Isomerase mutant with improved acid resistance

Abstract

The invention discloses an isomerase mutant with improved acid resistance, and belongs to the technical field of enzyme engineering. The enzyme activities of the mutants GALEM1 and GALEM2 provided by the invention at the pH value of 6.5 are respectively 1.98 times and 2.14 times of the enzyme activities of WT. The half-life periods of the mutants GALEM1 and GALEM2 under the condition of pH 6.5 are 6.9 h and 8.9 h respectively, and are prolonged by 0.1 h and 2.1 h respectively compared with WT. GALEM2 and sucrose synthase SusyM6 are cascaded to produce UDP-Gal from UDP and sucrose, after optimization, the total reaction time is shortened to only 1.25 h, 24.5 mM of UDP-Gal can be produced, and the final STY is 12 g / L / h.

Description

Technical Field

[0001] The present invention relates to an isomerase mutant with improved acid resistance and belongs to the technical field of enzyme engineering. Background Art

[0002] UDP-galactose (UDP-Gal) is the most common galactose donor, which can provide galactose donors for many active glycosides, such as thiogalactoside, α-galactosylceramide, quercetin-3-O-galactoside. UDP-Gal is also a key sugar donor for the production of prebiotic galactans and human milk oligosaccharides, including 2'-fucosyllactose, lacto-N-tetraose and 3'-sialyllactose. These galactose-containing carbohydrates have immunomodulatory functions, antagonize pathogens, promote brain development, intestinal prebiotics and neuromodulation, and are widely used as food ingredients, such as dietary fiber supplements. However, the low yield and high price of commercial UDP-Gal have hindered the efforts for large-scale and low-cost production of active galactose-containing carbohydrates, and there is a desire for effective and cost-effective synthesis of UDP-Gal.

[0003] Epimerization plays a crucial role in carbohydrate synthesis and can achieve interconversion by reversing the stereostructure of the sugar asymmetric center. Carbohydrate epimerase is 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 are only catalytically active towards activated nucleotide sugars, and there are fewer enzymes that can catalyze free sugars. Epimerases acting on nucleotide sugar substrates provide a more diverse catalytic combination, including oxidation / reduction and elimination / reproduction, so they can expand the scope of substrate isomerization conversion.

[0004] UDP-galactose 4-epimerase can catalyze the direct conversion of nucleotide glucose into nucleotide galactose. However, the activity of UDP-galactose 4-epimerase is relatively low. Therefore, improving the activity of UDP-galactose 4-epimerase can achieve the rapid synthesis of nucleotide mannose. UDP-glucose also belongs to a high-cost substrate, and sucrose synthase (Susy M6 ) can achieve the efficient synthesis of UDP-galactose from sucrose and UDP, but the optimal reaction pH of sucrose synthase is relatively low. In addition, a relatively low pH is also beneficial for industrial production. Therefore, developing a UDP-galactose 4-epimerase with simultaneously improved acid resistance and enzyme activity will be beneficial to the economical and low-cost production of nucleotide galactose, thereby reducing the price of nucleotide galactose, and is expected to provide an ideal donor substrate raw material for the synthesis of galactose-containing active substances. Summary of the Invention

[0005] In view of the deficiencies of the above-mentioned prior art, the present invention provides an isomerase mutant with improved acid resistance, aiming to solve the technical problem of low activity of UDP-galactose 4-epimerase under low pH conditions.

[0006] The first technical solution provided by the present invention is a UDP-galactose 4-epimerase mutant, which is obtained by mutating the lysine at position 29 of the UDP-galactose 4-epimerase parent into glutamate, resulting in R29E, named GALE M1 , and the amino acid sequence of the parent is shown in SEQ ID NO.1.

[0007] The present invention also provides a UDP-galactose 4-epimerase mutant, which is obtained by mutating the lysine at position 233 of the GALE M1 into glutamate, resulting in R29E-R233E, named GALE M2 .

[0008] In some embodiments, the source of the UDP-galactose 4-epimerase is Ganoderma lucidum.

[0009] In some embodiments, the nucleotide sequence encoding the UDP-galactose 4-epimerase is shown in SEQ ID NO.2.

