(a) -DTEase-203-237, and the amino acid sequences of the proteins are SEQ ID NO:4 and SEQ ID NO:5 respectively.
4. Expression of wild plants and mutants
The recombinant Escherichia coli were cultured in LB medium to E.coli BL21-pET29(a) -DTEase, E.coli BL21-pET29(a) -DTEase-153-211 and E.coli BL21-pET29(a) -DTEase-203-237, overnight at 37 ℃ and 200rpm, inoculated in 5% inoculum size into the fermentation medium, and expanded to the OD of the cells6000.3mM IPTG was added thereto, the mixture was induced overnight at low temperature, and whole cells were harvested by centrifugation as a catalyst.
The seed culture medium and the fermentation culture medium are as follows: 5g/L yeast extract powder, 10g/L tryptone, 10g/L NaCl10g/L, sterilizing at 121 ℃ for 15min, cooling, and adding 100mg/L kanamycin.
Example 2
In a 1L system, 700g of D-fructose and 144g of boric acid are weighed, water is added to adjust the pH value to be 7.5, whole cells of E.coli BL21-pET29(a) -DTEase-203-237 are added to reach the concentration of 30g/L, and the system is reacted for 1h at the temperature of 80 +/-1 ℃. Samples were taken every 10min for yield analysis.
The conversion rate of substrate fructose reaches 72% at 50min, the concentration of product allulose reaches 504g/L, and the production intensity is 607.2 g/L.h, which is 1.54 times of that of wild plants.
In a 1L system, 700g of D-fructose and 144g of boric acid are weighed, water is added to adjust the pH value to be 7.5, and whole cells of E.coli BL21-pET29(a) -DTEase-153-211 are added to reach the concentration of 30g/L, and the system is reacted for 1h at the temperature of 70 +/-1 ℃. Samples were taken every 10min for yield analysis.
After 1 hour, the conversion rate of the substrate fructose reaches 66.1%, the concentration of the product allulose reaches 462.7g/L, and the production intensity is 462.7 g/L.h, which is 1.17 times that of the wild strain.
Example 3 thermal stability
The optimum temperature of each cell is shown in FIG. 2. The optimum temperature of the mutant E.coli BL21-pET29(a) -DTEase-153-211 was increased from 60 ℃ to 75 ℃ and E.coli BL21-pET29(a) -DTEase-203-237 was increased to 85 ℃.
The whole cells of E.coli BL21-pET29(a) -DTEase, E.coli BL21-pET29(a) -DTEase-153-211 and E.coli BL21-pET29(a) -DTEase-203-237 are respectively placed at 60 ℃ and 90 ℃ for incubation for a certain time, and the reaction is carried out at the optimal temperature of each cell by using 100g/L of D-fructose solution every 1-2 h, and the activity change is detected by using the reaction transformation effect of the cells which are not incubated as a control.
The reaction results are shown in the left and right panels of FIG. 3. The mutant has excellent thermal stability, wherein the E.coli BL21-pET29(a) -DTEase-153-211 still maintains 93 percent of activity after being incubated for 1h at 90 ℃, and the residual enzyme activity is still 37 percent after being incubated for 12 h.
Half-life:
TABLE 2 half-lives of wild strains and mutant strains at 60 ℃ and 90 ℃
Example 4 efficient Synthesis of D-psicose in an aqueous System
100g/L of D-fructose was dissolved in 50mM pH7.5 phosphate buffer and pH7.5 water, respectively, and whole cells of E.coli BL21-pET29(a) -DTEase-203-237 or E.coli BL21-pET29(a) -DTEase-153-211 were added to a final concentration of 30g/L, and the reaction was carried out at the respective optimum temperatures. Samples were taken periodically for yield analysis.
When purified water was used as the reaction solution, the conversion of E.coli BL21-pET29(a) -DTEase-203-237 cells to D-psicose production in the buffer system was 116.9%, and the conversion of E.coli BL21-pET29(a) -DTEase-153-211 cells to D-psicose production in the buffer system was 108.2%.
Example 5 Borate addition increases psicose conversion
The conversion rate of the psicose can be greatly improved by adding the borate into the reaction system. The epimerase catalyzes the reaction of fructose to psicose, which is a bidirectional reaction. When the concentration of the two kinds of sugar reaches a certain proportion in the reaction system, the reaction is in an equilibrium state. After the borate is complexed with the psicose, the free psicose in the reaction system is reduced, the reaction is shifted to the right, and the conversion rate of the psicose is improved. Boric acid was added to a reaction system of 100g/L so that the molar ratios of boric acid to fructose were 0:1, 0.3:1, 0.5:1,1:1 and 2:1, respectively, and whole cells of E.coli BL21-pET29(a) -DTEase-203-237 were added to a concentration of 30g/L, and the reaction was carried out at a pH of 7.5 and 80. + -. 1 ℃.
