UDP / GDP-glucose epimerase for catalytic synthesis of UDP / GDP-mannose

Abstract

The invention discloses UDP / GDP-glucose epimerase for catalytic synthesis of UDP / GDP-mannose, and belongs to the technical field of enzyme engineering. The half-life periods of the mutants Mut3 and Mut4 provided by the invention at 50 DEG C are respectively 58.51 times and 60.30 times of those of WT. The specific enzyme activities of mutants Mut3 and Mut4 at 40 DEG C are respectively 108.69 U / mg and 77.44 U / mg, which are respectively improved by 73.21 times and 52.32 times compared with WT (Wolfram Titanate). Under the optimal condition, the conversion rates of the Mut3 to the UDP-Glc and the GDP-Glc respectively reach 55.38 percent and 23.84 percent. The conversion rates of the Mut4 on UDP-Glc and GDP-Glc are respectively up to 55.63% and 27.06%.

Description

Technical Field

[0001] The invention relates to a UDP / GDP-glucose epimerase for catalyzing and synthesizing UDP / GDP-mannose, belonging to the technical field of enzyme engineering. Background Art

[0002] Nucleotide sugar donors play an important role in all life systems. As an activated form of monosaccharides, they can be further utilized by glycosyltransferases, providing activated monosaccharides for the biosynthesis of oligosaccharides, polysaccharides and glycoconjugates. They are also used in many fields such as pharmaceuticals, cosmetics and food industries. Common sugar nucleotides include UDP-Glc, UDP-GlcNAc, UDP-GlcA, UDP-Gal, UDP-GalNAc, UDP-Xyl, GDP-L-Fuc, GDP-Man and CMP-Neu5Ac. At present, the activated form of mannose is mainly GDP-mannose, and there is also a small amount of UDP-mannose. Among them, the mannose donor GDP-Man is a key metabolite required for protein glycosylation and glycosylphosphatidylinositol anchor synthesis, and is involved in the synthesis of manno-oligosaccharides, mannans and mannose glyceric acid. In addition, the rare sugar donor UDP-Man of mannose was first discovered in Mycobacteriumsmegmatis. Subsequently, UDP-Man was also identified in mouse tissues and organs, and it was revealed that UDP-Man may play a unique role in the glycosylation of specific mammalian organs (including the brain). However, the current reaction to obtain nucleotide-mannose is complex and costly.

[0003] Dimerization plays a vital role in carbohydrate synthesis, and can achieve mutual conversion by reversing the stereostructure of the asymmetric center of the sugar. Carbohydrate dimerases are a class of enzymes that can achieve this reaction. In recent years, carbohydrate isomerases have received increasing attention in the production of rare sugars and their derivatives. The vast majority of these enzymes only have catalytic activity on activated nucleotide sugars, and fewer enzymes can catalyze free sugars. Dimerases acting on nucleotide sugar substrates provide a richer catalytic combination, including oxidation / reduction and elimination / reproduction, so they can expand the scope of substrate isomerization conversion.

[0004] C2 nucleotide sugar isomerases mainly include UDP-acetylglucosamine 2-isomerase and CDP-paratose 2-isomerase. Among them, although CDP-paratose 2-isomerase catalyzes the direct conversion of nucleotide glucose into nucleotide mannose, its activity is extremely low. Improving the enzymatic activity of CDP-paratose 2-isomerase has the potential to achieve rapid synthesis of nucleotide mannose. The development of CDP-paratose 2-isomerase with improved thermal stability and enzyme activity will be conducive to the economical and low-cost production of nucleotide mannose, thereby reducing the price of nucleotide mannose and providing a more ideal donor substrate raw material for the synthesis of mannose-based active substances. Summary of the invention

[0005] The technical problem to be solved by the present invention is the problem of low activity of CDP-paratose 2-isomerase.

