High-temperature-resistant D-psicose-3-epimerase mutant and application thereof in preparation of D-psicose

Abstract

The invention discloses a high-temperature-resistant D-psicose-3-epimerase mutant and application of the high-temperature-resistant D-psicose-3-epimerase mutant in preparation of D-psicose. The mutant is obtained by performing single mutation or multiple mutations on the 17th site, the 118th site, the 121st site, the 165 site, the 197th site and the 249th site of an amino acid sequence shown in SEQ ID NO.2. The invention further discloses a preparation method of the high-temperature-resistant D-psicose-3-epimerase mutant. The mutant obtained through screening can tolerate the high temperature of 45-70 DEG C, the Tm value of the mutant enzyme is increased, the half-life period of the enzyme is prolonged, the affinity and conversion efficiency of the enzyme to a substrate D-psicose are improved, a relatively rare enzyme library is expanded, then the production cost is reduced, and the industrial application value of the D-psicose-3-epimerase is further improved.

Description

(I) Technical field

[0001] The invention relates to a thermostable D-psicose-3-epimerase mutant and application thereof in the preparation of D-psicose. (II) Background technology

[0002] As a functional rare sugar, allulose has 70% of the sweetness of sucrose, but only 0.31% of the calories of sucrose. It is considered an ideal substitute for sucrose and fructose syrup. At the same time, in 2011, allulose was approved by the U.S. Food and Drug Administration (FDA) as a safe food additive, and was further excluded from the "added sugar" and "total sugar" labels in 2019. Allulose has specific physiological mechanisms in the prevention of obesity, treatment of atherosclerosis, anti-hyperlipidemia, anti-inflammation, antioxidant, anti-hyperglycemia and neuroprotection. In addition, D-allulose can also improve food gel behavior, increase flavor and reduce oxidation reactions during food processing and storage. Therefore, it is widely used in beverages, healthy foods and other fields, and has broad prospects.

[0003] Due to its rarity, allulose is difficult to obtain in large quantities from nature. Chemical synthesis has problems such as undesirable side reactions, difficult purification, and serious pollution, and no breakthrough has been made in industrial application. In contrast, biological pathways have the advantages of high specificity and environmental friendliness. According to the Izumoring strategy, D-psicose can be obtained by D-psicose-3-isomerase-catalyzed D-fructose isomerization, so D-psicose-3-isomerase has become the research focus of scholars. However, wild-type D-psicose-3-isomerase often has problems such as low catalytic activity and poor thermal stability, which cannot meet the needs of industrial production processes. (III) Summary of the invention

[0004] The present invention aims to provide a thermostable D-psicose-3-epimerase (ClDAE) mutant and its application in the preparation of D-psicose, thereby expanding the source of D-psicose-3-epimerase, effectively improving the thermal stability and conversion efficiency of the enzyme, improving the production efficiency of D-psicose, and increasing the industrial application potential of D-psicose-3-epimerase.

[0005] The technical solution adopted by the present invention is:

[0006] The present invention provides a thermostable D-psicose-3-epimerase mutant, wherein the mutant is obtained by subjecting the 17th, 118th, 121st, 165th, 197th and 249th positions of the amino acid sequence shown in SEQ ID NO.2 to single mutation or multiple mutation.

[0007] Furthermore, the D-psicose-3-epimerase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated into one of the following: (1) asparagine at position 17 is mutated into arginine; (2) glycine at position 118 is mutated into lysine; (3) histidine at position 121 is mutated into aspartic acid; (4) threonine at position 165 is mutated into cysteine; (5) isoleucine at position 197 is mutated into phenylalanine; (6) phenylalanine at position 249 is mutated into tyrosine; (7) histidine at position 121 is mutated into aspartic acid and phenylalanine at position 249 is mutated into tyrosine, the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.3.

[0008] Due to the particularity of the amino acid sequence, any variant of the polypeptide of the amino acid sequence shown in SEQ ID NO.4, such as its conservative variant, biologically active fragment or derivative, as long as the fragment or polypeptide variant of the polypeptide has a homology of more than 95% with the aforementioned amino acid sequence, belongs to the scope of protection of the present invention. The change may include the deletion, insertion or substitution of amino acids in the amino acid sequence; for conservative changes of the variant, the replaced amino acid has a similar structure or chemical property to the original amino acid, such as replacing isoleucine with leucine, and the variant may also have non-conservative changes, such as replacing glycine with tryptophan.