The reaction effect is shown in the figure, when the ratio of the addition amount of the boric acid to the fructose concentration is 0.5 mol: the conversion rate of allulose reaches the highest value at 1mol, and reaches 73.2%.
Comparative example 1
Efficient synthesis of D-psicose in water system
100g/L of D-fructose was dissolved in 50mM phosphate buffer pH7.5 and water pH7.5, respectively, and E.coli BL21-pET29(a) -DTEase whole cells were added to a final concentration of 30g/L, and the reaction was carried out at the respective optimum temperatures. Samples were taken periodically for yield analysis.
When purified water was used as the reaction solution, the conversion of E.coli BL21-pET29(a) -DTEase into D-psicose in the buffer system was 99.6%.
Comparative example 2
Response Effect of wild plants
700g of D-fructose and 144g of boric acid were weighed in a 1L system, added with water to adjust the pH to 7.5, and E.coli BL21-pET29(a) -DTEase whole cells were added, and the system was reacted at 60. + -. 1 ℃ for 1 hour. Samples were taken periodically for yield analysis.
The conversion of psicose reached 56.5% (1h), corresponding to a concentration of 395.5g/L, with a production strength of 395.5 g/L/h.
Comparative example 3
Effect of other mutants
Other mutants obtained by random mutagenesis were expressed in E.coli BL21 to obtain E.coli BL21-pET29(a) -DTEase-222-285, E.coli BL21-pET29(a) -DTEase-235-290, E.coli BL21-pET29(a) -DTEase-247-310, and E.coli BL21-pET29(a) -DTEase-252-325, and the cells were obtained by culturing in the above-mentioned manner.
In a 1L system, 700g of D-fructose and 144g of boric acid are weighed, water is added to adjust the pH value to be 7.5, and whole cells of E.coli BL21-pET29(a) -DTEase-222-285, E.coli BL21-pET29(a) -DTEase-235-290, E.coli BL21-pET29(a) -DTEase-247-310 and E.coli BL21-pET29(a) -DTEase-252-325 are added, and the system is reacted for 1h at the temperature of 60 +/-1 ℃. Samples were taken periodically for yield analysis.
The conversion rate of the psicose is only 12.3-13.7%, the corresponding concentration is 86.1-92 g/L, and the generation intensity is 78.2-86.1 g/L/h.
In summary, comparative and example data analysis of the present application shows that: the obtained mutants E.coli BL21-pET29(a) -DTEase-153-211 and E.coli BL21-pET29(a) -DTEase-203-237 can obviously improve the production strength when applied to the synthesis process of D-psicose, while the mutants E.coli BL21-pET29(a) -DTEase-222-285, E.coli BL21-pET29(a) -DTEase-235-290, E.coli BL21-pET29(a) -DTEase-247-310 and E.coli BL21-pET29(a) -DTEase-252-325. The same positive effect was not achieved after mutation, indicating that random mutation may possibly, but not necessarily, result in better performing mutants.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and those skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Sequence listing
<110> Daliano Yi biological shares Ltd
<120> epimerase mutant, coding gene, amino acid sequence and application thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 885
<212> DNA
<213> codon-optimized epimerase gene (none)
<400> 1
atgaacccga tcggtatgca ctacggtttc tggtctcaca actgggatga aatcgcgtac 60
atcccgctga tggaaaaact ggcatggctg ggttttgata tctgcgaagt tgcgtctgct 120
gaatggggtt attacgatga tgctcgtctg cgtgaactga aagcttgcgc tgatcataat 180
