Glycosyltransferase mutant and application thereof

Abstract

The invention discloses a glycosyl transferase mutant and application thereof. The mutant is obtained by performing single mutation or multiple mutation on 58th, 181th or 371th amino acid of an amino acid sequence as shown in SEQ ID NO.4, or performing single mutation or multiple mutation on 54th, 152th, 241th or 272th amino acid of an amino acid sequence as shown in SEQ ID NO.6. The invention further discloses a preparation method of the glycosyl transferase mutant. According to the invention, homologous modeling and sequence homology analysis are utilized to carry out rational transformation on glycosyltransferase, and the glycosyltransferase mutant with obviously improved enzyme activity is obtained through screening. Compared with a wild type, the relative enzyme activities of the mutants UGTM1-3 and UGTM2-4 are respectively improved by 2.88 times and 3.60 times compared with those before modification, so that the mutant UGTM1-3 and UGTM2-4 have great industrial application potential.

Description

(I) Technical field

[0001] The invention belongs to the technical field of genetic engineering and enzymology research, and relates to a glycosyltransferase mutant and application thereof. (II) Background technology

[0002] Monk fruit (Siraitia grosvenorii), belonging to the dicotyledonous plant family Cucurbitaceae, is also known as Han Guo, Luo Huangzi, and false bitter melon, and is known as the "Chinese magic fruit". Monk fruit is rich in content and high in nutrition. As an important raw material of traditional Chinese medicine, it has been widely used in the medical and health field in China. In addition, because mature monk fruit extract has natural low-calorie glycoside components, it is widely used in health foods and beverages in business, becoming an ideal substitute for non-sugar sweeteners.

[0003] Mogroside is the main terpene active substance in monk fruit and the main source of the sweetness of monk fruit. Mogroside is a general term for sugar compounds with mogroside alcohol as aglycone. In 1975, American researcher Lee first reported that the main active ingredient in monk fruit extract is tetracyclic triterpenoid saponin. With the in-depth research on monk fruit and the advancement of related technologies, more and more types of mogrosides have been successfully isolated and identified. Mogroside V is high in content and sweetness. At a concentration of 1 part per 10,000, its sweetness value is about 425 times that of sucrose.

[0004] The synthesis methods of mogrosides include: plant extraction, chemical synthesis and biosynthesis. At present, the main way to obtain mogrosides is through plant extraction. Although the plant extraction method is efficient, the types and components of mogrosides obtained vary greatly due to the influence of the environment and seasons, the quality is greatly reduced, and the preparation process is complicated. The finished products are expensive and cannot meet market demand; the chemical synthesis method has high costs, and the process is complicated with multiple chemical protection and deprotection steps, many by-products, high energy consumption, and serious environmental pollution; the production of mogrosides using synthetic biology has the advantages of low cost, mild reaction conditions, green environmental protection, outstanding position selectivity and stereoselectivity.

[0005] Glycosyltransferases (UGT, EC 2.4.xy) are a class of enzymes that catalyze glycosyl transfer reactions. Most glycosylation reactions in nature are mediated by glycosyltransferases, which can catalyze the transfer of monosaccharides from donors to acceptors and generate glycosidic bonds. Glycosylation of acceptor aglycones usually occurs on the nucleophilic oxygen atom of the hydroxyl group, as well as on the nucleophilic nitrogen, sulfur and carbon atoms. Sugar donors include nucleotide sugars, disaccharides, oligosaccharides, etc.

[0006] In 2016, Israeli researchers Itkin et al. analyzed the transcriptome and genome data of monk fruit and comprehensively analyzed the biosynthetic pathway of mogroside V. First, glycosyltransferase UGT720-269-1 added sugars to the C24 and C3 positions of the main chain of monk fruit alcohol to generate Ⅰ-A1 and ⅡE; then glycosyltransferase UGT94-289-3 continued to add sugars to the side chains at C3 and C24 positions, thus obtaining simonoside Ⅰ and mogroside V with higher sweetness. Later studies reported that the M7 (T79Y / L48M / R28H / L109I / S15A / M76L / H47R) mutant obtained after the mutation of UGT74AC1 in the Luo Han Guo family was found to specifically glycosylate the C3 and C24 of the main chain of Luo Han Guo alcohol to generate IIE. Unlike UGT720, the M7 mutant first glycosylated the C3 site of Mogrol to generate IE, and then added a sugar group at the C24 site to generate IIE. The enzyme activity of the UGTMS1-M7 mutant (S34A / F77L / V146A / T344V / A313V / M360L / A391V) modified by Li Jiao et al. showed a 73- to 400-fold increase in other substrates such as IIIA, IIIE, IVA, IVE, Sia I and V. Currently, there are reports of engineered yeast strains that can synthesize mogroside from scratch, but there is insufficient research on the biosynthetic pathway of glycosylation modification of mogroside. Therefore, using glycosyltransferase to synthesize mogroside is a method with application potential.

[0007] Due to the large market demand for mogrosides, the traditional plant extraction method causes a shortage and waste of natural resources, and the low efficiency of chemical synthesis methods limits the supply of mogroside alcohol and its glycosylated derivatives. Therefore, the enzymatic glycosylation of mogroside alcohol has more potential for application development. However, the heterologous recombinant expression of plant glycosyltransferases using a prokaryotic expression system has low solubility, low catalytic activity, and poor catalytic specificity for substrates. Protein modification of them to obtain mutants with excellent catalytic activity is an effective method to solve the problems currently faced in the enzymatic preparation of mogroside V. (III) Summary of the invention

[0008] The purpose of the present invention is to provide a glycosyltransferase mutant and its application, which effectively improves the enzyme activity of the glycosyltransferase and solves the problem of low catalytic activity in the prior art.