[0009] The present invention also provides a gene encoding the D-psicose-3-epimerase mutant.

[0010] Due to the particularity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO.3, as long as it has more than 95% homology with the polynucleotide, belongs to the scope of protection of the present invention. The variant of the polynucleotide refers to a polynucleotide sequence with one or more nucleotide changes. The variant of this polynucleotide can be an allelic variant or an abiotic variant, including a substitution variant, a deletion variant and an insertion variant. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of encoding amino acids.

[0011] The present invention also provides a recombinant plasmid containing the D-psicose-3-epimerase mutant encoding gene, and a recombinant genetic engineering bacterium constructed by the recombinant plasmid; the recombinant plasmid uses the vector pET28a as a basic vector; the host cell of the engineering bacterium is preferably Escherichia coli, and can also be Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae or lactic acid bacteria.

[0012] The present invention also provides an application of the D-psicose-3-epimerase mutant in the preparation of D-psicose by microbial catalytic D-fructose isomerization. The application comprises: using wet bacteria obtained by fermentation induction culture of recombinant Escherichia coli containing the D-psicose-3-epimerase mutant gene or pure enzyme solution extracted by ultrasonic crushing of wet bacteria as an enzyme source, using D-fructose as a substrate, using cobalt ions as a catalyst, and using a pH 6.5 buffer as a reaction medium to form a reaction system, reacting at 50 to 80° C. (preferably 70° C.) and 1000 r / min, and obtaining a mixture of D-fructose and D-psicose after the reaction is complete.

[0013] Furthermore, in the reaction system, the amount of wet bacteria is 2.5-10 g / L, preferably 2.5 g / L; the final concentration of cobalt ions is 0.1-10 mM, preferably 1 mM; the initial concentration of substrate is 50-700 g / L, preferably 50 g / L. The cobalt ions are added in the form of CoCl2 aqueous solution.

[0014] Furthermore, the wet bacterial cells were prepared as follows: recombinant Escherichia coli containing a D-psicose-3-epimerase mutant encoding gene was inoculated into an LB liquid culture medium containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37°C and 200 r / min until OD 600 =0.8~1.0, obtain seed solution; transfer the seed solution into LB liquid medium containing kanamycin at a final concentration of 50 μg / mL at a volume concentration of 1~5%, and culture at 37°C and 180r / min until OD 600 =0.6-0.8, add IPTG with a final concentration of 0.1 mM, induce culture at 28°C and 180 r / min for 12-14 h to obtain induced culture bacterial suspension, centrifuge the induced culture bacterial suspension, and collect wet bacteria.

[0015] Further, the pure enzyme solution is prepared according to the following steps: 1g of wet bacteria is suspended in 10mL of pH 6.5, 50mM sodium phosphate buffer, ultrasonically disrupted at 120W for 30min, ultrasonicated for 2s, paused for 2s, centrifuged, and the supernatant is taken to obtain a crude enzyme solution; the crude enzyme solution is purified using a nickel-NTA affinity chromatography column (Bio-Scale Mini Profinity IMAC pre-packed column, 40mm length×12.6mm inner diameter), the chromatography column is first equilibrated with an equilibrium buffer, the crude enzyme solution is loaded at a rate of 1mL / min for 4 column volumes, and then the eluent is used to elute at a rate of 2mL / min, and the ultraviolet detector and the conductivity detector are used for monitoring, and the corresponding eluent is collected when the signal of the ultraviolet detector and the signal of the conductivity detector rise at the same time, and the collection is stopped when the signal of the conductivity detector remains unchanged and the signal of the ultraviolet detector decreases, and the collected enzyme solution is the pure enzyme solution; the equilibrium buffer composition: 50mMPB buffer, 300mM NaCl, 20mM imidazole, pH 8.0; the eluent composition: 50 mM PB buffer, 300 mM NaCl, 500 mM imidazole, pH 8.0.

[0016] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0017] (1) The present invention screened out a thermostable D-psicose-3-epimerase mutant that can withstand a high temperature of 45 to 70°C, thereby increasing the Tm value of the mutant enzyme and prolonging the half-life of the enzyme. 1 / 2 The value was 2295min, which was 38.25 times that of the wild-type ClDAE; the Tm value increased from 58.5℃ of the wild-type ClDAE to 71.3℃, an increase of 12.8℃.