[0006] The present invention provides a CDP-paratose 2-isomerase mutant. The mutant is obtained by mutating the threonine at position 144 of the CDP-paratose 2-isomerase having an amino acid sequence as shown in SEQ ID NO.1 to isoleucine, the tyrosine at position 152 to phenylalanine, the glutamine at position 157 to proline, the tyrosine at position 203 to proline, the threonine at position 227 to histidine, the isoleucine at position 228 to valine, the serine at position 229 to phenylalanine, and the isoleucine at position 338 to tryptophan, thereby obtaining T144I / Y152F / Q157P / Y203P / T227H / I228V / S229F / I338W, which is named Mut3.

[0007] Or the threonine at position 144 of the CDP-paratose 2-isomerase having an amino acid sequence as shown in SEQ ID NO.1 is mutated to isoleucine, the tyrosine at position 152 is mutated to phenylalanine, the glutamine at position 157 is mutated to proline, the tyrosine at position 203 is mutated to proline, the threonine at position 227 is mutated to glutamate, the isoleucine at position 228 is mutated to valine, the serine at position 229 is mutated to phenylalanine, and the isoleucine at position 338 is mutated to tryptophan to obtain T144I / Y152F / Q157P / Y203P / T227E / I228V / S229F / I338W, which is named Mut4.

[0008] In one embodiment of the present invention, the source of the CDP-paratose 2-isomerase is: Salmonella typhi.

[0009] In one embodiment of the present invention, the nucleotide sequence encoding the CDP-paratose 2-isomerase is shown as SEQ ID NO.2.

[0010] The present invention also provides a gene encoding the CDP-paratose 2-isomerase mutant.

[0011] The invention also provides a recombinant vector carrying the gene.

[0012] The present invention also provides a recombinant cell expressing the CDP-paratose 2-isomerase mutant, or carrying the gene, or carrying the recombinant vector.

[0013] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0014] In one embodiment of the present invention, the recombinant cell uses Escherichia coli as an expression host.

[0015] The present invention provides a method for improving the thermal stability and / or enzyme activity of CDP-paratose 2-isomerase, wherein the method comprises the following steps: mutating the threonine at position 144 of the CDP-paratose 2-isomerase having an amino acid sequence as shown in SEQ ID NO.1 to isoleucine, mutating the tyrosine at position 152 to phenylalanine, mutating the glutamine at position 157 to proline, mutating the tyrosine at position 203 to proline, mutating the threonine at position 227 to histidine, mutating the isoleucine at position 228 to valine, mutating the serine at position 229 to phenylalanine, and mutating the isoleucine at position 338 to tryptophan;

[0016] Or the threonine at position 144 of the CDP-paratose 2-isomerase having an amino acid sequence as shown in SEQ ID NO.1 is mutated to isoleucine, the tyrosine at position 152 is mutated to phenylalanine, the glutamine at position 157 is mutated to proline, the tyrosine at position 203 is mutated to proline, the threonine at position 227 is mutated to glutamate, the isoleucine at position 228 is mutated to valine, the serine at position 229 is mutated to phenylalanine, and the isoleucine at position 338 is mutated to tryptophan.

[0017] The present invention also provides a method for preparing UDP-Man, which comprises using the mutant or the recombinant cell or the recombinant CDP-mannosyl 2-isomerase expressed by the recombinant cell as a catalyst and UDP-Glc as a substrate to react and prepare UDP-Man.

[0018] The structural formula of UDP-Man (C15H24N2O17P2) is:

[0019]

[0020] The structural formula of uridine diphosphate glucose (UDP-Glc, C15H24N2O17P2) is:

[0021]

[0022] The structural formula of guanosine diphosphate mannose (GDP-Man, C16H25N5O16P2) is:

[0023]

[0024] The structural formula of guanosine diphosphate glucose (GDP-Glc, C16H25N5O16P2) is:

[0025]

[0026] In one embodiment of the present invention, the mutant or recombinant CDP-paratose 2-isomerase is added in an amount of 2.5-5 g / L.

[0027] In one embodiment of the present invention, the reaction conditions are: at a temperature of 30-60° C. and a substrate concentration of 10-50 mM, and the reaction is carried out for 4-6 hours.

[0028] In one embodiment of the present invention, the reaction conditions are: PB buffer (pH 8.0), reaction temperature 50° C., 50 mM substrate, reaction time 4-6 h.