[0009] The technical solution adopted by the present invention is:

[0010] The present invention provides a glycosyltransferase mutant, which is obtained by subjecting the 58th, 181st or 371st amino acid of the amino acid sequence shown in SEQ ID NO.4 to single mutation or multiple mutation, or subjecting the 54th, 152nd, 241st or 272nd amino acid of the amino acid sequence shown in SEQ ID NO.6 to single mutation or multiple mutation.

[0011] Furthermore, the glycosyltransferase mutant is obtained by mutating the valine at position 58 of the amino acid sequence shown in SEQ ID NO.4 into leucine, the serine at position 181 into phenylalanine, and the alanine at position 371 into serine, and is recorded as mutant UGTM1-3. The amino acid sequence of the mutant UGTM1-3 is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0012] Furthermore, the glycosyltransferase mutant is obtained by mutating the proline at position 54 of the amino acid sequence shown in SEQ ID NO.6 into alanine, the glycine at position 152 into threonine, the proline at position 241 into glutamate, and the serine at position 272 into histidine, and is recorded as mutant UGTM2-4. The amino acid sequence of the mutant UGTM2-4 is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7.

[0013] The present invention also provides a gene encoding the glycosyltransferase mutant, a recombinant expression plasmid constructed by the encoding gene, and a recombinant genetically engineered bacterium transformed by the recombinant expression plasmid. The recombinant expression plasmid can be directly sent to the company for synthesis, or the recombinant expression plasmid that can express the glycosyltransferase mutant of the present invention by inserting the target gene into the expression vector by any conventional means known to those skilled in the art is within the protection scope of the present invention, and the vector used to construct the recombinant expression plasmid is preferably pET28a. The host cell of the recombinant genetically engineered bacterium is E.coliBL21 (DE3). The recombinant genetically engineered bacterium is constructed as follows: the recombinant expression plasmid is transferred into E.coli BL21 (DE3) competent cells by the heat shock method, and the transformation product is evenly coated on an LB solid culture medium containing 100 μg / mL ampicillin, and cultured overnight at 37°C, and a single clone is picked for sequencing verification to obtain the recombinant genetically engineered bacterium.

[0014] The present invention also provides an application of the glycosyltransferase mutant in catalyzing the preparation of mogroside IIE from mogrol. The glycosyltransferase mutant is obtained by single or multiple mutations of the 58th, 181st or 371st amino acid of the amino acid sequence shown in SEQ ID NO.4, and the mutant UGTM1-3 is preferred.

[0015] Furthermore, the application method is: using the pure enzyme liquid obtained by ultrasonic extraction of wet bacteria obtained by fermentation and culture of the recombinant genetically engineered bacteria containing the glycosyltransferase mutant encoding gene as a catalyst, using mogroside alcohol and uridine diphosphate glucose (UDPG) as substrates, adding MgCl2, and constructing a reaction system with a pH 6-8 buffer as a reaction medium, reacting at 30-50°C to obtain mogroside IIE.

[0016] Furthermore, in the reaction system, the amount of catalyst is 30-70 mg / L, preferably 50 mg / L, based on the protein content of the pure enzyme solution; the final concentration of the added mogrosanol is 0.1-1 mM, preferably 0.5 mM; the final concentration of the added UDPG is 5-15 mM, preferably 10 mM; and the final concentration of the added MgCl2 is 5-15 mM, preferably 10 mM.

[0017] Furthermore, the buffer is 50 mM Tris-HCl (pH 8.0).

[0018] The present invention also provides an application of the glycosyltransferase mutant in catalyzing the preparation of mogroside V from mogroside IIE. The glycosyltransferase mutant is obtained by single or multiple mutations of the amino acid at position 54, position 152, position 241 or position 272 of the amino acid sequence shown in SEQ ID NO.6, and the mutant UGTM2-4 is preferred.

[0019] Furthermore, the application method is: using the pure enzyme liquid extracted by ultrasonic extraction of wet bacteria obtained by fermentation culture of the recombinant genetically engineered bacteria containing the glycosyltransferase mutant encoding gene as a catalyst, using Mogroside Ⅱ E and UDPG as substrates, adding MgCl2, and constructing a conversion system with a pH 6-8 buffer as a reaction medium, and carrying out a glycosylation reaction at 30-50°C to obtain mogroside V.

[0020] Furthermore, in the conversion system, the amount of catalyst used is 30-70 mg / L, preferably 50 mg / L, based on the protein content of the pure enzyme solution; the mogroside IIE is added to a final concentration of 0.1-1 mM, preferably 0.5 mM; the UDPG is added to a final concentration of 5-15 mM, preferably 10 mM; the MgCl2 is added to a final concentration of 5-15 mM, preferably 10 mM. The buffer is 50 mM Tris-HCl (pH 8.0).

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the present invention uses homology modeling and sequence homology analysis to rationally transform glycosyltransferases, and screens out glycosyltransferase mutants with significantly improved enzyme activity. Compared with the wild type, the relative enzyme activities of mutants UGTM1-3 and UGTM2-4 are increased by 2.88 times and 3.60 times respectively compared with before transformation, and have great industrial application potential. (IV) Description of the drawings

[0022] Figure 1 , Schematic diagram of the docking structure of UGTM1 and Mogrol.