[0010] The second technical solution provided by the present invention is a gene encoding the UDP-galactose 4-epimerase mutant described in the first technical solution.

[0011] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0012] In some embodiments, the recombinant vector uses plasmid PTIG as the expression vector.

[0013] The fourth technical solution provided by the present invention is a recombinant cell expressing the UDP-galactose 4-epimerase mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant vector described in the third technical solution.

[0014] In some embodiments, the recombinant cell uses bacteria or fungi as the expression host.

[0015] In some embodiments, the recombinant cell uses Escherichia coli as the expression host.

[0016] The fifth technical solution provided by the present invention is a method for improving the acid resistance and / or enzyme activity of UDP-galactose 4-epimerase. The method is to perform any one of the following mutations on the UDP-galactose 4-epimerase with the amino acid sequence shown in SEQ ID NO. 1:

[0017] (1) Mutating the lysine at position 29 to glutamate;

[0018] (2) Mutating the lysine at position 29 to glutamate and mutating the lysine at position 233 to glutamate.

[0019] The sixth technical solution provided by the present invention is a method for preparing uridine diphosphate galactose (UDP-Gal). The method is to use the mutant described in the first technical solution as a catalyst and use uridine diphosphate glucose (UDP-Glc) as a substrate to form a reaction system, and react to prepare UDP-Gal.

[0020] In some embodiments, the uridine diphosphate glucose is obtained by using UDP and sucrose as substrates and catalyzed by sucrose synthase.

[0021] Structural formula of uridine diphosphate galactose (UDP-Gal, C 15 H 24 N2O 17 P2):

[0022]

[0023] Structural formula of uridine diphosphate glucose (UDP-Glc, C 15 H 24 N2O 17 P2):

[0024]

[0025] In some embodiments, the addition amount of the catalyst is 0.1-5 g / L.

[0026] In some embodiments, the reaction conditions are: at a temperature of 10-50 °C and a substrate concentration of 1-10 mM, the pH of the system is 5-9, and the reaction is carried out for 1-10 min.

[0027] In some embodiments, the reaction conditions are: PB buffer (pH 5.0-9.0), reaction temperature 20-50 °C, 1-10 mM substrate reacts for 1-10 min.

[0028] In some embodiments, the reaction conditions are: MOPS buffer (pH 5.0-9.0), reaction temperature 20-50 °C, 1-10 mM substrate reacts for 1-10 min.

[0029] In some embodiments, the UDP-galactose 4-epimerase mutant is obtained by expression of the recombinant cell described in the fourth technical solution.

[0030] The seventh technical solution provided by the present invention is the application of the mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the method described in the fifth technical solution, or the method described in the sixth technical solution in the preparation of uridine diphosphate galactose UDP-Gal or a product containing uridine diphosphate galactose UDP-Gal.

[0031] The present invention also provides the application of the above mutant, or the above gene, or the above recombinant vector, or the above recombinant cell, or the above preparation method in the preparation of food, health products and cosmetics.

[0032] The technical effects of the present invention are as follows:

[0033] (1) By means of genetic engineering, the UDP-galactose 4-epimerase (GALE) derived from Ganoderma lucidum is modified in the present invention, and transformants with improved acid resistance and enzyme activity are obtained through single-point mutation and combinatorial mutation, which can achieve efficient synthesis of UDP-Gal at the optimal temperature.

[0034] (2) The half-life of GALE M1 at pH 6.5 is 6.9 h, which is comparable to that of WT. The enzyme activity with UDP-Glc as the substrate at pH 6.5 is 0.98 times higher than that of WT.

[0035] (3) The half-life of GALE M2 at pH 6.5 is 8.9 h, which is 2.1 h longer than that of WT. The enzyme activity with UDP-Glc as the substrate at pH 6.5 is 1.14 times higher than that of WT.