[0018] (2) The mutant provided by the present invention improves the affinity and conversion efficiency of the enzyme for the substrate D-psicose. When the substrate concentration is 50 g / L and the reaction temperature is 70°C, the enzyme activity of the wild-type ClDAE reaches 2324.6 U / (g·wet cellweight) and the conversion rate reaches 30%; the enzyme activity of the mutant ClDAE / H121D / F249Y reaches 2960.6 U / (g·wet cellweight) and the conversion rate reaches 34.1%.

[0019] (3) The mutant obtained in the present invention significantly improves the thermal stability and catalytic ability of D-psicose-3-epimerase, expands the relatively scarce enzyme library, thereby reducing production costs, and further improving the industrial application value of D-psicose-3-epimerase. (IV) Description of the drawings

[0020] Figure 1 This is the SDS-PAGE electrophoresis diagram of the dialyzed enzyme solution of Example 3; Lane M: Marker, Lane 1: wild-type enzyme ClDAE, Lane 2: ClDAE / H121D, Lane 3: ClDAE / F249Y, Lane 4: ClDAE / H121D / F249Y.

[0021] Figure 2 This is a bar graph showing the relative enzyme activities of the single-site mutant (A) and the double-site mutant (B) in Example 4.

[0022] Figure 3 The thermal stability diagrams of the wild-type enzyme ClDAE (A) and the mutant enzyme ClDAE / H121D / F249Y (B) at different temperatures in Example 6.

[0023] Figure 4 This is the optimal reaction temperature for the wild-type enzyme ClDAE and the mutant enzyme ClDAE / H121D / F249Y in Example 7.

[0024] Figure 5 The T of the wild-type enzyme ClDAE and the mutant enzyme ClDAE / H121D / F249Y pure enzyme in Example 8 m picture.

[0025] Figure 6 The figure is the conversion rate of the mutant enzyme ClDAE / H121D / F249Y in Example 9 at different substrate concentrations. (V) Specific implementation methods

[0026] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0027] LB liquid medium (g / L) composition: tryptone 10, yeast powder 5, NaCl 10, the solvent is ultrapure water; LB solid medium is added with 20g / L agar; high pressure sterilization; before use, add kanamycin with a final concentration of 50μg / mL.

[0028] Example 1: Construction of single-site mutants of D-psicose-3-epimerase

[0029] (1) Construction of wild-type recombinant bacteria

[0030] The wild-type D-psicose-3-epimerase ClDAE (Genbank number: MBQ7726265.1) from Clostridia bacterium in Genbank was artificially synthesized, with an amino acid sequence of SEQ ID NO.2 and a nucleotide sequence of SEQ ID NO.1, and was ligated between the NcoI and XhoI restriction sites of the vector pET28a to construct a recombinant vector pET28a / ClDAE. The wild-type recombinant bacteria E. coli BL21 (DE3) / pET28a / ClDAE were constructed using the method in step (3), and the corresponding enzyme was recorded as the wild-type enzyme ClDAE.

[0031] SEQ ID NO.2

[0032] MKNPVGIYYAYWKEWNADFIPYIKKVKKLGF

[0033] DVLEINAGSITEMTSAQRITLASAAKEEGIKL

[0034] TYCIGLPAQYDVSSSDDSIRKNGIAFAKTMLD

[0035] GIHEMGGDTLGGIVYATWPMQGVPHYDEKCRM

[0036] RERSLASLREIAPLAEEYGISYCLEIVNRFEQ

[0037] CILNTAEEGVAYVKELGSPNVKLLLDTFHMNI

[0038] EEDDIAAAIRSAKGVLGHFHMGECNRKTPGTG

[0039] RMPWSDIFAALRDIDYQGAVVMEPFMKTGGQV

[0040] GKDIRVFRDLSCGADEITMDRLAAQAA AFVKE

[0041] QLVLE.

[0042] (2) Design of mutation primers

[0043] Primers for site-directed mutagenesis were designed based on the gene sequence of ClDAE, and single mutations were introduced into amino acids 17, 118, 165, 166, 197, 121, 249, and 154, respectively.