[0023] Figure 2 , Schematic diagram of the molecular docking structure of UGTM2 and MogrosideⅡE.

[0024] Figure 3 , Schematic diagram of recombinant plasmid pET28a-UGTM1.

[0025] Figure 4 , bar graph of relative enzyme activity of UGTM1 mutants.

[0026] Figure 5 , bar graph of relative enzyme activity of UGTM1 mutants.

[0027] Figure 6 , Schematic diagram of recombinant plasmid pET28a-UGTM2.

[0028] Figure 7 , bar graph of relative enzyme activity of UGTM2 mutants.

[0029] Figure 8 , bar graph of relative enzyme activity of UGTM2 mutants.

[0030] Fig. 9 , SDS-PAGE of glycosyltransferase mutants; M: protein marker; lanes 1 and 2 are: UGTM1-3 crude enzyme solution and UGTM1-3 pure enzyme solution; lanes 3 and 4 are: UGTM2-4 crude enzyme solution and UGTM2-4 pure enzyme solution.

[0031] Fig.10, UGTM1, and UGTM1-3 product analysis; b represents the mass ratio of UGTM1 substrate and product; c represents the mass ratio of UGTM1-3 substrate and product.

[0032] Fig.11 , UGTM2, and UGTM2-4 product analysis; b represents the mass ratio of UGTM2 substrate and product; c represents the mass ratio of UGTM2-4 substrate and product. (V) Specific implementation methods

[0033] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0034] Unless otherwise specified, the materials and reagents used in the examples can be obtained from conventional sales channels;

[0035] The experimental methods used in the examples are conventional methods unless otherwise specified.

[0036] The LB medium composition was: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the solvent was deionized water, and the pH value was natural;

[0037] LB solid medium composition: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 20g / L, solvent is deionized water, pH value is natural.

[0038] 50 mM Tris-HCL (pH 8.0) buffer: Dissolve 6.05 g of Tris in ultrapure water, adjust the pH to 8.0 with HCl, and then add dilute to 1 L using a volumetric flask.

[0039] Buffer A: 50 mM Tris-HCl, 300 mM NaCl, 25 mM imidazole, pH 8.0.

[0040] Buffer B: 50 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 8.0.

[0041] 20 mM PBS buffer: 20 mM Na2HPO4, 20 mM NaH2PO4, 0.9% NaCl, pH 6.0.

[0042] binding buffer: 50mM Tris-HCl, 300mM NaCl, pH8.0.

[0043] Example 1: Screening of active sites of glycosyltransferase mutants

[0044] 1. Construction of UGTM1 and UGTM2 gene models and molecular docking

[0045] The glycosyltransferase gene UGT74AC1 from Monk Fruit reported by Li J team (Li J, Yang JG, Mou SC, et al. Efficient O-glycosylation of triterpenes enabled by protein engineering of plant glycosyltransferase UGT74AC1 [J]. Chemicals & Chemistry, 2020.) M7(T79Y / L48M / R28H / L109I / S15A / M76L / H47R) The whole gene was synthesized by GenScript (Nanjing) Co., Ltd. using the UGTM1 as a template, and the nucleotide sequence was shown in SEQ ID NO.3, and the amino acid sequence of the encoded enzyme was shown in SEQ ID NO.4.

[0046] The glycosyltransferase gene UGT94-298-3 from Monk Fruit reported by Li J team (Li J, Mou SC, Yang JG, et al. Glycosyltransferase engineering and multi-glycosylation routes development facilitating synthesis of high-intensity sweetener mogrosides[J]. iScience, 2022, 25(10): 105222-105222.) M7(S34A / F77L / V146A / T344V / A313V / M360L / A391V) The whole gene was synthesized by GenScript (Nanjing) Co., Ltd. using the gene as a template and named UGTM2. The nucleotide sequence is shown in SEQ ID NO.5 and the amino acid sequence of the encoded enzyme is shown in SEQ ID NO.6.

[0047] The crystal structure of UGTM1 has been reported in the literature, and its protein structure can be downloaded from the PDB database (ID: 6L8W). The software Swiss-model (https: / / swissmodel.expasy.org / ) was used to perform online modeling on UGTM2 (amino acid sequence as shown in SEQ ID NO.6), and the protein with the highest homology was selected as the model. The PROCHECK server (http: / / services.mbi.ucla.edu / PROCHECK / ) was used for structural evaluation. Both glycosyltransferases first introduced the UDPG in the 2ACW crystal structure into the UDPG binding site of UGTs by superposition. Molecular docking was performed after protonation and energy optimization of the substrate using MOE software to determine the key amino acids in the substrate pocket that regulate its conformation, providing ideas for subsequent experimental analysis. The docking results were visualized using PyMOL. See the schematic diagram of the molecular docking of UGTM1 and mogrosanol for details. Figure 1 Schematic diagram of UGTM2 and MogrosideⅡE molecular docking is shown in Figure 2 .

[0048] 2. Active site screening

[0049] ClustalW (https: / / embnet.vital-it.ch / software / ClustalW.html) and ESPript3 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi) were used for multiple sequence alignment. MOE software was used to infer the amino acids in the active sites, and virtual mutation sites can reduce the adverse mutations generated during the mutation process. Snapgene4.2.6 software was used for primer design, and BioEdit software was used for sequencing result comparison.