ggtctgggta ttacctactc tattggcctg gaagcgaaat atgatctggc gtctgatgat 240
ccggctgttc gtgaaaacgg catccgtcac gttacccgta ttctggaatc tatgccaaaa 300
gttggtgcag ctattctgaa cggcgtgtct tacgcgggtt ggcaggcgtt accggatcac 360
ggcatcaccc tggatgaaaa acgtcgtaaa gaagaactgg cgctggaatc tatgagccgt 420
ctgatgaaag ttgctgaaga ttgcggtgtt ctgtattgtt gtgaagttgt gaaccgtttt 480
gaacagtatt tactgaacac cgctaaagaa ggtgttgaat tcgttaaacg tctgggtagc 540
ccgaacgctc gtgttctgct ggataccttc cacatgaaca tcgaagaaga ttctatggtt 600
gatgctattt tagaagcggg cccgtggctg ggtcatttcc atgttggtga aaacaaccgt 660
cgtccggcgg gtagcaccaa ccgtctgccg tggaaagata tggcggcggc tctgaaacag 720
gttaactacc agggtgcgat tgttatggaa ccgttcgttc tgatgggtgg caccattccg 780
tatgatatca aagtttggcg tgatctgtct ggtggtgcgg gtgaagcggg cctggatgaa 840
atggcgggtc gtgcgtgtcg ttttctgaaa gaactgaccg cgtaa 885
<210> 2
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<212> DNA
<213> epimerase mutant E.coli BL21-pET29(a) -DTEase-153-211 base sequence (none)
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atgaacccga tcggtatgca ctacggtttc tggtctcaca actgggatga aatcgcgtac 60
atcccgctga tggaaaaact ggcatggctg ggttttgata tctgcgaagt tgcgtctgct 120
gaatggggtt attacgatga tgctcgtctg cgtgaactga aagcttgcgc tgatcataat 180
ggtctgggta ttacctactc tattggcctg gaagcgaaat atgatctggc gtctgatgat 240
ccggctgttc gtgaaaacgg catccgtcac gttacccgta ttctggaatc tatgccaaaa 300
gttggtgcag ctattctgaa cggcgtgtct tacgcgggtt ggcaggcgtt accggatcac 360
ggcatcaccc tggatgaaaa acgtcgtaaa gaagaactgg cgctggaatc tatgagccgt 420
ctgatgaaag ttgctgaaga ttgcggtgtt ctgtattgtt gtgaagttgt gaaccgtttt 480
gaacagtatt tactgaacac cgctaaagaa ggtgttgaat tcgttaaacg tctgggtagc 540
ccgaacgctc gtgttctgct ggataccttc cacatgaaca tcgaagaaga ttctatggtt 600
gatgctattt tagaagcggg cccgtggctg tgccatttcc atgttggtga aaacaaccgt 660
cgtccggcgg gtagcaccaa ccgtcgctcg tggaaagata tggcggcgtg cctgaaacag 720
gttaactacc agggtgcgat tgttatggaa ccgttcgttc tgatgggtgg caccattccg 780
tatgatatca aagtttggcg tgatctgtct ggtggtgcgg gtgaagcggg cctggatgaa 840
atggcgggtc gtgcgtgtcg ttttctgaaa gaactgaccg cgtaa 885
<210> 3
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<213> epimerase mutant E.coli BL21-pET29(a) -DTEase-203-237 base sequence (none)
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atgaacccga tcggtatgca ctacggtttc tggtctcaca actgggatga aatcgcgtac 60
atcccgctga tggaaaaact ggcatggctg ggttttgata tctgcgaagt tgcgtctgct 120
gaatggggtt attacgatga tgctcgtctg cgtgaactga aagcttgcgc tgatcataat 180
ggtctgggta ttacctactc tattggcctg gaagcgaaat atgatctggc gtctgatgat 240
ccggctgttc gtgaaaacgg catccgtcac gttacccgta ttctggaatc tatgccaaaa 300
gttggtgcag ctattctgaa cggcgtgtct tacgcgggtt ggcaggcgtt accggatcac 360
ggcatcaccc tggatgaaaa acgtcgtaaa gaagaactgg cgctggaatc tatgagccgt 420
ctgatgaaag ttgctgaaga ttgcggtgtt ctgtattgtt gtgaagttgt gaaccgtttt 480
gaacagtatt tactgaacac cgctaaagaa ggtgttgaat tcgttaaacg tctgggtagc 540
ccgaacgctc gtgttctgct ggataccttc cacatgaaca tcgaagaaga ttctatggtt 600
gatgcttgct tagaagcggg cccgtggctg ggtcatttcc atgttggtga aaacaaccgt 660
cgtccggcgg gtagcaccaa ccgtctgccg tggaaagata tggcggcgtg cctgaaacag 720
gttaactacc agggtgcgat tgttatggaa ccgttcgttc tgatgggtgg caccattccg 780
tatgatatca aagtttggcg tgatctgtct ggtggtgcgg gtgaagcggg cctggatgaa 840
atggcgggtc gtgcgtgtcg ttttctgaaa gaactgaccg cgtaa 885
<210> 4
<211> 294
<212> PRT