[0044] Forward primer N17R: CCATTCTTTTTCCCAATATGCATAA

[0045] Reverse primer N17R: TGGGAAAAAGAATGG CGT GCAGA (underlined bases are mutated)

[0046] Forward primer G118K: CTGCATCGGCCAGGTTGCATAAACA

[0047] Reverse primer G118K: TGGCCGATGCAG AAA GTTCCGCAT (underlined bases are mutated)

[0048] Forward primer A165C: GTTCAGAATACACTGTTCAAAACGA

[0049] Reverse primer A165C: TGTATTCTGAACACC TGT GAAGAAG (underlined mutated bases)

[0050] Forward primer A166C: GGTGTTCAGAATACACTGTTCAAAA

[0051] Reverse primer A166C: TGTATTCTGAACACC TGT GAAGAAG (underlined mutated bases)

[0052] Forward primer I197F: ATCATCTTCTTCAATATTCATATGA

[0053] Reverse primer I197F: ATTGAAGAAGATGAT TTT GCGGCCG (underlined bases are mutated)

[0054] Forward primer H121D: CGGAACACCCTGCATCGGCC

[0055] Reverse primer H121D: ATGCAGGGTGTTCCG GAT TATGATG (underlined mutated bases)

[0056] Forward primer F249Y: CGGTTCCATAACAACTGCACCCTG

[0057] Reverse primer F249Y: GTTGTTATGGAACCG TAT ATGAAAAC (underlined bases are mutated)

[0058] Forward primer I154M: TTCCAGACAATAACTAATACCATATTC

[0059] Reverse primer I154M: AGTTATTGTCTGGAA ATG GTTAATC (underlined mutated bases)

[0060] (3) Single-site mutation

[0061] Rapid PCR technology was used to perform PCR amplification with the recombinant vector pET28a / ClDAE as a template.

[0062] The PCR reaction system (50 μL) was: 2×Phanta Max Buffer 25 μL, dNTPs 1 μL, forward primer 2 μL (5 μmol / μL), reverse primer 2 μL (5 μmol / μL), template DNA 1 μL (20 ng / μL), Phanta Max Super-Fidelity DNA Polymerase 1 μL, and ddH2O was added to 50 μL.

[0063] The PCR reaction program is: pre-denaturation at 95°C for 5 minutes; 30 cycles (denaturation at 95°C for 10 seconds, annealing at 55-60°C for 30 seconds, extension at 72°C for 5.5 minutes); extension at 72°C for 10 minutes, and finally insulation at 16°C. The PCR product was verified by 0.9% agarose gel electrophoresis. The result showed that the gene fragment consistent with the size of the target vector was amplified, and the next step of the experiment could be carried out.

[0064] After the PCR product was digested with DpnI at 37℃ for 3h, 3μL of the PCR product was taken and added to 100μL of E.coliBL21 (DE3) competent cell suspension in an ice bath, and then placed on ice for 30min. The transformation product was heat-shocked at 42℃ for 90s, and then quickly placed on ice for 5min. 600μL of LB liquid culture medium was added to the tube, and cultured at 37℃, 150r / min for 50min, centrifuged at 12000r / min for 1min, 400μL of supernatant was discarded, and then the remaining supernatant was resuspended to obtain a resuspension. 200μL of the above resuspension was applied to LB solid culture medium containing a final concentration of 50μg / mL kanamycin resistance. After the bacterial solution was completely absorbed by the culture medium, it was inverted and cultured at 37℃ for 12h. The recombinant strain had kanamycin resistance and could grow on a plate containing 50μg / mL kanamycin. The colonies were picked and inoculated into 10 mL of LB liquid culture medium containing kanamycin resistance at a final concentration of 50 μg / mL, and cultured at 37° C. for 12 h to obtain the respective bacterial suspensions.