[0050] Combining the above analysis software and experimental screening results, the active sites of UGTM1 are valine at position 58, serine at position 181, and alanine at position 371. The active sites of UGTM2 are proline at position 54, glycine at position 152, proline at position 241, and serine at position 272.

[0051] Example 2: Screening and strain construction of glycosyltransferase mutants UGTM1-3

[0052] 1. Construction of starting plasmid

[0053] The UGTM1 gene synthesized in Example 1 was inserted into the multiple cloning site of the pET28a vector, and a 6×His-tag was introduced at the N-terminus and the C-terminus to obtain the recombinant expression plasmid pET28a-UGTM1 containing the glycosyltransferase gene. The plasmid map is shown in Figure 3 .

[0054] 2. Screening of glycosyltransferase mutants UGTM1-3

[0055] (1) Screening of mutation site V58L

[0056] Based on the amino acid sequence, structure and active site of UGTM1, 9 UGT sequences were searched from NCBI, which were from Momordica grosvenori (UGT94-289-1, UGT94-289-2, UGT94-289-3, UGT720-269-1, UGT720-269-2, UGT720-269-3), Stevia (UGT85C2, UGT74G1) and Rice (EUGT11). Through sequence alignment analysis, according to the consistency and difference in their evolutionary process, 10 sites (K32, V36, S37, V58, I60, S67, L80, K86, L91, F107) were finally selected from the amino acid sequence of UGTM1 shown in SEQ ID NO.4 for site-directed mutagenesis.

[0057] Plasmid pET28a-UGTM1 was used as a template, and the primers in Table 1 were used for site-directed PCR reaction. After sequencing was correct, the enzyme activity was measured according to the method of Examples 4 and 5. The results are shown in Figure 4 shown.

[0058] PCR reaction program: pre-denaturation at 98°C for 5 min, then enter temperature cycle at 98°C for 10 sec; 57°C for 15 sec; 72°C for 2 min; a total of 35 cycles, with a termination temperature of 4°C.

[0059] PCR reaction system: 1 μL of 1 ng / μL pET28a-UGTM1 plasmid as template, 1 μL of 10 μM primer F and primer R, 25 μL of 2× PrimeSTAR HSDNA Polymerase high-fidelity DNA polymerase, and 22 μL of ultrapure water.

[0060] Table 1. Mutation primers

[0061]

[0062] Figure 4The results showed that compared with UGTM1 (0.08U / g), the specific enzyme activity of mutant V58L was 0.12U / g, which was increased by 1.50 times. The relative enzyme activities of mutants K32R, S37T, V58F, and I60F did not change. The specific enzyme activities of mutants V36L, S37Y, S67L, L80M, L80Y, and L91F were 0.04U / g, 0.04U / g, 0.07U / g, 0.03U / g, 0.05U / g, and 0.04U / g, respectively, which were reduced to 0.50 times, 0.50 times, 0.87 times, 0.38 times, 0.63 times, and 0.50 times of the initial enzyme, respectively. K86A, L91A, and F107C mutants lost their catalytic activity. Therefore, mutant V58L was selected as the dominant mutant.

[0063] (2) Screening of mutation sites A371S and S181F

[0064] Using MOE software, the amino acids at three sites (S181, W370, A371) around the substrate Mogrol were virtually mutated (Table 2). Plasmid pET28a-UGTM1 was used as a template, and the primers in Table 3 were used to perform a site-directed PCR reaction using the PCR reaction program and system in step (1). The relative enzyme activity of the mutant was then detected using the methods of Examples 4 and 5. The results are shown in Table 2. Figure 5 .

[0065] Table 2 MOE virtual mutation design

[0066]

[0067] Table 3. Mutation primers

[0068]

[0069] Figure 5 The results showed that compared with the enzyme activity of UGTM1 (0.08U / g), mutants with improved enzyme activity were obtained: S181A, S181F, W370F and A371S, with specific enzyme activities of 0.10U / g, 0.12U / g, 0.11U / g and 0.12U / g, respectively, which were increased by 1.25 times, 1.50 times, 1.38 times and 1.50 times, respectively.

[0070] (3) UGTM1 combined mutations

[0071] The positive single mutants with improved UGTM1 enzyme activity obtained in steps (1)-(2) were combined and superimposed, and then the relative enzyme activity was detected using Examples 4 and 5. The results are shown in Table 4. On the basis of V58L, the S181F, W370F, and A371S sites were superimposed and mutated to first obtain double mutants V58L\S181F, V58L\W370F, and V58L\A371S, with specific enzyme activities of 0.15U / g, 0.13U / g, and 0.20U / g, respectively, and the relative enzyme activities were increased by 1.88 times, 1.63 times, and 2.50 times, respectively. Then, the superposition was continued on the basis of the double mutation to obtain triple mutants V58L\S181F\W370F, V58L\S181F\A371S, and V58L\W370F\A371S, respectively. The enzyme activity of the triple mutant V58L\S181F\A371S was significantly improved compared with the forward double mutation, with a specific enzyme activity of 0.23U / g, which was 2.88 times the initial enzyme activity. The triple mutant V58L\S181F\A371S was named UGTM1-3, and the enzyme activities of the other triple mutants were lower than those of UGTM1-3, with specific enzyme activities of 0.17U / g and 0.07U / g, respectively.