<213> epimerase mutant E.coli BL21-pET29(a) -DTEase-153-211 amino acid sequence (none)
<400> 4
Met Asn Pro Ile Gly Met His Tyr Gly Phe Trp Ser His Asn Trp Asp
1 5 10 15
Glu Ile Ala Tyr Ile Pro Leu Met Glu Lys Leu Ala Trp Leu Gly Phe
20 25 30
Asp Ile Cys Glu Val Ala Ser Ala Glu Trp Gly Tyr Tyr Asp Asp Ala
35 40 45
Arg Leu Arg Glu Leu Lys Ala Cys Ala Asp His Asn Gly Leu Gly Ile
50 55 60
Thr Tyr Ser Ile Gly Leu Glu Ala Lys Tyr Asp Leu Ala Ser Asp Asp
65 70 75 80
Pro Ala Val Arg Glu Asn Gly Ile Arg His Val Thr Arg Ile Leu Glu
85 90 95
Ser Met Pro Lys Val Gly Ala Ala Ile Leu Asn Gly Val Ser Tyr Ala
100 105 110
Gly Trp Gln Ala Leu Pro Asp His Gly Ile Thr Leu Asp Glu Lys Arg
115 120 125
Arg Lys Glu Glu Leu Ala Leu Glu Ser Met Ser Arg Leu Met Lys Val
130 135 140
Ala Glu Asp Cys Gly Val Leu Tyr Cys Cys Glu Val Val Asn Arg Phe
145 150 155 160
Glu Gln Tyr Leu Leu Asn Thr Ala Lys Glu Gly Val Glu Phe Val Lys
165 170 175
Arg Leu Gly Ser Pro Asn Ala Arg Val Leu Leu Asp Thr Phe His Met
180 185 190
Asn Ile Glu Glu Asp Ser Met Val Asp Ala Ile Leu Glu Ala Gly Pro
195 200 205
Trp Leu Cys His Phe His Val Gly Glu Asn Asn Arg Arg Pro Ala Gly
210 215 220
Ser Thr Asn Arg Leu Pro Trp Lys Asp Met Ala Ala Ala Leu Lys Gln
225 230 235 240
Val Asn Tyr Gln Gly Ala Ile Val Met Glu Pro Phe Val Leu Met Gly
245 250 255
Gly Thr Ile Pro Tyr Asp Ile Lys Val Trp Arg Asp Leu Ser Gly Gly
260 265 270
Ala Gly Glu Ala Gly Leu Asp Glu Met Ala Gly Arg Ala Cys Arg Phe
275 280 285
Leu Lys Glu Leu Thr Ala
290
<210> 5
<211> 294
<212> PRT
<213> epimerase mutant E.coli BL21-pET29(a) -DTEase-203-237 amino acid sequence (none)
<400> 5
Met Asn Pro Ile Gly Met His Tyr Gly Phe Trp Ser His Asn Trp Asp
1 5 10 15
Glu Ile Ala Tyr Ile Pro Leu Met Glu Lys Leu Ala Trp Leu Gly Phe
20 25 30
Asp Ile Cys Glu Val Ala Ser Ala Glu Trp Gly Tyr Tyr Asp Asp Ala
35 40 45
Arg Leu Arg Glu Leu Lys Ala Cys Ala Asp His Asn Gly Leu Gly Ile
50 55 60
Thr Tyr Ser Ile Gly Leu Glu Ala Lys Tyr Asp Leu Ala Ser Asp Asp
65 70 75 80
Pro Ala Val Arg Glu Asn Gly Ile Arg His Val Thr Arg Ile Leu Glu
85 90 95
Ser Met Pro Lys Val Gly Ala Ala Ile Leu Asn Gly Val Ser Tyr Ala
100 105 110
Gly Trp Gln Ala Leu Pro Asp His Gly Ile Thr Leu Asp Glu Lys Arg
115 120 125
Arg Lys Glu Glu Leu Ala Leu Glu Ser Met Ser Arg Leu Met Lys Val
130 135 140
Ala Glu Asp Cys Gly Val Leu Tyr Cys Cys Glu Val Val Asn Arg Phe
145 150 155 160
Glu Gln Tyr Leu Leu Asn Thr Ala Lys Glu Gly Val Glu Phe Val Lys
165 170 175
Arg Leu Gly Ser Pro Asn Ala Arg Val Leu Leu Asp Thr Phe His Met
180 185 190
Asn Ile Glu Glu Asp Ser Met Val Asp Ala Cys Leu Glu Ala Gly Pro
195 200 205
Trp Leu Gly His Phe His Val Gly Glu Asn Asn Arg Arg Pro Ala Gly
210 215 220
Ser Thr Asn Arg Leu Pro Trp Lys Asp Met Ala Ala Cys Leu Lys Gln
225 230 235 240
Val Asn Tyr Gln Gly Ala Ile Val Met Glu Pro Phe Val Leu Met Gly
245 250 255
Gly Thr Ile Pro Tyr Asp Ile Lys Val Trp Arg Asp Leu Ser Gly Gly
260 265 270
Ala Gly Glu Ala Gly Leu Asp Glu Met Ala Gly Arg Ala Cys Arg Phe
275 280 285
Leu Lys Glu Leu Thr Ala
290
<210> 6
<211> 28
<212> DNA
<213> random primer-F (none)
<400> 6
gatatacata tgaacccgat cggtatgc 28
<210> 7
<211> 22
<212> DNA
<213> random primer-R (none)
<400> 7
ggtgctcgag cgcggtcagt tc 22
Claims (10)
1. An epimerase mutant characterized in that: the amino acid sequence of the protein for coding the mutant is shown as SEQ ID NO.5 or SEQ ID NO. 4.