[0036] (4) The application of the mutant enzyme can achieve high-level synthesis of UDP-Gal under low pH conditions. Under the conditions of pH 5 - 8, reaction temperature 30 - 60 °C, 80 - 120 mM UDP and 1 - 2 M sucrose, react for 0.5 - 2 h; after terminating the reaction, add 10 - 50 U of CIAP (nucleotide degrading enzyme) and react for 0.1 - 0.5 h under the conditions of pH 5 - 8 and reaction temperature 30 - 60 °C; after terminating the reaction, react for 1 - 4 h under the conditions of pH 5.0 - 8.0, reaction temperature 20 - 50 °C and 80 - 120 mM UDP-Glc. GALE M2The highest yield of UDP-Gal was 15 g / L. Under the conditions of pH 5.0 - 8.0, reaction temperature 20 - 50 °C, and 80 - 120 mM substrate, WT reacted for 1 - 4 h, and the highest yield of UDP-Gal only reached 6.7 g / L. Brief Description of the Drawings

[0037] Figure 1 is the relative enzyme activity of the single-point mutant under the conditions of pH 6.5 (A) and pH 5.5 (B).

[0038] Figure 2 is the relative enzyme activity of the single-point mutant under the conditions of pH 6.5 (A) and pH 5.5 (B).

[0039] Figure 3 is the relative enzyme activity of the single-point mutant under the conditions of pH 6.5 (A) and pH 5.5 (B).

[0040] Figure 4 is for the method of obtaining the combined mutant (A), WT, GALE M1 and GALE M2 's relative enzyme activity (B), and the relative enzyme activity of the combined mutant under the conditions of pH 6.5 (C) and pH 5.5 (D).

[0041] Figure 5 is for WT, GALE M1 and GALE M2 's optimal pH (A)(B)(C) and half-life at pH 6.5 (D)(E)(F).

[0042] Figure 6 is for Susy M6 and GALE M2 cascade production of UDP-Gal.

[0043] Figure 7 is a protein gel diagram. Detailed Embodiments

[0044] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0045] Testing method:

[0046] Enzyme activity assay method: 0.5 g / L of the enzyme was incubated in 50 mmol / L PB buffer (pH 8.0) containing 4 mmol / L UDP-Glc with a total volume of 100 μL at 30 °C for 5 min.

[0047] Definition of enzyme activity: The amount of enzyme required to convert 1 μmol of UDP-Glc in 1 minute is defined as 1 enzyme activity unit (U).

[0048] Detection of the optimal pH of the enzyme: PB buffers with different pH values were used. The enzyme activity of the pure enzyme was measured at pH 5.5, 6.5, 8.0, and 9.0 respectively to explore the optimal pH of the enzyme.

[0049] Half-life detection: PB buffers with different pH values were used. After incubating the pure enzyme for different times at pH 5.5, 6.5, 8.0, and 9.0 respectively, its enzyme activity was detected to explore the half-life of the enzyme.

[0050] Determination method of products: The determination of UDP-Glc and UDP-Gal used a Waters T3 column as the chromatographic column, with a mobile phase of 30 mM triethylamine acetate (pH 6.0), a flow rate of 1.0 ml / min, and a UV detector.

[0051] Raw materials used in the examples:

[0052] The culture media involved in the following examples are as follows:

[0053] LB medium: Tryptone 10 g / L (Thermo Scientific TM Oxoid), yeast extract 5 g / L (ThermoScientific TM Oxoid), sodium chloride (NaCl) 10 g / L (Sinopharm), pH 7.4; adding 2% agar to the LB liquid medium makes it the LB solid medium.

[0054] TB medium: Tryptone 12 g / L (Thermo Scientific TM Oxoid), yeast extract 24 g / L (Thermo Scientific TM Oxoid), K2HPO4 12.54 g / L KH2PO4 2.31 g / L (Sinopharm), glycerol 5 g / L (Sinopharm).

[0055] The solutions involved in the following examples are as follows:

[0056] PB buffer: 2.299 g of Na2HPO4 (Sinopharm), 0.455 g of NaH2PO4 (Sinopharm), made up to 1 L.

[0057] Solution A (equilibration solution): In the above PB solution, add 0.3404 g / L imidazole (5 mM) (Aladdin), 29.22 g / L NaCl (Sinopharm) (0.5 M), and adjust the pH to 7.4.

[0058] Solution B (eluent): In the above PB solution, add 34.075 g / L imidazole (500 mM) (Aladdin) and 29.22 g / L NaCl (0.5 M) (Sinopharm Chemical Reagent Co., Ltd.), and adjust the pH to 7.4.