[0072] Table 4 Relative enzyme activities of UGTM1 combination mutants

[0073]

[0074] 3. Construction of recombinant genetically engineered bacteria

[0075] The recombinant mutant plasmid pET28a-UGTM1-3 was extracted and transferred into the competent expression strain E. coli BL21 (DE3) by heat shock method. The strain was cultured at 37°C for 12 h in LB medium containing 100 μg / mL Amp. The positive transformants were picked and identified to obtain the recombinant genetically engineered bacteria containing mutant UGTM1-3.

[0076] Example 3: Screening and strain construction of glycosyltransferase mutant UGTM2-4

[0077] 1. Starting plasmid

[0078] The UGTM2 gene synthesized in Example 1 was inserted into the multiple cloning site of the pET28a vector, and a 6×His-tag was introduced at the N-terminus and the C-terminus to obtain the recombinant expression plasmid pET28a-UGTM2. The plasmid map is shown in Figure 6 .

[0079] 2. Screening of glycosyltransferase UGTM2-4 and construction of engineered bacteria

[0080] (1) Single-site mutation screening

[0081] The method of Example 2 was used to modify different sites of UGTM2 to improve the enzyme activity.

[0082] according to Figure 2 Based on the molecular docking results, site-directed mutagenesis was performed on the selected sites, and positive mutants were screened by measuring the relative enzyme activity. According to the characteristics and structure of amino acids, amino acid categories with different characteristics (non-polar, polar, acidic, basic, aliphatic, aromatic and sulfur-containing) were selected, such as alanine (Ala), threonine (Thr), glutamic acid (Glu), histidine (His), phenylalanine (Phe) and methionine (Met) and other suitable strategic amino acids.

[0083] First, alanine scanning was performed on Y20, P54, K55, V238, P241, S272, M92, P93, L123, G152, and G193, and the relative enzyme activity was detected according to Examples 4 and 5. The results are shown in FIG. Figure 7 The dominant mutation sites P54, G152, and P241 were screened, among which the enzyme activity of P54A was 0.09U / g, which was 1.78 times higher than that of UGTM2 (0.05U / g).

[0084] Secondly, P54, G152, P241, and S272 were subjected to half-saturation mutations. P54, G152, P241, and S272 were mutated to P54T, P54E, P54H, P54F, P54M, G152T, G152E, G152H, G152F, G152M, P241T, P241E, P241H, P241F, P241M, S272T, S272E, S272H, S272F, and S272M, respectively. The relative enzyme activity was detected according to Examples 4 and 5. The results are as follows. Figure 8 , and obtained positive single mutants: G152T, P241E, S272H. The specific enzyme activities were 0.13U / g, 0.08U / g, and 0.06U / g, respectively, which were 2.60 times, 1.61 times, and 1.20 times higher than those of UGTM2 (0.05U / g).

[0085] (2) UGTM2 combined mutations

[0086] The single mutants P54A, G152T, P241E, and S272H with improved UGTM2 activity were combined and superimposed, and the results of enzyme activity determination are shown in Table 5. Among them, the quadruple mutant was named UGTM2-4, and the highest specific enzyme activity was 0.18 U / g, which was 3.60 times the initial enzyme activity.

[0087] Table 5 Relative enzyme activities of UGTM2 combination mutants

[0088]

[0089] 3. Construction of recombinant genetically engineered bacteria

[0090] The recombinant mutant plasmid pET28a-UGTM2-4 was extracted and transferred into the competent expression strain E. coli BL21 (DE3) by heat shock method. The strain was cultured at 37°C for 12 h in LB medium containing 100 μg / mL Amp. The positive transformants were picked and identified to obtain the recombinant genetically engineered bacteria containing mutant UGTM2-4.

[0091] Example 4: Expression and purification of glycosyltransferase mutant proteins

[0092] 1. Inducible expression of glycosyltransferase mutant genes

[0093] The engineered strains containing the glycosyltransferase mutants UGTM1-3 and UGTM2-4 constructed in Examples 2 and 3 were inoculated into LB medium containing 100 μg / mL Amp and shaken at 37° C. and 180 rpm for 12 h to obtain seed solution.

[0094] The seed solution was transferred to LB medium containing 100 μg / mL Amp at a 2% (v / v) inoculation rate, and shaken at 37°C, 180 rpm for 2 h until OD600 was 0.6-0.8. Isopropylthiogalactoside (IPTG) was added at a final concentration of 0.1 mM, and the expression was induced at 20°C, 180 rpm for 12 h.

[0095] 2. Purification of glycosyltransferase mutants

[0096] Step (1) The culture solution is centrifuged at 8000rpm and 4°C for 10min in a high-speed refrigerated centrifuge, the supernatant is discarded and the cells are retained, and the cells are resuspended and washed with ultrapure water, centrifuged at 8000rpm and 4°C for 10min, the supernatant is removed, the cells are collected and weighed. 1g of wet cells are weighed, resuspended in 20mL of 50mM Tris-HCl (pH 8.0) buffer, placed on ice, the power is 200W, working for 2s and resting for 4s, after ultrasonic disruption for 30min, the cell disruption solution is centrifuged at 4°C and 10000rpm for 10min, and the supernatant is the crude enzyme solution; in order to remove the impurities in the crude enzyme solution, the crude enzyme solution is first removed by a 0.45μm filter membrane, and the filtrate is collected.

[0097] Ni 2+ The affinity column was cleaned with ultrapure water and binding buffer, and Ni 2+The collected filtrate was loaded at a flow rate of 2 mL / min. Buffer A was used to elute the impurities until the UV absorbance reached the baseline equilibrium. Then, buffer B was used to elute the target protein. When the UV absorbance began to rise, the eluate was collected until the UV absorbance returned to the baseline and the collection was stopped. This was the target protein. Finally, the Ni was flushed with binding buffer. 2+ The affinity chromatography column was equilibrated to the baseline and protected with 20% ethanol.