[0065] The bacterial solution was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing and the sequencing results were aligned correctly, obtaining the single-site mutant recombinant bacteria E. coli BL21 (DE3) / pET28a / ClDAE / N17R, E. coli BL21 (DE3) / pET28a / ClDAE / G118K, E. coli BL21 (DE3) / pET28a / ClDAE / T165C, E. coli BL21 (DE3) / pET28a / ClDAE / A166C, E. coli BL21 (DE3) / pET28a / ClDAE / I197F, E. coli BL21 (DE3) / pET28a / ClDAE / H121D, E. coli BL21 (DE3) / pET28a / ClDAE / F249Y, E. coli BL21(DE3) / pET28a / ClDAE / I154M, and the corresponding enzymes were recorded as mutant enzymes ClDAE / N17R, ClDAE / G118K, ClDAE / T165C, ClDAE / A166C, ClDAE / I197F, ClDAE / H121D, ClDAE / F249Y, and ClDAE / I154M.

[0066] Example 2: Construction of a double-site mutant of D-psicose-3-epimerase

[0067] Using the single-site mutant constructed in Example 1 as a template, random pairwise combinations were performed using the method of Example 1 to obtain double-site mutant recombinant bacteria:

[0068] E.coli BL21(DE3) / pET28a / ClDAE / I197F / T165C、

[0069] E.coli BL21(DE3) / pET28a / ClDAE / I197F / N17R、

[0070] E.coli BL21(DE3) / pET28a / ClDAE / I197F / G118K、

[0071] E.coli BL21(DE3) / pET28a / ClDAE / H121D / N17R、

[0072] E.coli BL21(DE3) / pET28a / ClDAE / H121D / I197F、

[0073] E.coli BL21(DE3) / pET28a / ClDAE / H121D / F249Y、

[0074] E.coli BL21(DE3) / pET28a / ClDAE / G118K / N17R、

[0075] E. coli BL21(DE3) / pET28a / ClDAE / A166C / N17R and

[0076] E. coli BL21(DE3) / pET28a / ClDAE / A166C / G118K, and the corresponding mutant enzymes were recorded as mutant enzymes ClDAE / I197F / T165C, ClDAE / I197F / N17R, ClDAE / I197F / G118K, ClDAE / H121D / N17R, ClDAE / H121D / I197F, ClDAE / H121D / F249Y, ClDAE / G118K / N17R, ClDAE / A166C / N17R and ClDAE / A166C / G118K.

[0077] Example 3: Expression and purification of D-psicose-3-epimerase

[0078] (1) The strains constructed in Example 1 and Example 2 were inoculated into 10 mL of LB liquid medium containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37°C and 200 r / min until OD 600 =0.8-1.0, to obtain seed solution; the seed solution was transferred into a fresh 100 mL LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 1%, and cultured at 37°C and 180 r / min until the bacterial concentration OD 600 =0.6-0.8, add IPTG at a final concentration of 0.1 mM, then induce and culture at 28°C and 180 r / min for 12 h, centrifuge at 4°C and 8000 r / min for 10 min, and collect the wet cells.

[0079] (2) 1 g of wet bacterial cells was suspended in 10 mL of 50 mM sodium phosphate buffer (pH 6.5), and ultrasonically disrupted (120 W, ultrasonic for 2 s, pause for 2 s) for 30 min. The cells were centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant was collected to obtain a crude enzyme solution.

[0080] (3) The crude enzyme solution was purified using a nickel-NTA affinity chromatography column (Bio-Scale Mini Profinity IMAC prepacked column, 40 mm length × 12.6 mm inner diameter). The column was first equilibrated with an equilibration buffer (50 mM PB buffer, 300 mM NaCl, 20 mM imidazole, pH 8.0). The crude enzyme solution was loaded at a rate of 1 mL / min for 4 column volumes, and then eluted with an eluent (50 mM PB buffer, 300 mM NaCl, 500 mM imidazole, pH 8.0) at a rate of 2 mL / min. The solution was monitored using an ultraviolet detector and a conductivity detector. When the signals of the ultraviolet detector and the conductivity detector increased at the same time, the corresponding eluate was collected. When the conductivity detector signal remained unchanged and the ultraviolet detector signal decreased, the collection was stopped. This was the pure enzyme solution. The protein concentration of the pure enzyme solution was tested using a BCA kit.

[0081] (4) The pure enzyme solution collected in step (3) was dialyzed overnight in 50 mM sodium phosphate buffer (pH 6.5) with a molecular weight cutoff of 10 KDa. 15 μL of the dialyzate was added with 5 μL of 4×SDS buffer and mixed. The mixture was heated in a boiling water bath for 15 min and 5 μL was taken for SDS-PAGE electrophoresis analysis. Figure 1 ), with ClDAE as the control, a protein band with a molecular weight of about 33 kDa was obtained, indicating that the target protein was successfully expressed.