[0029] The present invention also provides a method for preparing guanosine diphosphate mannose GDP-Man, which comprises using the mutant or the recombinant cell or the recombinant CDP-beratose 2-isomerase expressed by the recombinant cell as a catalyst and guanosine diphosphate glucose GDP-Glc as a substrate to react and prepare GDP-Man.

[0030] In one embodiment of the present invention, the mutant or recombinant CDP-paratose 2-isomerase is added in an amount of 2.5-5 g / L.

[0031] In one embodiment of the present invention, the reaction conditions are: at a temperature of 30-60° C. and a substrate concentration of 10-50 mM, and the reaction is carried out for 4-6 hours.

[0032] In one embodiment of the present invention, the reaction conditions are: PB buffer (pH 8.0), reaction temperature 50° C., 50 mM substrate, reaction time 4-6 h.

[0033] The present invention also provides the use of the mutant or the recombinant cell or the recombinant CDP-paratose 2-isomerase expressed by the recombinant cell in the preparation of uridine diphosphate mannose UDP-Man or guanosine diphosphate mannose GDP-Man or a product containing uridine diphosphate mannose UDP-Man or a product containing guanosine diphosphate mannose GDP-Man.

[0034] The present invention also provides the use of the mutant, or the gene, or the recombinant vector, or the recombinant cell, or the preparation method in the preparation of food, health products and cosmetics.

[0035] Beneficial Effects

[0036] (1) The present invention transforms CDP-paratose 2-isomerase (stTyvE) from Salmonella typhi by genetic engineering, obtains transformants with improved thermal stability and enzyme activity through single-point mutation and combined mutation, and can achieve efficient synthesis of UDP / GDP-Man at the optimal temperature.

[0037] (2) The half-life of Mut3 at 50℃ was 1.96h, which was 58.51 times that of WT. The enzyme activity at 40℃ with UDP-Glc as substrate was 73.21 times higher than that of WT, with a specific activity of 108.69mU / mg, and the conversion rate of UDP-Glc reached 55.38%. The conversion rate of GDP-Glc reached 23.84%.

[0038] (3) The half-life of Mut4 at 50℃ was 2.02h, which was 60.30 times that of WT. The enzyme activity at 40℃ with UDP-Glc as substrate was 52.32 times higher than that of WT, with a specific activity of 77.44mU / mg, and the conversion rate of UDP-Glc reached 55.63%. The conversion rate of GDP-Glc reached 27.60%, among which WT had no activity detected for GDP-Glc, and the conversion rate of UDP-Glc was only 4.26%.

[0039] (4) The mutant enzymes can be used to achieve high-level synthesis of UDP-Man and GDP-Man. Under the conditions of pH 8.0, reaction temperature 50℃, and 50mM UDP-Glc, the reaction lasted for 4-6h. The maximum UDP-Man yield of Mut3 was 6.22g / L, and the GDP-Man yield was 1g / L; the maximum UDP-Man yield of Mut4 was 11.03g / L, and the GDP-Man yield was 3g / L. Under the conditions of pH 8.0, reaction temperature 50℃, and 50mM substrate, the maximum UDP-Man yield of WT was only 0.17g / L after 4-6h reaction, and it was unable to catalyze the synthesis of GDP-Man. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : Reaction curve of mutant enzyme Mut3.

[0041] Figure 2 : Reaction curve of mutant enzyme Mut4.

[0042] Figure 3 : Protein gel electrophoresis of mutant enzyme Mut3 and mutant enzyme Mut4; wherein, 1: maker; 2: mutant enzyme Mut3; 3: mutant enzyme Mut4. DETAILED DESCRIPTION

[0043] The culture medium involved in the following examples is as follows:

[0044] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl), pH 7.4; LB solid medium is prepared by adding 2% agar to LB liquid medium.

[0045] TB medium: tryptone 12 g / L, yeast extract 24 g / L, K2HPO4 12.54 g / L KH2PO4 2.31 g / L, glycerol 5 g / L.