[0098] The purified target protein is desalted by dialysis. Before dialysis, the dialysis bag (3.5kDa) needs to be leak-checked to ensure good sealing of the dialysis bag. The target protein is placed in a 20mM pH8.0 phosphate buffer in a dialysis bag, and the solution is changed every 6 hours. The dialysis is performed for 12 hours, and low temperature (ice bath 0°C) treatment is ensured throughout the desalination process. Finally, the intercepted liquid is collected as the pure enzyme liquid, and pure enzyme liquid UGTM1-3 and pure enzyme liquid UGTM2-4 are obtained respectively, which are divided into 1.5mL EP tubes, quickly frozen with liquid nitrogen, and stored in an ultra-low temperature refrigerator at -80°C for standby use. In the same way, pure enzyme liquid UGTM1 and pure enzyme liquid UGTM2 are prepared.

[0099] 3. SDS-PAGE analysis

[0100] The SDS-PAGE verification process is as follows: first, take 30μL of sample and mix it with 10μL 4×Protien Loading buffer, boil it at 100℃ for 10min, and then centrifuge it at 12000rpm for 2min to remove possible impurities and insoluble substances. Finally, take an appropriate amount of sample and protein marker and add them to the prefabricated polyacrylamide gel wells. Perform electrophoresis at 160V for 40min. After the electrophoresis, stain it with Coomassie Brilliant Blue and then decolorize it with a decolorizer. Finally, place the gel on an imager to observe the electrophoresis results.

[0101] The crude enzyme solution and the pure enzyme solution obtained in step (2) were tested by SDS-PAGE, and the results are shown in Fig. 9 The bands in the lanes were uniform, and the sizes of the protein bands were consistent with the molecular weights of UGTM1 (51.32 kDa) and UGTM2 proteins (50.59 kDa), respectively, and could be used for subsequent experiments.

[0102] Example 5: Enzyme activity measurement and enzymatic characterization of glycosyltransferase mutants

[0103] 1. Glycosyltransferase mutant specific enzyme activity measurement method

[0104] (1) Determination of UGTM1-3 enzyme activity

[0105] Definition of enzyme activity unit (U): The amount of enzyme required to convert 1 micromole of substrate per minute is one enzyme activity unit (U).

[0106] Reaction system: 0.5 mM Mogrol, 10 mM UDPG, 10 mM MgCl2, pure enzyme solution UGTM1-3 prepared in Example 3 with a protein content of 50 mg, and 50 mM Tris-HCl (pH 8.0) to make up to 300 μL.

[0107] After reacting at 40℃ for 10min, the enzyme was immediately inactivated by heating at 95℃ for 5min, and the same volume of methanol was added for dilution. The supernatant was filtered with a 0.22μm organic filter membrane. The filtrate was analyzed by high performance liquid chromatography (HPLC) system to analyze Mogrol and MogrosideⅡE in the obtained sample. Each reaction was repeated three times. Under the same conditions, pure enzyme solution UGTM1 was used as a control. The results showed that the enzyme activity of UGTM1-3 was 230U / mg; the enzyme activity of UGTM1 was 80U / mg, and the relative enzyme activity was increased by 2.88 times.

[0108] HPLC conditions: The treated samples were detected and analyzed using Aglient 1260 liquid phase. Analysis method: C18 column (250 mm × 4.6 mm, 5 μm); mobile phase A (acetonitrile + 0.1% formic acid) and mobile phase B (water + 0.1% formic acid); injection volume 5 μL; elution conditions: 0-20 min, 10%-90% A + 90%-10% B gradient elution; 20-22 min, 10% A + 90% B elution; 1 mL / min, wavelength 210 nm, column temperature 30 ° C.

[0109] (2) Determination of UGTM2-4 enzyme activity

[0110] Definition of enzyme activity unit (U): The amount of enzyme required to convert 1 micromole of substrate per minute is one enzyme activity unit (U).

[0111] Reaction system: 0.5 mM Mogroside Ⅱ E, 10 mM UDPG, 10 mM MgCl2, pure enzyme solution UGTM2-4 prepared by the method of Example 3 with a protein content of 50 mg, and 50 mM Tris-HCl (pH 8.0) to make up to 300 μL.

[0112] After reacting at 40℃ for 10min, the enzyme was immediately inactivated by heating at 95℃ for 5min, and the same volume of methanol was added for dilution. The supernatant was filtered with a 0.22μm organic filter membrane after centrifugation at 12000×g for 5min. The obtained samples MogrosideⅡE and Mogroside V were analyzed by high performance liquid chromatography (HPLC). Each reaction was repeated three times. Under the same conditions, pure enzyme solution UGTM2 was used as a control. The results showed that the enzyme activity of UGTM2-4 was 180U / mg; the enzyme activity of UGTM2 was 50U / mg, and the relative enzyme activity was increased by 3.60 times.

[0113] HPLC conditions were the same as in step (1).

[0114] 2. Kinetic parameter detection

[0115] (1) Kinetic parameters of UGTM1 and UGTM1-3

[0116] The kinetic parameters of UGTM1 and its mutants UGTM1-3 using Mogrol as substrate were determined.