[0082] Example 4: Mutant Screening

[0083] Screening method: The crude enzyme solution prepared in Example 3 was diluted 100 times with PB buffer solution (pH 6.5), and 100 μL was taken and heat-treated at 65° C. for 60 min. After ice bathing for 10 min, it was added to 50 mM PB buffer solution (pH 6.5), and then an aqueous solution of 1 mM CoCl2 and a final concentration of 50 g / L of D-fructose were added to form a 1 ml reaction system. The reaction was carried out at 70° C. and 1000 r / min for 10 min. Sampling was performed, ice bathing was performed for 10 min, and boiling water bathing was performed for 15 min to terminate the reaction. The supernatant was centrifuged at 12000 r / min for 1 min, and diluted 10 times with pure water; filtered through a 0.22 μm water membrane, and the concentrations of D-fructose and D-psicose were detected using the HPLC liquid chromatograph described in Example 5, and the relative enzyme activity was calculated.

[0084] The crude enzyme activity of wild-type ClDAE was used as the control group and defined as 100% to screen mutants with superior thermal stability. Figure 2As shown in A, after 60 min of heat treatment, the mutants with effective single-point mutations are ClDAE / N17R, ClDAE / G118K, ClDAE / T165C, ClDAE / A166C, ClDAE / I197F, ClDAE / H121D, ClDAE / F249Y, and ClDAE / I154M. The single-point mutants were randomly superimposed, and the results were shown in Figure 2 As shown in B, the thermal stability of double-site mutants ClDAE / I197F / T165C, ClDAE / I197F / N17R, ClDAE / I197F / G118K, ClDAE / H121D / N17R, ClDAE / H121D / I197F, ClDAE / H121D / F249Y, ClDAE / G118K / N17R, ClDAE / A166C / N17R and ClDAE / A166C / G118K were all improved. Among them, the thermal stability of double-site mutants ClDAE / I197F / T165C, ClDAE / I197F / N17R, ClDAE / H121D / N17R, ClDAE / H121D / I197F and ClDAE / H121D / F249Y was significantly improved. The mutant ClDAE / H121D / F249Y was selected as the catalytic enzyme for subsequent experiments.

[0085] Example 5: Determination of specific enzyme activity of dominant mutants

[0086] The engineering bacteria of the advantageous mutants shown in Table 1 were selected for comparative enzyme activity test.

[0087] Reaction system: 2.5 mg of wet bacteria prepared by the method of Example 3 was added to 50 mM PB buffer solution (pH 6.5), and then a final concentration of 1 mM CoCl2 aqueous solution and a final concentration of 50 g / L D-fructose were added to form a 1 mL reaction system, the amount of wet bacteria added was 2.5 mg / mL, and the reaction was carried out at 70°C and 1000 r / min for 10 min. Sampling was performed, ice bath for 10 min, boiling water bath for 15 min to terminate the reaction, and centrifuged at 12000 r / min for 1 min. The supernatant was taken and diluted 10 times with pure water; 0.22 μm water membrane was filtered, and the concentrations of D-fructose and D-psicose were detected by HPLC liquid chromatograph.

[0088] Enzyme activity definition: At 70°C and pH 6.5, the amount of enzyme required to isomerize D-fructose to produce 1 μmol D-psicose per minute is defined as one enzyme activity unit (U).

[0089] The specific enzyme activity of D-psicose-3-epimerase was expressed as the enzyme activity unit per gram of wet cell weight (U / g·wet cell weight), and the results are shown in Table 1. The activity of the D-psicose-3-epimerase mutant ClDAE / H121D / F249Y was 1.27 times that of the wild-type ClDAE, reaching 2960.6 U / (g·wet cell weight).

[0090] Table 1: Enzyme activities of D-psicose-3-epimerase and its mutants

[0091]

[0092] HPLC detection conditions: Agilent 1260HPLC chromatograph, Agilent autosampler, sugar-park column, Agilent differential detector, 500 mg / L EDTA disodium calcium aqueous solution as mobile phase, column temperature set at 80°C, flow rate at 0.5 mL / min, external standard method, the yield of D-psicose was determined based on the peak retention time and peak area. The retention time of D-fructose was 13.4 min, and the retention time of D-psicose was 20.18 min.