[0046] The solutions involved in the following examples are as follows:

[0047] PB buffer: 2.299 g Na2HPO4, 0.455 g NaH2PO4, make up to 1 L.

[0048] Solution A (balanced solution): Add 0.3404 g / L imidazole (5 mM) and 29.22 g / L NaCl (0.5 M) to the above PB solution and adjust the pH to 7.4.

[0049] Solution B (eluent): Add 34.075 g / L imidazole (500 mM) and 29.22 g / L NaCl (0.5 M) to the above PB solution and adjust the pH to 7.4.

[0050] The detection methods involved in the following embodiments are as follows:

[0051] Enzyme activity assay method: 0.5 g / L enzyme was incubated in 50 mmol / L PB buffer (pH 8.0) with a total volume of 200 μL containing 10 mmol / L UDP-Glc at 40°C for 10 min.

[0052] Definition of enzyme activity: The amount of enzyme required to convert 1 μmol UDP-Glc within 1 min is 1 enzyme activity unit (U).

[0053] Calculation of specific enzyme activity: specific enzyme activity (mU / mg) = enzyme activity (mU) / protein content (mg)

[0054] Enzyme thermal stability test: The enzyme activity of pure enzyme was measured at 50°C, with the enzyme activity at 0 min as the highest value of 100%. The pure enzyme was kept at this temperature, and the remaining enzyme activity was measured at regular intervals to explore the thermal stability of the enzyme.

[0055] Product determination method: UDP-Glc and UDP-Man were determined using a Waters T3 column, a mobile phase of 30 mM triethylamine acetate (pH 6.0), a flow rate of 1.0 ml / min, and a UV detector.

[0056] Example 1: Construction of CDP-Poratose 2-isomerase (stTyvE) and its mutants

[0057] (1) Construction of a recombinant plasmid containing the stTyvE gene

[0058] The CDP-paratose 2-isomerase from Salmonella typhi was selected as the wild type, the amino acid sequence of the CDP-paratose 2-isomerase is shown in SEQ ID NO.1, and the nucleotide sequence encoding the CDP-paratose 2-isomerase is shown in SEQ ID NO.2.

[0059] SEQ ID NO.1:

[0060] MKLLITGGCGFLGSNLASFALSQGIDLIVFDNLSRKGATDNLHWLSSLGNFEFVHGDIRNKNDVTRLITKYMPDSCFHLAGQVAMTTSIDNPCMDFEINVGGTLNLLEAVRQYNSNCNIIYSSTNKVYGDLEQYKYNETETRYTCVDKPNGYDESTQLDFHSPYGCSKG AADQYMLDYARIFGLNTVVFRHSSMYGGRQFATYDQGWVGWFCQKAVEIKNGINKPFTISGNGKQVRDVLHAEDMISLYFTALANVSKIRGNAFNIGGTIVNSSLLELFKLLEDYCNIDMRFTNLPVRESDQRVFVADIKKITNAIDWSPKVSAKDGVQKMYDWTSSI

[0061] The nucleotide sequence SEQ ID NO.2 was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0062] SEQ ID NO.2:

