Glycosyltransferase mutants and uses thereof

Abstract

The application discloses a glycosyltransferase mutant and application thereof, and the mutant is obtained by single mutation or multiple mutations of amino acids at positions 58, 181 or 371 of the amino acid sequence shown in SEQ ID NO. 4 or single mutation or multiple mutations of amino acids at positions 54, 152, 241 or 272 of the amino acid sequence shown in SEQ ID NO. 6. The glycosyltransferase is rationally modified by homologous modeling and sequence homology analysis, and a glycosyltransferase mutant with obviously improved enzyme activity is screened. Compared with the wild type, the relative enzyme activity of the mutants UGTM1-3 and UGTM2-4 is respectively improved by 2.88 times and 3.60 times compared with that before modification, and the mutants have great industrial application potential.

Description

(I) Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzymology research technology, and relates to a glycosyltransferase mutant and its application. (II) Background Technology

[0002] Monk fruit (Siraitia grosvenorii), belonging to the Cucurbitaceae family of dicotyledonous plants, is also known as Han Guo, Luo Huang Zi, and Jia Ku Gua, and is often called the "Chinese miracle fruit." Rich in nutrients, monk fruit is an important raw material in traditional Chinese medicine and is widely used in the medical and health fields in China. Furthermore, because mature monk fruit extract contains natural low-calorie glycosides, it is widely used commercially in health foods and beverages, becoming an ideal alternative to sugar-free sweeteners.

[0003] Mogrosides are the main terpenoid active substances in monk fruit (Siraitia grosvenorii), and are the primary source of its sweetness. Mogrosides are a collective term for sugar compounds with mogroside as the aglycone. In 1975, American researcher Lee first reported that the main active ingredient in monk fruit extract was a tetracyclic triterpenoid saponin. With the deepening of research on monk fruit and the advancement of related technologies, an increasing number of mogrosides have been successfully isolated and identified. Mogroside V is particularly high in content and sweetness; at a concentration of 0.01%, its sweetness value is approximately 425 times that of sucrose.

[0004] The synthesis methods of mogrosides include plant extraction, chemical synthesis, and biosynthesis. Currently, the main route to obtain mogrosides is through plant extraction. Although plant extraction is highly efficient, it is greatly affected by environmental and seasonal factors, resulting in significant differences in the types and components of the obtained glycosides, leading to a substantial reduction in quality. Furthermore, the preparation process is complex, and the resulting products are expensive, failing to meet market demand. Chemical synthesis is costly, complex, involves multiple chemical protection and deprotection steps, produces numerous byproducts, consumes a large amount of energy, and causes serious environmental pollution. Synthetic biosynthesis of mogrosides offers advantages such as low cost, mild reaction conditions, environmental friendliness, and outstanding positional and stereoselectivity.

[0005] Glycosyltransferases (UGT, EC 2.4.xy) are a class of enzymes that catalyze glycosylation reactions. The vast majority of glycosylation reactions in nature are mediated by glycosyltransferases, which catalyze the transfer of monosaccharides from donors to acceptors, forming glycosidic bonds. Glycosylation of acceptor aglycones typically occurs at the nucleophilic oxygen atom of the hydroxyl group, as well as at 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 *Siraitia grosvenorii* (monk fruit) to comprehensively elucidate the biosynthetic pathway of mogroside V. First, glycosyltransferase UGT720-269-1 adds glycosyl groups to the C24 and C3 positions of the mogroside backbone, generating I-A1 and IIE, respectively. Subsequently, glycosyltransferase UGT94-289-3 continues to add glycosyl groups to the branches at the C3 and C24 positions, thus obtaining the highly sweet symmenidine I and mogroside V. Subsequent studies reported that the M7 (T79Y / L48M / R28H / L109I / S15A / M76L / H47R) mutant obtained by the UGT74AC1 mutation of the mogrol family specifically glycosylated the C3 and C24 sites of the mogrol alcohol backbone to generate IIE. Unlike UGT720, the M7 mutant first glycosylated the C3 site of Mogrol to generate IE, and then added a glycosyl 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 reported yeast strains that synthesize mogrosides de novo, but research on the biosynthetic pathways of mogroside glycosylation modification is insufficient. Therefore, the synthesis of mogrosides using glycosyltransferases is a method with application potential.

[0007] Due to the large market demand for mogrosides, traditional plant extraction methods lead to the waste of natural resources, and the low efficiency of chemical synthesis methods limit the supply of mogrool and its glycosylated derivatives. Therefore, enzymatic catalysis of mogrool glycosylation has greater potential for application development. However, the heterologous recombinant expression of plant glycosyltransferases using prokaryotic expression systems suffers from low solubility, low catalytic activity, and poor substrate specificity. Modifying these proteins 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 this invention is to provide a glycosyltransferase mutant and its application, which effectively improves the enzyme activity of glycosyltransferase and solves the problem of low catalytic activity in the prior art.

[0009] The technical solution adopted in this invention is:

[0010] The present invention provides a glycosyltransferase mutant, which is obtained by single or multiple mutations of amino acids at positions 58, 181, or 371 of the amino acid sequence shown in SEQ ID NO.4, or by single or multiple mutations of amino acids at positions 54, 152, 241, or 272 of the amino acid sequence shown in SEQ ID NO.6.