[0059] The primer information involved is shown in Table 1:

[0060] Table 1 Primers for mutant construction

[0061]

[0062]

[0063]

[0064] The sucrose synthase Susy involved in the following examples M6 has been published in the literature "Synthesis of value-added uridine 5'-diphosphate-glucose from sucrose applying an engineered sucrose synthase counteracts the activity-stability trade-off" (citation directory: Liting, Zhao, Zhongbao, Ma, Linpei, Zhang et al. Synthesis of value-added uridine 5'-diphosphate-glucose from sucrose applying an engineered sucrose synthase counteracts the activity-stability trade-off. [J]. Food Chem, 2024, 464:0.).

[0065] The calf intestinal alkaline phosphatase CIAP involved in the following examples is from Thermo Fisher Scientific. Enzyme activity definition: At 37 °C, one unit hydrolyzes 1 μmol of 4-nitrophenyl phosphate in 1 minute.

[0066] Example 1: Construction of UDP-galactose 4-epimerase (GALE) and its mutants

[0067] (1) Construction of a recombinant plasmid containing the gene encoding GALE

[0068] The UDP-galactose 4-epimerase derived from Ganoderma lucidum was selected as the wild type. The amino acid sequence of the UDP-galactose 4-epimerase is shown in SEQ ID NO.1, and the nucleotide sequence encoding the UDP-galactose 4-epimerase is shown in SEQ ID NO.2.

[0069] SEQ ID NO.1:

[0070] MSELKRVLVTGGAGYIGSHVIFALQQTRRYKVISVDNHHNSSPKALERVAKIARDALPADASVQDKDSAEIDVHTVDLTKPDQIRSVFAKYGKGGIWGVIHIAAYKAVGESTEIPLTYYENNVSATVYLLQVAGEFDCTRVVYSSSATVYGTPPVIPIPETTRLEAHSPYGKTKVMCETIISDLCAAEPKRWRGLSLRYFNPGGAHPSGDIGEAPIGRPGNLFPLLAAIAVGRQPNDLKVFGNDYPTPDGTCVRDYLHVLDLAKGHLLALDALAPESKVFDDCPTDARYKAYNLGRGKGMSVLQIVEAMRAETKFDFKYEIVGRRRGDVPDLTADPALAEAQLGFKADKDLETMCRDLWNWQTKNPKGYDTD

[0071] The nucleotide sequence SEQ ID NO.2 was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0072] SEQ ID NO.2:

[0073] ATGTCGGAACTCAAACGAGTGCTAGTCACCGGGGGTGCAGGCTACATCGGCTCGCACGTCATTTTCGCGCTGCAGCAGACGCGGAGATACAAGGTCATCTCCGTCGACAACCACCACAACTCGTCCCCCAAGGCCCTCGAGCGCGTCGCAAAGATCGCGCGCGACGCCCTCCCCGCCGACGCGTCCGTGCAGGACAAGGACAGCGCCGAGATCGACGTGCACACCGTGGACCTCACCAAGCCCGACCAGATCCGCTCCGTGTTCGCCAAGTACGGCAAGGGCGGCATCTGGGGCGTCATCCACATCGCGGCGTACAAGGCCGTCGGGGAGTCGACGGAGATCCCGCTGACGTACTACGAGAACAACGTGTCCGCGACGGTCTACCTCCTCCAGGTCGCGGGCGAGTTCGACTGCACGCGCGTCGTGTACTCGTCCTCCGCGACCGTGTACGGCACCCCGCCCGTCATCCCCATCCCCGAGACGACCCGGCTCGAGGCGCACTCGCCGTACGGCAAGACCAAGGTCATGTGCGAGACCATCATCTCCGACTTGTGCGCGGCAGAGCCCAAGCGGTGGCGTGGGCTGTCGCTGCGGTACTTCAACCCGGGGGGTGCGCACCCGTCTGGCGACATCGGCGAGGCCCCCATCGGCCGCCCGGGCAACCTGTTCCCCCTCCTCGCTGCTATTGCGGTCGGACGCCAACCGAACGACCTCAAGGTCTTCGGCAATGACTACCCTACTCCTGATGGGACCTGCGTGCGGGACTACTTGCACGTCCTCGACCTCGCGAAGGGTCACTTGCTCGCTCTGGACGCGCTCGCGCCCGAGTCGAAGGTGTTCGACGACTGCCCGACTGACGCGCGCTACAAGGCGTACAACCTCGGGCGTGGCAAAGGCATGAGTGTGCTCCAGATCGTCGAGGCGATGCGCGCAGAGACCAAGTTCGACTTCAAGTACGAGATCGTCGGTCGCAGGCGTGGGGACGTTCCCGACCTGACGGCGGACCCCGCCCTTGCGGAGGCACAGCTGGGCTTCAAGGCGGACAAGGACCTCGAGACGATGTGCCGTGACCTTTGGAACTGGCAGACGAAGAACCCCAAGGGGTACGATACCGAC