[0063] ATGAAGCTTTTAATTACCGGTGGATGTGGCTTCCTTGGGAGTAATCTTGCCTCCTTTGCTTTAAGTCAA

[0064] GGGATTGATTTAATTGTATTCGATAATCTATCACGTAAAGGTGCAACAGATAATTTACATTGGTTATCCT

[0065] CCTTAGGAAACTTTGAGTTTGTACATGGTGATATTCGCAACAAAAATGATGTTACAAGATTAATAACTA

[0066] AGTATATGCCTGATAGCTGTTTTCATCTTGCAGGTCCAAGTGGCAATGACTACATCTATTGACAATCCTTG

[0067] TATGGATTTTGAAATTAATGTAGGTGGAACTTTAAATTACTTGAGGCAGTACGGCAGTATAATTCAAA

[0068] TTGTAATATAATTATTCATCAACAAATAAAGTATACGGCGATCTTGAGCAATATAAATACAATGAAACA

[0069] GAAACTAGATACACTTGTGTAGATAAGCCTAATGGATATGATGAGAGCACACAATTAGATTTCCACTCA

[0070] CCATATGGTTGTTCAAAAGGTGCTGCAGACCAATACATGCTTGATTATGCAAGGATTTTTGGTTTGAAT

[0071] ACAGTGGTGTTCAGGCATTCATCAATGTATGGTGGGAGACAGTTTGCTACTTATGATCAAGGCTGGGT

[0072] AGGTTGGTTTTGTCAAAAGCGGTTGAAATTAAAAATGGTATTAATAAACCCTTCACTATTCTGGTAA

[0073] TGGTAAGCAAGTTAGGGATGTTTTGCATGCTGAAGATATGATTTCGTTATATTTCACTGCCTTGGCAAAT

[0074] GTATCAAAAATTAGGGGGAACGCTTTTAATATTGGTGGTACCATTGTCAACAGCCTATCATTACTTGAA

[0075] TTATTCAAATTGCTTGAAGATTATTGCAACATAGATATGAGGTTCACTAATTTACCTGTAAGGGAAAGT

[0076] GATCAGCGTGTTTTTGTTGCAGATATTAAAAAAATCACTAATGCAATTGACTGGAGCCCGAAAGTCTC

[0077] GGCAAAAGATGGTGTCCAGAAAATGTATGATTGGACTAGTTCTATATGA

[0078] Select Nco I and Xho I on pET28a as the sites for inserting the target gene, and amplify the target gene, upstream primer: TTAAGAAGGAGATATACCATGGGCATGAAGCTTTTAATT, downstream primer TGGACTAGTTCTATACTCGAGCACCACCACCACCACCAC, so that the target gene has a restriction site. Double-digest pET28a and purify it to obtain a linear plasmid fragment. The target gene with restriction sites and the linear plasmid fragment are connected together by homologous recombination. The system for homologous recombination contains: 1μL restriction linear plasmid, 1μL target fragment, 2μL Buffer, 1μL homologous recombination enzyme, 5μL H2O. Homologous recombination is carried out at 37℃ for 30min. Then, the connection product is stored at -20℃.

[0079] The ligation product was transformed into the competent cell of Escherichia coli DE3 (BL21), and after culturing at 37°C for 1 hour, the bacterial solution was spread on a plate LB solid culture medium containing kanamycin resistance. After overnight culture, a single colony was picked, amplified and cultured, and the plasmid was extracted. Colony PCR was performed with universal primers, and the plasmid with a positive PCR result was sequenced and verified, and finally the correct CDP-paratose 2-isomerase gene recombinant plasmid: pET28a-WT-stTyvE and recombinant strain: E. coli DE3 (BL21) / pET28a-WT-stTyvE were obtained.

[0080] (2) Construction of recombinant plasmid of CDP-paratose 2-isomerase mutant

[0081] Using the pET28a-WT-stTyvE plasmid as a template, the whole plasmid PCR was carried out using the primers in Table 1, and the eight mutation sites were introduced in sequence to obtain: pET28a-Mut3 plasmid and pET28a-Mut4 plasmid, wherein Mut3 is T144I / Y152F / Q157P / Y203P / T227H / I228V / S229F / I338W, and Mut4 is T144I / Y152F / Q157P / Y203P / T227E / I228V / S229F / I338W. The primers involved are shown in Table 1.

[0082] Table 1 Primers for mutant construction

[0083]

[0084] The recombinant plasmids pET28a-Mut3 and pET28a-Mut4 were digested with DpnI for about 2 hours, and transferred into competent E. coli BL21 (DE3) by heat shock method. Monoclonal colonies were selected and cultured overnight in LB medium at 37°C and 200 rpm. Plasmids were extracted from the monoclonal strain culture and sent to a sequencing company for sequencing. The recombinant strains with correct sequencing results were E. coli BL21 (DE3) / pET28a-Mut3 and E. coli BL21 (DE3) / pET28a-Mut4.

[0085] Among them, Mut3 is T144I / Y152F / Q157P / Y203P / T227H / I228V / S229F / I338W;

[0086] Mut4 is T144I / Y152F / Q157P / Y203P / T227E / I228V / S229F / I338W.