[0011] Furthermore, the glycosyltransferase mutant was obtained by mutating valine at position 58 of the amino acid sequence shown in SEQ ID NO.4 to leucine, serine at position 181 to phenylalanine, and alanine at position 371 to serine, and is designated as mutant UGTM1-3. The amino acid sequence of 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 was obtained by mutating proline at position 54 of the amino acid sequence shown in SEQ ID NO.6 to alanine, glycine at position 152 to threonine, proline at position 241 to glutamic acid, and serine at position 272 to histidine, and is designated as mutant UGTM2-4. The amino acid sequence of 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] This invention also provides a coding gene for the glycosyltransferase mutant, a recombinant expression plasmid constructed from the coding gene, and a recombinant genetically engineered bacterium transformed from the recombinant expression plasmid. The recombinant expression plasmid can be directly synthesized by a company, or a recombinant expression plasmid capable of expressing the glycosyltransferase mutant of this invention can be obtained by inserting the target gene into an expression vector using any conventional method known to those skilled in the art. The vector used to construct the recombinant expression plasmid is preferably pET28a. The host cell of the recombinant genetically engineered bacterium is E. coli BL21(DE3). The recombinant genetically engineered bacterium is constructed as follows: the recombinant expression plasmid is transformed into E. coli BL21(DE3) competent cells using a heat shock method; the transformation product is evenly spread on LB solid medium containing 100 μg / mL ampicillin; after overnight culture at 37°C, single clones are picked and sequenced for verification to obtain the recombinant genetically engineered bacterium.

[0014] The present invention also provides the application of the glycosyltransferase mutant in the catalytic preparation of mogroside IIE from mogrol, wherein the glycosyltransferase mutant is obtained by single or multiple mutations of amino acids at positions 58, 181 or 371 of the amino acid sequence shown in SEQ ID NO.4, with mutant UGTM1-3 being preferred.

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

[0016] Furthermore, in the reaction system, the amount of catalyst used, based on the protein content of the pure enzyme solution, is 30-70 mg / L, preferably 50 mg / L; the final concentration of the added mogroside 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 solution is 50 mM Tris-HCl (pH 8.0).

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

[0019] Furthermore, the method of application is as follows: using the pure enzyme solution obtained by ultrasonic extraction of wet bacterial cells from recombinant genetically engineered bacteria containing the glycosyltransferase mutant encoding gene through fermentation culture as a catalyst, using Mogroside IIE and UDPG as substrates, adding MgCl2, and using a buffer solution of pH 6-8 as the reaction medium to construct a transformation system, and carrying out a glycosylation reaction at 30-50℃ to obtain mogroside V.

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

[0021] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following: This invention utilizes homology modeling and sequence homology analysis to rationally modify 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, showing great potential for industrial application. (iv) Description of the attached drawings

[0022] Figure 1 A schematic diagram of the docking structure between UGTM1 and Mogrol.

[0023] Figure 2 Schematic diagram of the molecular docking structure of UGTM2 and Mogroside IIE.

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

[0025] Figure 4 UGTM1 mutant relative enzyme activity bar graph.

[0026] Figure 5 UGTM1 mutant relative enzyme activity bar graph.

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

[0028] Figure 7 UGTM2 mutant relative enzyme activity bar graph.

[0029] Figure 8 UGTM2 mutant relative enzyme activity bar graph.

[0030] Figure 9 SDS-PAGE image of glycosyltransferase mutant; M: protein marker; lanes 1 and 2: crude enzyme solution of UGTM1-3 and pure enzyme solution of UGTM1-3; lanes 3 and 4: crude enzyme solution of UGTM2-4 and pure enzyme solution of UGTM2-4.

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

[0032] Figure 11 UGTM2 and UGTM2-4 product analysis; b represents the mass ratio of substrate and product in UGTM2; c represents the mass ratio of substrate and product in UGTM2-4. (V) Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

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

[0035] Unless otherwise specified, the experimental methods used in the implementation series are all conventional methods;

[0036] The LB medium consisted of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, deionized water as solvent, and natural pH.

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

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

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

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

[0041] 20mM PBS buffer: 20mM Na2HPO4, 20mM NaH2PO4, with 0.9% NaCl added, 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 and molecular docking of UGTM1 and UGTM2 gene models

[0045] The glycosyltransferase gene UGT74AC1 derived from *Siraitia grosvenorii* reported by Li J's 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) Using UGTM1 as a template, the entire genome was synthesized by GenScript (Nanjing) Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO.3. The amino acid sequence encoding the enzyme is shown in SEQ ID NO.4.