[0074] Select EcoR I and Hind III on PTIG as the sites for inserting the target gene, and amplify the target gene. Forward primer: CCGGAATTCATGTCGGAACTCAAACGAGTG, reverse primer: CCCAAGCTTCTAGTCGGTATCGTACCCCTTGGGGTTC, so that the target gene carries restriction enzyme sites. Double digest PTIG and purify it to obtain a linear plasmid fragment. Connect the target gene with the restriction enzyme sites and the linear plasmid fragment together through homologous recombination. The system for homologous recombination contains: 1 μL of enzyme-digested linear plasmid, 1 μL of target fragment, 2 μL of Buffer, 1 μL of homologous recombination enzyme, and 5 μL of H2O. The homologous recombination is carried out at 37 °C for 30 min. Then, store the ligation product at -20 °C.

[0075] Transform the ligation product into Escherichia coli DE3 (BL21) competent cells. After culturing at 37 °C for 1 h, spread the bacterial solution on a solid LB medium plate containing ampicillin antibiotic resistance. After overnight culture, pick single colonies, amplify the culture, extract the plasmid, perform colony PCR with universal primers, sequence and verify the plasmids with positive PCR results, and finally obtain the correct recombinant plasmid of UDP-galactose 4-epimerase gene: PTIG-GALE and the recombinant strain: E. coli DE3 (BL21) / PTIG-GALE.

[0076] (2) Construction of recombinant plasmid of UDP-galactose 4-epimerase mutant

[0077] Using the PTIG-GALE plasmid as a template, perform whole plasmid PCR with the primers in Table 1. First, screen single-point mutants with increased enzyme activity relative to WT under the condition of pH 6.5, and obtain ten forward mutants, namely Q234E, Q342E, R29E, R29D, R233E, K264E, K264D, K51D, K66E, and K239E. Their relative enzyme activities are increased by 151.6%, 118.3%, 168.21%, 122.6%, 131.73%, 119.6%, 119.4%, 106.5%, 109.7%, and 103.7% respectively

[0078] ( Figures 1-3 )。

[0079] Ten mutation sites were introduced sequentially through a specific strategy, and finally PTIG-GALE was obtained: M1 Plasmid GALE M1 is R29E; PTIG-GALE M2 Plasmid GALE M2 is R29E-R233E, and the relative enzyme activities are increased by 168.21% and 214.26% respectively ( Figure 4 ).

[0080] The recombinant plasmid GALE M2 was digested with DpnI for about 2 h, and then transferred into competent Escherichia coli BL21(DE3) by heat shock method. Monoclonal colonies were selected and cultured overnight at 37 °C and 200 rpm in LB medium. Plasmids were extracted from the monoclonal strain cultures and sent to a sequencing company for sequencing. The recombinant strain containing the successfully mutated mutants was the one with correct sequencing results: E. coli BL21(DE3) / GALE M2 . Among them, GALE M2 is R29E-R233E.

[0081] (3) Induction expression and purification of the target protein

[0082] The monoclonal strain E. coli BL21(DE3) / GALE with correct sequencing M2 was cultured in a shake flask in LB medium for 12 h for activation, then inoculated into fresh TB medium and cultured at 37 °C and 200 rpm until the OD reached 0.8. IPTG with a final concentration of 0.1 mM was added, and the culture was continued at 16 °C and 200 rpm for 20 h. After the culture was completed, the precipitate was collected by centrifugation. The precipitate was treated with a cell disruptor and then centrifuged to collect the supernatant to obtain a crude enzyme solution. The pure enzyme was obtained by nickel column purification method.