[0087] (3) Induced expression and purification of target protein

[0088] The correctly sequenced monoclonal strains E.coli BL21(DE3) / pET28a-Mut3 and E.coli BL21(DE3) / pET28a-Mut4 were cultured in LB medium for 12 hours for activation, and then inoculated into fresh TB medium, cultured at 37°C, 200rpm until OD was 0.8, and the inducer IPTG was added with a final concentration of 0.1mM, and cultured at 16°C, 200rpm for 20 hours. After the culture was completed, the precipitate was centrifuged and the precipitate was treated with a cell disruptor and centrifuged to obtain the supernatant to obtain the crude enzyme solution, and the pure enzyme was obtained by nickel column purification.

[0089] The method of nickel column purification is as follows:

[0090] The nickel column was first equilibrated with equilibrium solution A for 15 min, and then -1 The sample was injected at a flow rate of 100 mM. After the injection, the column was flushed with liquid A for 15 min. Then, the mixing ratio of liquid B to liquid A was adjusted so that the imidazole concentration in the eluent was 100 mM. Then, the impurity protein was eluted with the eluent. The imidazole concentration in the eluent was adjusted to 250 mM, and the target protein was eluted and collected. The eluent containing the target protein (CDP-paratose 2-isomerase mutant) collected above was desalted and concentrated by ultrafiltration tube, and the concentrate was stored at 4°C for subsequent experiments.

[0091] The pure enzyme solutions of Mut3 and Mut4 were prepared respectively, and the gel electrophoresis patterns were as follows: Figure 3 As shown, the results showed that the molecular weight was 33 kDa and it was a single band, and the purity of the enzyme was high.

[0092] According to the above method, the wild-type WT pure enzyme solution was prepared.

[0093] Example 2: Properties of Mutants Mut3 and Mut4

[0094] The enzymatic properties of the pure enzyme solutions of Mut3 and Mut4 prepared in Example 1 were tested respectively, as follows:

[0095] (1) Detection of relative enzyme activity and specific enzyme activity

[0096] 0.5 g / L enzyme was incubated in 50 mmol / L PB buffer (pH 8.0) containing 10 mmol / L UDP-Glc in a total volume of 200 μL at 40°C for 10 min. The amount of enzyme required to convert 1 μmol UDP-Glc within 1 min is 1 enzyme activity unit (U). WT enzyme activity was used as a control.

[0097] The relative enzyme activity assay results showed:

[0098] The relative enzyme activity of mutant Mut3 with UDP-Glc as substrate at 40°C was 73.21 times higher than that of WT. The specific enzyme activity of mutant Mut3 was 108.69 mU / mg, while the specific enzyme activity of wild-type CDP-paratose 2-isomerase as a control was only 1.48 mU / mg.

[0099] The relative enzyme activity of mutant Mut4 using UDP-Glc as substrate at 40°C was 52.32 times higher than that of WT, and the specific enzyme activity of mutant Mut4 was 77.44 mU / mg.

[0100] (2) Thermal stability testing

[0101] The enzyme activity of the pure enzyme was measured at 50°C, with the enzyme activity at 0 min as the highest value of 100%. The pure enzyme was kept at this temperature and the remaining enzyme activity was measured at regular intervals to explore the thermal stability of the enzyme.

[0102] The results show:

[0103] The stability of the mutant pure enzyme was measured at 50°C, and the fitting analysis results showed that the half-life of Mut3 was 1.96h, which was 58.51 times that of WT. The half-life of the wild-type WT was 2.01min.

[0104] The half-life of Mut4 was 2.02 h, which was 60.30 times that of WT.

[0105] It can be seen that the mutant successfully overcame the loss of enzyme activity caused by stability and achieved simultaneous improvement of stability and enzyme activity.