[0117] Reaction system: different concentrations of Mogrol (0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM), pure enzyme solution UGTM1 or mutant UGTM1-3 prepared by the method of Example 3 with a protein content of 50 mg, 10 mM UDPG, 10 mM MgCl2, and the system was supplemented to 300 μL with 50 mM Tris-HCl (pH 8.0) buffer.

[0118] React for 10 minutes at 40°C and pH 8.0. After the reaction, the temperature was raised to 95°C and maintained for 5 minutes to effectively terminate the reaction process. An equal volume of methanol was added, centrifuged at 12000×g for 5 minutes, and HPLC detection was performed after filtration with a 0.22μm organic filter membrane. The detection method is the same as that described in step 1. The initial reaction velocities of UGTM1 and UGTM1-3 at different concentrations of mogrosanol were determined, and the Km and Vmax values ​​of UGTM1 and UGTM1-3 were obtained by nonlinear fitting using the Michaelis-Menten equation. The catalytic constant (kcat) and catalytic efficiency (kcat / Km) were then calculated using the formula. The results are shown in Table 6. The Km and kcat values ​​of UGTM1 are 96.57μM and 2.54min, respectively. -1 The Km and kcat values ​​of UGTM1-3 were 72.98 μM and 5.52 min, respectively. -1 , indicating that the mutant UGTM1-3 has a further enhanced affinity for the substrate. The catalytic rates kcat / Km of UGTM1 and UGTM1-3 are 26.35min -1 mM-1 、75.68min -1 mM -1 These results showed that UGTM1-3 had high catalytic activity and affinity for substrates.

[0119] Table 6 Kinetic parameters of UGTM1 and UGTM1-3

[0120]

[0121] (2) Kinetic parameters of UGTM2 and UGTM2-4

[0122] Reaction system: use different concentrations of Mogroside IIE of 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, and 0.6mM, pure enzyme solution UGTM2 or mutant UGTM2-4 prepared by the method of Example 3 with a protein content of 50mg, 10mM UDPG, 10mM MgCl2, and make up the system to 300μL with 50mM Tris-HCl (pH 8.0) buffer.

[0123] The kinetic parameters were analyzed using the method in step (1). The results are shown in Table 7. m , k cat The values ​​were 136.12 μM and 1.33 min, respectively. -1 , K of UGTM2-4 m , k cat The values ​​were 116.01 μM and 3.75 min, respectively. -1 , indicating that the mutant UGTM2-4 has a further enhanced affinity for the substrate. The catalytic rates k of UGTM2 and UGTM2-4 cat / K m 9.77min respectively -1 mM -1 、32.32min -1 mM -1 These results showed that UGTM2-4 had high catalytic activity and substrate affinity.

[0124] Table 7 Kinetic parameters of UGTM2 and UGTM2-4

[0125]

[0126] 3. Measurement of the optimal temperature of glycosyltransferase mutants

[0127] According to the reaction system of enzyme activity determination in step 1 above, the following temperatures were selected for the experiment: 25°C, 30°C, 35°C, 40°C, 45°C and 50°C to explore the optimal temperature of the reaction. The enzyme activity measured at the optimal temperature was set to 100%, and the relative enzyme activity measured was calculated based on the highest enzyme activity as a reference value. It was found that the optimal catalytic temperature of UGTM1-3 was 45°C, while that of UGTM2-4 was 40°C. Within the range of 40°C-50°C, both enzymes can maintain good catalytic activity, maintaining a level above 80%. Therefore, 40°C was determined as the optimal reaction temperature for subsequent experiments.

[0128] 4. Measurement of the optimal pH of glycosyltransferase mutants

[0129] In order to explore the optimal pH of glycosyltransferase, different pH buffers were prepared, including 50mM CH3COOH-CH3COONa buffer (pH 5.0-6.0), 50mM Tris-HCl buffer (pH 7.0-9.0) and 50mM NaH2PO4-Na2HPO4 buffer (pH 8.0-10.0). According to the reaction system of enzyme activity determination in step 1 above, buffers with different pH values ​​were used as reaction system solvents to calculate the enzyme activity. The enzyme activity measured at the optimal pH was set as 100%, and the relative enzyme activity measured was calculated based on the highest enzyme activity as a reference value. The results showed that the enzyme activity change trends of UGTM1-3 and UGTM2-4 under different pH conditions were basically the same. The optimal pH value of both is 8.0.

[0130] Example 6: Glycosylation reaction of substrates by glycosyltransferase mutants

[0131] 1. Glycosyltransferase mutants UGTM1-3

[0132] Reaction system: 0.5 mM Mogrol, 10 mM UDPG, 10 mM MgCl2, protein content 50 mg, pure enzyme solution UGTM1-3 or pure enzyme solution UGTM1 prepared in Example 3, 50 mM Tris-HCl (pH 8.0) supplemented to 300 μL.

[0133] After reacting at 40°C for 10 min, the enzyme was immediately inactivated by heating at 95°C for 5 min, followed by dilution with the same volume of methanol, centrifugation at 12000×g for 5 min, and filtration of the supernatant with a 0.22 μm organic filter membrane. The obtained sample was analyzed by HPLC under the same HPLC conditions as in Example 5. The results are shown in Fig.10UGTM1 or its mutant UGTM1-3 catalyzed the conversion of Mogrol to Mogroside ⅠE and finally generated Mogroside ⅡE. It was found that UGTM1 catalyzed Mogrol to generate 29.78% of Mogroside ⅡE, 27.75% of Mogrol and 42.47% of Mogroside ⅠE were not completely converted. However, UGTM1-3 catalyzed the synthesis of 64.05% of Mogroside ⅡE, 10.27% of Mogrol and 25.68% of Mogroside ⅠE were not completely converted. This shows that UGTM1-3 has higher catalytic activity and affinity for substrates than UGTM1.