[0083] The method of nickel column purification is as follows:

[0084] First, the nickel column was equilibrated with equilibration buffer A for 15 min, and then the sample was injected at a flow rate of 1 mL·min -1 . After the injection was completed, the column was rinsed with solution A again for 15 min. Subsequently, by adjusting the mixing ratio of solution B and solution A, the concentration of imidazole in the eluent was made to be 75 mM, and then the impurities were eluted with the eluent. Then, the concentration of imidazole in the eluent was adjusted to 500 mM, and the target protein was eluted and collected. The eluent containing the target protein (UDP-galactose 4-epimerase mutant) collected above was desalted and concentrated through an ultrafiltration tube, and the concentrated solution was stored at 4 °C for subsequent experiments.

[0085] GALE was prepared separately M2The pure enzyme solution has a gel electrophoresis pattern as shown in Figure 7 The results show that the molecular weight is 39 kDa and it is a single band, indicating a relatively high purity of the enzyme.

[0086] According to the above method, the wild-type WT pure enzyme solution was prepared.

[0087] Example 2: Properties of Mutant GALE M2

[0088] The enzymatic properties of the GALE M1 and GALE M2 pure enzyme solutions prepared in Example 1 were detected respectively, and the specific results are as follows:

[0089] (1) Detection of the Optimal pH

[0090] 0.5 g / L of the enzyme was reacted in 50 mmol / L PB buffer (pH 5.5, 6.5, 8.0, 9.0) containing 4 mmol / L UDP-Glc with a total volume of 100 μL at 30 °C for 5 min. The amount of enzyme required to convert 1 μmol of UDP-Glc within 1 min was defined as 1 enzyme activity unit (U), with the enzyme activity of WT as the control.

[0091] The relative enzyme activity measurement results show that:

[0092] The relative enzyme activities of mutant GALE M1 and GALE M2 with UDP-Glc as the substrate at pH 6.5 were increased by 0.89 times and 1.14 times respectively compared to WT, but the optimal pH was still 8.0 ( Figure 5 ).

[0093] (2) Detection of the Half-life

[0094] The enzyme activity of the pure enzyme was measured at 30 °C, and the enzyme activity at 0 min was taken as the highest value of 100%. The pure enzyme was continuously stored at pH 5.5, 6.5, 8.0, and 9.0 respectively, and the remaining enzyme activity was measured at regular intervals to explore the acid resistance of the enzyme.

[0095] The results show that:

[0096] The half-life of the mutant pure enzyme at 30 °C was measured, and the fitting analysis results show that the half-life of GALE M1 was 6.9 h, which was not much different from that of WT; the half-life of GALE M2 was 8.9 h, which was 2.1 h longer than that of WT, and the half-life of wild-type WT was 6.8 h ( Figure 5 ).

[0097] It can be seen that the mutator has successfully overcome the problem of improving acid resistance, achieving simultaneous improvement in stability and enzyme activity.

[0098] Example 3: Application of UDP-galactose 4-epimerase mutant and sucrose synthase Susy M6 Cascade production of UDP-Gal.

[0099] The specific mode is as follows:

[0100] (A) Under the condition of adding 100 mM UDP and 1 M sucrose to 500 μL of phosphate buffer (pH 6.5), add sucrose synthase Susy with a final concentration of 240 U / L M6 React for 0.5 - 2 h; after terminating the reaction, adjust the pH to 8.0 and react at a reaction temperature of 50 °C for 0.1 - 0.5 h; after terminating the reaction, add GALE with a final concentration of 2 g / L and react at a reaction temperature of 30 °C for 0.5 - 4 h.

[0101] (B) Under the condition of adding 100 mM UDP and 1 M sucrose to 500 μL of phosphate buffer (pH 6.5) respectively, add sucrose synthase Susy with a final concentration of 240 U / L M6 React for 0.5 - 2 h; after terminating the reaction, add GALE with a final concentration of 2 g / L M2 and react at a reaction temperature of 30 °C for 0.5 - 4 h.