[0106] (3) Conversion rate detection

[0107] After adding pure enzyme solution and substrate solution (UDP-Glc or GDP-Glc) to 50mmol / L PB buffer containing pH 8.0, at 40℃, when the enzyme concentration is 0.5g / L and the substrate is 2mM, the reaction is balanced after 10h of reaction. The reaction system is 200μL, and the conversion rate (%) = molar amount of product after reaction equilibrium / (molar amount of product after reaction equilibrium + molar amount of substrate after reaction equilibrium) × 100%.

[0108] The results show:

[0109] The conversion rates of UDP-Glc and GDP-Glc by Mut3 reached 55.38% and 23.84%, respectively.

[0110] The conversion rates of UDP-Glc and GDP-Glc by Mut4 reached 55.63% and 27.06%, respectively.

[0111] However, WT showed no activity towards GDP-Glc and the conversion rate towards UDP-Glc was only 4.26%.

[0112] Example 3: Synthesis of UDP-Man using CDP-paratose 2-isomerase mutants Mut3 and Mut4

[0113] The specific steps are as follows:

[0114] (1) After adding 10-50 mM UDP-Glc (10 mM, 20 mM, 30 mM, 40 mM, 50 mM) to 10 mL phosphate buffer (pH 8.0), the mutant enzyme Mut3 (pure enzyme obtained in Example 1) was added respectively. The concentration of the mutant enzyme was 2.5 g / L ( Figure 1 ), react at 50°C for 8h, take 10μl samples at different time periods, and measure the contents of UDP-Glc and UDP-Man.

[0115] (2) After adding 10-50 mM UDP-Glc (10 mM, 20 mM, 30 mM, 40 mM, 50 mM) to 10 mL of phosphate buffer (pH 8.0), the mutant enzyme Mut4 (pure enzyme obtained in Example 1) was added respectively. The concentration of the mutant enzyme was 5 g / L ( Figure 2 ), react at 50°C for 8h, take 10μl samples at different time periods, and measure the contents of UDP-Glc and UDP-Man.

[0116] (3) After adding 10-50 mM UDP-Glc (10 mM, 20 mM, 30 mM, 40 mM, 50 mM) to 10 mL of phosphate buffer (pH 8.0), the wild-type enzyme WT (pure enzyme obtained in Example 1) was added respectively. The concentration of the wild-type enzyme was 5 g / L. The reaction was carried out at 50° C. for 8 h. 10 μl of samples were taken at different time periods of the reaction, and the contents of UDP-Glc and UDP-Man therein were determined.

[0117] The results show:

[0118] The reaction reached equilibrium in 4-6 hours. The maximum production of UDP-Man of the mutant enzyme Mut3 was 6.22 g / L at 50 mM substrate; the maximum production of UDP-Man of the mutant enzyme Mut4 was 11.03 g / L at 50 mM substrate; and the maximum production of UDP-Man of the wild-type enzyme WT was only 0.117 g / L at 50 mM substrate.

[0119] In addition, sucrose synthase can be used to carry out a cascade reaction with Mut3 or Mut4.

[0120] Example 4: Synthesis of GDP-Man using CDP-paratose 2-isomerase mutants Mut3 and Mut4

[0121] As can be seen from Example 2, the wild-type enzyme has no catalytic activity for the substrate GDP-Glc, so the wild-type enzyme cannot synthesize GDP-Man. In the following examples, only the mutant enzyme is used to react and prepare GDP-Man. The specific steps are as follows:

[0122] (1) 20 mM GDP-Glc and 2.5 g / L Mut3 (pure enzyme obtained in Example 1) were added to 10 mL phosphate buffer (pH 8.0), and the reaction was carried out at 50° C. for 8 h. 10 μl samples were taken at different time periods to measure the GDP-Glc and GDP-Man contents therein.

[0123] (2) 20 mM GDP-Glc and 5 g / L Mut4 (pure enzyme obtained in Example 1) were added to 10 mL phosphate buffer (pH 8.0), and the reaction was carried out at 50° C. for 8 h. 10 μl samples were taken at different time periods to measure the GDP-Glc and GDP-Man contents therein.

[0124] The results show:

[0125] The reaction reached equilibrium in 4-6 hours, and the GDP-Man production of the mutant enzyme Mut3 was 1 g / L, and the GDP-Man production of the mutant enzyme Mut4 was 3 g / L.