[0093] Example 6: Determination of thermal stability of D-psicose-3-epimerase and its mutants

[0094] The wild-type ClDAE crude enzyme solution (protein concentration 0.03 mg / mL) prepared by the method of Example 3 was placed at 55, 60, and 65°C, respectively, and samples were taken at 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, and 360 min. The mutant enzyme ClDAE / H121D / F249Y crude enzyme solution (protein concentration 0.03 mg / mL) was placed at 60, 65, and 70°C for heat treatment, and samples were taken at 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 24 h, 26 h, 32 h, 36 h, and 40 h. The relative enzyme activity was detected by the method of Example 5. The results are shown in Table 5. Figure 3 .

[0095] The results showed that the half-life of wild-type ClDAE at 65°C was only about 60 min, while the mutant ClDAE / H121D / F249Y had a half-life of only about 60 min at 65°C. 1 / 2 The value is 2295min. At the same time, the half-life of the mutant enzyme at 70℃ can reach about 240min. It can be found that the thermal stability of the mutant enzyme is significantly improved and can withstand high temperatures of 45-70℃.

[0096] Example 7: Determination of the optimal reaction temperature of wild-type ClDAE and mutant ClDAE / H121D / F249Y

[0097] 100 μL (protein concentration: 0.03 mg / mL) of crude enzyme solution of wild-type ClDAE and mutant enzyme ClDAE / H121D / F249Y prepared by the method of Example 3 was used as the conversion enzyme instead of the wet bacteria in Example 5. The enzyme activity was tested at different temperatures (45, 50, 55, 60, 65, 70, 75, 80, 85°C). The amount of crude enzyme solution added was 0.03 g / mL in terms of protein concentration. The results are shown in Table 1. Figure 4 As shown. Figure 4 It can be seen that the optimal reaction temperatures of the wild-type ClDAE and the mutant enzyme ClDAE / H121D / F249Y are 65 and 70°C, respectively, and the optimal temperature of the mutant enzyme ClDAE / H121D / F249Y is increased by 5°C. At the same time, in the temperature range of 70°C-85°C, the enzyme activity of the wild-type ClDAE rapidly decreased, while the enzyme activity of the mutant enzyme ClDAE / H121D / F249Y remained stable, and the mutant enzyme was more heat-resistant.

[0098] Example 8: Determination of Tm Values ​​of D-psicose-3-epimerase and Its Mutants

[0099] The dialyzates of the wild-type ClDAE, mutant enzyme ClDAE / H121D, mutant enzyme ClDAE / F249Y, and mutant enzyme ClDAE / H121D / F249Y prepared by the method of Example 3 were measured for Tm values ​​using a circular dichroism spectrometer. The results are as follows: Figure 5 As shown, the Tm value of the mutant enzyme ClDAE / H121D / F249Y increased from 58.5°C of the wild-type ClDAE to 71.3°C.

[0100] Example 9: Preparation of D-psicose from D-fructose catalyzed by D-psicose-3-epimerase mutant

[0101] The mutant enzyme ClDAE / H121D / F249Y wet bacteria prepared by the method of Example 3 was used as a conversion enzyme, and D-fructose was used as a substrate to perform an isomerization reaction to prepare D-psicose. The specific operation was as follows:

[0102] Using 50mM PB buffer solution (pH6.5) as the reaction medium, different concentrations (50g / L, 100g / L, 200g / L, 300g / L, 400g / L, 500g / L, 600g / L, 700g / L) of D-fructose and a final concentration of 1mM CoCl2 aqueous solution were added, and then 2.5mg of wet bacteria (final concentration 2.5mg / mL) was added to form a 1mL reaction system. The reaction was carried out at 70°C and 1000r / min for 3h, and the supernatant was centrifuged at 12000r / min. After diluting the supernatant 10 times with pure water and filtering with a 0.22μm water membrane, the concentrations of D-fructose and D-psicose were detected by the HPLC liquid chromatograph described in Example 5, and the conversion rate was calculated. The results are shown in Figure 6 shown.

[0103] The conversion rate (%) was defined as the ratio of the D-psicose concentration to the initial D-fructose concentration at the time of reaction equilibrium sampling.