[0102] (C) Under the condition of adding 100 mM UDP and 1 M sucrose to 500 μL of phosphate buffer (pH 6.5) respectively, add sucrose synthase Susy with a final concentration of 240 U / L M6 React for 0.5 - 2 h; after terminating the reaction, add CIAP with a final concentration of 40 U / L and react for 0.5 h, then after terminating the reaction, add GALE with a final concentration of 2 g / L M2 and react at a reaction temperature of 30 °C for 0.5 - 4 h.

[0103] (D) Under the condition of adding 100 mM UDP and 1 M sucrose to 500 μL of phosphate buffer (pH 6.5) respectively, add sucrose synthase Susy with a final concentration of 240 U / L M6 React for 0.5 - 2 h; after terminating the reaction, add CIAP with a concentration of 40 U / L and react for 0.25 h, then after terminating the reaction, add GALE with a final concentration of 2 g / L M2 and react at a reaction temperature of 30 °C for 0.5 - 4 h.

[0104] Figure 6 The results show that in mode (A), the production of UDP-Gal is slow and finally reaches 11 mM. In mode (B), Susy M6The reaction reached equilibrium in 0.5 h in the first stage, and the yield of UDP-Glc reached 90.4 mM. The addition of GALE M2 After that, due to the inhibition of UDP, the synthesis of UDP-Gal increased slowly in the early stage and finally reached a yield of 26.1 mM after 2 h. In mode (C), GALE was added 15 min after CIAP treatment M2 to completely remove UDP. At this time, UDP-Gal was rapidly produced within 15 min and approached equilibrium within 30 min, and the STY could reach 31.1 g / L / h during the UDP-Gal synthesis stage. For the entire catalytic cycle, the total reaction time was shortened to only 1.75 h, and 25.5 mM UDP-Gal was produced at a final STY of 8.9 g / L / h. Therefore, the reaction system was further optimized. In mode (D), the reaction time in the first stage was reduced to 0.5 h, the total reaction time was shortened to only 1.25 h, 24.5 mM UDP-Gal was produced, and the final STY was 12 g / L / h.

[0105] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A UDP-galactose 4-epimerase mutant, characterized in that, The mutant is obtained by mutating the lysine R at position 29 of the UDP-galactose 4-epimerase parent with the amino acid sequence shown in SEQ ID NO.1 into glutamic acid E, and is named GALE M1 ; Alternatively, the mutant is obtained by mutating the lysine (R) at position 29 and the lysine (R) at position 233 of the UDP-galactose 4-epimerase parent having the amino acid sequence as shown in SEQ ID NO.1 into glutamic acid (E), and is named GALE M1 .

2. A gene encoding the UDP-galactose 4-epimerase mutant according to claim 1.

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

4. The UDP-galactose 4-epimerase mutant according to claim 1, or a recombinant cell containing the gene according to claim 2, or transformed with the recombinant vector according to claim 3.

5. A method for improving the acid resistance and / or enzyme activity of UDP-galactose 4-epimerase, characterized in that, The method is to perform any one of the following mutations on the UDP-galactose 4-epimerase with the amino acid sequence shown in SEQ ID NO.1: (1) Mutating the lysine at position 29 to glutamate; (2) Mutating the lysine at position 29 to glutamate and mutating the lysine at position 233 to glutamate.

6. A method for preparing uridine diphosphate galactose (UDP-Gal), characterized in that, The method is to use the mutant according to claim 1 as a catalyst to form a reaction system with uridine diphosphate glucose (UDP-Glc) as a substrate, and react to prepare UDP-Gal.

7. The preparation method according to claim 6, characterized in that, The uridine diphosphate glucose is obtained by using sucrose synthase to catalyze with UDP and sucrose as substrates.

8. The preparation method according to claim 6, characterized in that, The addition amount of the catalyst is 0.1-5 g / L, and the substrate concentration is 1-10 mM.

9. The preparation method according to claim 6, wherein The reaction temperature is 20-50 °C, the reaction pH is 5.0-9.0, and the reaction time is 1-10 min.

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 3, or the method according to claim 5, or the method according to any one of claims 6-9 in the preparation of uridine diphosphate galactose UDP-Gal or a product containing uridine diphosphate galactose UDP-Gal.

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