[0126] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A CDP-paratose 2-isomerase mutant, characterized in that The mutant is obtained by mutating the threonine at position 144 of the CDP-paratose 2-isomerase with an amino acid sequence as shown in SEQ ID NO.1 to isoleucine, the tyrosine at position 152 to phenylalanine, the glutamine at position 157 to proline, the tyrosine at position 203 to proline, the threonine at position 227 to histidine, the isoleucine at position 228 to valine, the serine at position 229 to phenylalanine, and the isoleucine at position 338 to tryptophan; Or the threonine at position 144 of the CDP-paratose 2-isomerase having an amino acid sequence as shown in SEQ ID NO.1 is mutated to isoleucine, the tyrosine at position 152 is mutated to phenylalanine, the glutamine at position 157 is mutated to proline, the tyrosine at position 203 is mutated to proline, the threonine at position 227 is mutated to glutamic acid, the isoleucine at position 228 is mutated to valine, the serine at position 229 is mutated to phenylalanine, and the isoleucine at position 338 is mutated to tryptophan to obtain the obtained protein.

2. A gene encoding the CDP-paratose 2-isomerase mutant according to claim 1.

3. A recombinant vector carrying the gene according to claim 2.

4. A recombinant cell expressing the CDP-paratose 2-isomerase mutant of claim 1, or carrying the gene of claim 2, or carrying the recombinant vector of claim 3.

5. The recombinant cell according to claim 4, characterized in that The recombinant cell uses bacteria or fungi as expression hosts.

6. A method for improving the thermal stability and / or enzyme activity of CDP-paratose 2-isomerase, characterized in that: The method comprises the following steps: mutating the threonine at position 144 of the CDP-paratose 2-isomerase having an amino acid sequence as shown in SEQ ID NO.1 to isoleucine, mutating the tyrosine at position 152 to phenylalanine, mutating the glutamine at position 157 to proline, mutating the tyrosine at position 203 to proline, mutating the threonine at position 227 to histidine, mutating the isoleucine at position 228 to valine, mutating the serine at position 229 to phenylalanine, and mutating the isoleucine at position 338 to tryptophan; Or the threonine at position 144 of the CDP-paratose 2-isomerase having an amino acid sequence as shown in SEQ ID NO.1 is mutated to isoleucine, the tyrosine at position 152 is mutated to phenylalanine, the glutamine at position 157 is mutated to proline, the tyrosine at position 203 is mutated to proline, the threonine at position 227 is mutated to glutamate, the isoleucine at position 228 is mutated to valine, the serine at position 229 is mutated to phenylalanine, and the isoleucine at position 338 is mutated to tryptophan.

7. A method for preparing uridine diphosphate mannose UDP-Man or guanosine diphosphate mannose GDP-Man, characterized in that: The method comprises the following steps: using the mutant according to claim 1 or the recombinant cell according to claim 4 or 5 or the recombinant CDP-paratose 2-isomerase expressed by the recombinant cell according to claim 4 or 5 as a catalyst, and using uridine diphosphate glucose UDP-Glc or guanosine diphosphate glucose GDP-Glc as a substrate to react and prepare UDP-Man or GDP-Man.

8. The preparation method according to claim 7, characterized in that: The mutant or recombinant CDP-paratose 2-isomerase is added in an amount of 2.5-5 g / L; preferably, the reaction conditions are: a temperature of 30-60° C., a substrate concentration of 10-50 mM, and a reaction time of 4-6 hours.

9. Use of the mutant according to claim 1 or the recombinant cell according to claim 4 or 5 or the recombinant CDP-paratose 2-isomerase expressed by the recombinant cell according to claim 4 or 5 in the preparation of uridine diphosphate mannose UDP-Man or guanosine diphosphate mannose GDP-Man or a product containing uridine diphosphate mannose UDP-Man or a product containing guanosine diphosphate mannose GDP-Man.

10. Use of the mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4 or 5, or the preparation method according to claim 7 or 8 in the preparation of food, health products and cosmetics.

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