[0104] The results showed that the D-psicose-3-epimerase mutant catalyzed the conversion of D-fructose to D-psicose, with a maximum conversion rate of 34.1% at a substrate concentration of 50 g / L. At the same time, as the substrate concentration increased, the conversion rate could still be maintained above 30%, and the substrate inhibition effect was small.

[0105] Under the same conditions, the wild-type ClDAE wet bacteria prepared by the method of Example 3 was used as the conversion enzyme, and the conversion rate was 30% when the substrate concentration was 50 g / L.

Claims

1. A D-psicose-3-epimerase mutant, characterized in that: The mutant is obtained by performing single mutation or multiple mutations on positions 17, 118, 121, 165, 197 and 249 of the amino acid sequence shown in SEQ ID NO.

2.

2. The D-psicose-3-epimerase mutant according to claim 1, wherein The D-psicose-3-epimerase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated into one of the following: (1) asparagine at position 17 is mutated into arginine; (2) glycine at position 118 is mutated into lysine; (3) histidine at position 121 is mutated into aspartic acid; (4) threonine at position 165 is mutated into cysteine; (5) isoleucine at position 197 is mutated into phenylalanine; (6) phenylalanine at position 249 is mutated into tyrosine; (7) histidine at position 121 is mutated into aspartic acid and phenylalanine at position 249 is mutated into tyrosine. The amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.

3.

3. A recombinant genetically engineered bacterium containing the gene encoding the D-psicose-3-epimerase mutant according to claim 1.

4. Use of the D-psicose-3-epimerase mutant according to claim 1 in the preparation of D-psicose by microbial catalytic D-fructose isomerization.

5. The use according to claim 4, characterized in that The application comprises the following steps: using wet cells obtained by fermentation-induced culture of recombinant Escherichia coli containing a D-psicose-3-epimerase mutant gene, or pure enzyme solution extracted by ultrasonic crushing of the wet cells, as an enzyme source; using D-fructose as a substrate; using cobalt ions as a catalyst; and using a pH 6.5 buffer solution as a reaction medium to form a reaction system; reacting at 50-80° C. and 1000 rpm; and obtaining a mixture of D-fructose and D-psicose after the reaction is complete.

6. The use according to claim 5, characterized in that In the reaction system, the wet bacterial cell dosage is 2.5-10 g / L; the final cobalt ion concentration is 0.1-10 mM; and the initial substrate addition concentration is 50-700 g / L.

7. The use according to claim 5, characterized in that The wet cells were prepared as follows: recombinant Escherichia coli containing a D-psicose-3-epimerase mutant encoding gene was inoculated into LB liquid culture medium containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37° C. and 200 rpm until the OD 600 =0.8~1.0, obtain seed solution; The seed solution was transferred into LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 1-5% inoculum, and cultured at 37°C and 180 r / min until the OD 600 =0.6-0.8, add IPTG at a final concentration of 0.1 mM, and induce culture at 28°C and 180 r / min for 12-14 h to obtain an induced culture suspension, which is centrifuged to collect wet cells.

8. The use according to claim 5, characterized in that The pure enzyme solution was prepared according to the following steps: the wet bacteria were suspended in a pH 6.5, 50mM sodium phosphate buffer, ultrasonically disrupted at 120W for 30min, ultrasonicated for 2s, paused for 2s, centrifuged, and the supernatant was taken to obtain a crude enzyme solution; the crude enzyme solution was purified using a nickel-NTA affinity chromatography column, first equilibrated the chromatography column with an equilibration buffer, the crude enzyme solution was loaded at a rate of 1mL / min for 4 column volumes, and then eluted with an eluent at a rate of 2mL / min, and monitored by an ultraviolet detector and a conductivity detector. When the signal of the ultraviolet detector and the signal of the conductivity detector increased simultaneously, the corresponding eluent was collected, and when the signal of the conductivity detector remained unchanged and the signal of the ultraviolet detector decreased, the collection was stopped, and the collected enzyme solution was the pure enzyme solution; the equilibration buffer composition: 50mM PB buffer, 300mM NaCl, 20mM imidazole, pH 8.0; the eluent composition: 50mM PB buffer, 300mM NaCl, 500mM imidazole, pH 8.0.

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