2. The epimerase mutant according to claim 1, which is characterized in that: the gene sequence of the coding mutant is shown as SEQ ID NO.2 or 3, or the gene sequence of the coding mutant is a nucleotide sequence of the amino acid sequence of the coding claim 1.
3. Use of the epimerase mutant according to claim 1 for producing D-psicose.
4. Use according to claim 3, characterized in that: the application comprises the steps of using the epimerase mutant cell in a catalytic system for producing D-psicose to perform reaction; the catalytic system comprises 100-1000 g/L of fructose, 0-576 g/L of boric acid and 15-45 g/L of whole cells containing the epimerase mutant as described in claim 1.
5. Use according to claim 4, characterized in that: in the catalytic system for producing D-psicose, the whole cell concentration of the epimerase mutant as set forth in claim 1 is 25-35 g/L.
6. Use according to claim 4, characterized in that: the temperature of a catalytic system in the catalytic system for producing the D-psicose is 65-90 ℃; the reaction pH is 6.5-8.5; the concentration of boric acid is 72-288 g/L, and the concentration of D-fructose is 200-800 g/L.
7. Use according to claim 6, characterized in that: the system temperature in the catalytic system for producing the D-psicose is controlled within the range of 75-85 ℃.
8. Use according to claim 6, characterized in that: the reaction pH value in the catalytic system for producing the D-psicose is 7.0-8.0.
9. Use according to claim 6, characterized in that: the concentration of boric acid in a catalytic system for producing D-psicose is 72-216 g/L.
10. Use according to claim 6, characterized in that: the concentration of D-fructose in the catalytic system for producing D-psicose is 500-700 g/L.
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| CN202110226700.2A CN112921025B (en) | 2021-02-27 | 2021-02-27 | Mutant of epimerase, coding gene, amino acid sequence and application thereof |
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| CN112921025B true CN112921025B (en) | 2022-06-10 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710330A (en) * | 2014-01-03 | 2014-04-09 | 江南大学 | High-catalytic-activity mutant enzyme for D-allulose 3-epimerase and application thereof |
| CN103849612A (en) * | 2014-01-03 | 2014-06-11 | 江南大学 | 68th and 109th double mutant enzyme of D-psicose 3-epimerase and application thereof |
| CN103849613A (en) * | 2014-01-03 | 2014-06-11 | 江南大学 | Thermal stability improved mutant enzyme of D-psicose 3-epimerase and application thereof |
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2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710330A (en) * | 2014-01-03 | 2014-04-09 | 江南大学 | High-catalytic-activity mutant enzyme for D-allulose 3-epimerase and application thereof |
| CN103849612A (en) * | 2014-01-03 | 2014-06-11 | 江南大学 | 68th and 109th double mutant enzyme of D-psicose 3-epimerase and application thereof |
| CN103849613A (en) * | 2014-01-03 | 2014-06-11 | 江南大学 | Thermal stability improved mutant enzyme of D-psicose 3-epimerase and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| sugar phosphate isomerase/epimerase [Flavonifractor plautii];NCBI Reference Sequence: WP_007494289.1;《NCBI》;20170704;全文 * |
| 我国生物合成D-阿洛酮糖的研究及产业化进展;郭元亨等;《现代食品》;20200330(第06期);40-46页 * |
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