[0134] 2. Glycosyltransferase mutant UGTM2-4

[0135] Reaction system: 0.5 mM Mogroside Ⅱ E, 10 mM UDPG, 10 mM MgCl2, pure enzyme solution UGTM2-4 or pure enzyme solution UGTM2 prepared by the method of Example 3 with a protein content of 50 mg, and 50 mM Tris-HCl (pH 8.0) to make up to 300 μL.

[0136] After reacting at 40°C for 10 min, the enzyme was immediately inactivated by heating at 95°C for 5 min, followed by dilution with the same volume of methanol, centrifugation at 12000×g for 5 min, and filtration of the supernatant with a 0.22 μm organic filter membrane. The obtained sample was analyzed by HPLC under the same HPLC conditions as in Example 5. The results are shown in Fig.11 UGTM2 or its mutant UGTM2-4 catalyzed the conversion of Mogroside ⅡE to Mogroside ⅢA, which was further converted to Mogroside ⅣA or Siamenoside Ⅰ, and finally generated Mogroside V. In the UGTM2 reaction system, 47.94% of Mogroside ⅢA, 14.47% of Mogroside ⅣA and Siamenoside Ⅰ, and 8.75% of Mogroside V were catalytically synthesized. In the UGTM2-4 reaction system, 21.73% of Mogroside ⅢA, 28.59% of Mogroside ⅣA and Siamenoside Ⅰ, and 35.16% of Mogroside Ⅴ were catalytically synthesized. This shows that UGTM2-4 has higher catalytic activity and affinity for substrates than UGTM2.

[0137] The above is a detailed implementation method and a specific operation flow of the present invention, which are implemented on the premise of the technical solution of the present invention, but the protection scope of the present invention is not limited to the above embodiments.

Claims

1. A glycosyltransferase mutant, characterized in that: The mutant is obtained by single or multiple mutation of the 58th, 181st or 371st amino acid of the amino acid sequence shown in SEQ ID NO.4, or single or multiple mutation of the 54th, 152nd, 241st or 272nd amino acid of the amino acid sequence shown in SEQ ID NO.

6.

2. The glycosyltransferase mutant according to claim 1, characterized in that The glycosyltransferase mutant is obtained by mutating the 58th valine of the amino acid sequence shown in SEQ ID NO.4 into leucine, the 181st serine into phenylalanine, and the 371st alanine into serine.

3. The glycosyltransferase mutant according to claim 1, characterized in that The glycosyltransferase mutant is obtained by mutating the proline at position 54 of the amino acid sequence shown in SEQ ID NO.6 into alanine, the glycine at position 152 into threonine, the proline at position 241 into glutamic acid, and the serine at position 272 into histidine.

4. A recombinant genetically engineered bacterium constructed with the gene encoding the glycosyltransferase mutant according to claim 1.

5. Use of the glycosyltransferase mutant according to claim 1 in catalyzing the preparation of mogroside IIE from mogroside alcohol, characterized in that: The glycosyltransferase mutant is obtained by subjecting the 58th, 181st or 371st amino acid of the amino acid sequence shown in SEQ ID NO.4 to single mutation or multiple mutation.

6. The use according to claim 5, characterized in that The application method comprises the following steps: using a pure enzyme solution extracted from wet bacteria ultrasonically obtained by fermentation culture of a recombinant genetically engineered bacterium containing the gene encoding the glycosyltransferase mutant as a catalyst, using mogroside alcohol and uridine diphosphate glucose as substrates, adding MgCl2, and constructing a reaction system with a pH 6-8 buffer as a reaction medium, reacting at 30-50°C to obtain mogroside IIE.

7. The use according to claim 6, characterized in that In the reaction system, the amount of catalyst used is 30-70 mg / L based on the protein content of the pure enzyme solution, the final concentration of mogrosanol added is 0.1-1 mM, the final concentration of UDP-glucose added is 5-15 mM, and the final concentration of MgCl2 added is 5-15 mM.

8. Use of the glycosyltransferase mutant according to claim 1 in catalyzing the preparation of mogroside V from mogroside IIE, characterized in that: The glycosyltransferase mutant is obtained by subjecting the amino acid at position 54, position 152, position 241 or position 272 of the amino acid sequence shown in SEQ ID NO.6 to single mutation or multiple mutation.

9. The use according to claim 8, characterized in that The application method comprises the following steps: using a pure enzyme solution extracted from wet bacteria ultrasonically obtained by fermentation culture of a recombinant genetically engineered bacterium containing the glycosyltransferase mutant encoding gene as a catalyst, using mogroside Ⅱ E and uridine diphosphate glucose as substrates, adding MgCl2, and constructing a conversion system with a pH 6-8 buffer as a reaction medium, and carrying out a glycosylation reaction at 30-50°C to obtain mogroside V.

10. The use according to claim 9, characterized in that In the conversion system, the amount of catalyst used is 30-70 mg / L based on the protein content of the pure enzyme solution, the final concentration of mogroside Ⅱ E added is 0.1-1 mM, the final concentration of UDP-glucose added is 5-15 mM, and the final concentration of MgCl2 added is 5-15 mM.

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