[0046] The glycosyltransferase gene UGT94-298-3 from monk fruit reported by Li J's 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) Using UGTM2 as a template, the entire genome was synthesized by GenScript (Nanjing) Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO.5. The amino acid sequence encoding the 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). Swiss-model software (https: / / swissmodel.expasy.org / ) was used to model UGTM2 (amino acid sequence as shown in SEQ ID NO. 6) online, selecting the protein with the highest homology as the model. The PROCHECK server (http: / / services.mbi.ucla.edu / PROCHECK / ) was used for structural evaluation. Both glycosyltransferases first introduced UDPG from the 2ACW crystal structure into the UDPG binding site of UGTs via superposition. After substrate quantification and energy optimization using MOE software, molecular docking was performed to determine the key amino acids regulating the conformation within the substrate pocket, providing insights for subsequent experimental analysis. The docking results were visualized using PyMOL. A schematic diagram of the molecular docking between UGTM1 and mogroside is shown below. Figure 1 A schematic diagram of the molecular docking between UGTM2 and Mogroside IIE is shown below. Figure 2 .

[0048] 2. Screening of active sites

[0049] Multiple sequence alignment was performed using ClustalW (https: / / embnet.vital-it.ch / software / ClustalW.html) and ESPript3 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi). MOE software was used to infer amino acids at active sites, and dummy mutation sites can reduce unfavorable mutations generated during the mutation process. Snapgene 4.2.6 software was used for primer design, and BioEdit software was used for sequencing result alignment.

[0050] Based on the 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 mutant UGTM1-3

[0052] 1. Construction of the starter plasmid

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

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

[0055] (1) Screening of mutation site V58L

[0056] Based on the amino acid sequence, structure, and active sites of UGTM1, nine UGT sequences were searched on NCBI, originating from *Siraitia grosvenorii* (UGT94-289-1, UGT94-289-2, UGT94-289-3, UGT720-269-1, UGT720-269-2, UGT720-269-3), *Stevia repens* (UGT85C2, UGT74G1), and rice (EUGT11). Through sequence alignment analysis, based on their consistency and differences during evolution, ten sites (K32, V36, S37, V58, I60, S67, L80, K86, L91, F107) were selected from the amino acid sequence of UGTM1 shown in SEQ ID NO.4 for site-directed mutagenesis.

[0057] Using plasmid pET28a-UGTM1 as a template, site-directed PCR was performed using the primers in Table 1. After successful sequencing, enzyme activity was measured according to the methods in Examples 4 and 5. The results are shown in [Table 1]. Figure 4 As shown.

[0058] PCR reaction program: pre-denaturation at 98℃ for 5 min, followed by temperature cycling at 98℃ for 10 sec; 57℃ for 15 sec; 72℃ for 2 min; for a total of 35 cycles, with a termination temperature of 4℃.

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

[0060] Table 1. Mutant Primers

[0061]

[0062] Figure 4The results showed that compared with UGTM1 (0.08 U / g), the specific enzyme activity of mutant V58L was 0.12 U / g, an increase of 1.50-fold. The relative enzyme activities of mutants K32R, S37T, V58F, and I60F remained unchanged. The specific enzyme activities of mutants V36L, S37Y, S67L, L80M, L80Y, and L91F were 0.04 U / g, 0.04 U / g, 0.07 U / g, 0.03 U / g, 0.05 U / g, and 0.04 U / g, respectively, representing a decrease to 0.50-fold, 0.50-fold, 0.87-fold, 0.38-fold, 0.63-fold, and 0.50-fold of the initial enzyme activity. Mutants K86A, L91A, and F107C lost their catalytic activity. Therefore, mutant V58L was selected as the dominant mutant.

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

[0064] Using MOE software, virtual mutations were performed on the amino acids at three sites (S181, W370, and A371) surrounding the substrate Mogrol (Table 2). Using plasmid pET28a-UGTM1 as a template, site-directed PCR was performed using the primers in Table 3 according to the PCR reaction procedure and system in step (1). The relative enzyme activity of the mutants was then detected using the methods in 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 (0.08 U / g), mutants with increased enzyme activity were obtained: S181A, S181F, W370F and A371S, with specific enzyme activities of 0.10 U / g, 0.12 U / g, 0.11 U / g and 0.12 U / g, respectively, which were 1.25 times, 1.50 times, 1.38 times and 1.50 times.

[0070] (3) UGTM1 combinatorial mutations

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

[0072] Table 4. Relative enzyme activities of UGTM1 combined mutants

[0073]

[0074] 3. Construction of recombinant genetically engineered bacteria

[0075] The recombinant mutant plasmid pET28a-UGTM1-3 was extracted and transformed into competent cells of the expression strain E. coli BL21(DE3) by heat shock. The cells were cultured at 37°C for 12 h in LB medium containing 100 μg / mL Amp. The bacteria were picked and positive transformants were identified to obtain recombinant genetically engineered bacteria containing the 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, with a 6×His-tag introduced at both the N-terminus and C-terminus, resulting in the recombinant expression plasmid pET28a-UGTM2. (See plasmid map below.) Figure 6 .

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

[0080] (1) Screening for unit point mutations

[0081] Using the method in Example 2, different sites of UGTM2 were modified to enhance enzyme activity.

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

[0083] First, alanine scans were performed at positions Y20, P54, K55, V238, P241, S272, M92, P93, L123, G152, and G193. Relative enzyme activity was then measured according to Examples 4 and 5, and the results are shown below. Figure 7 The dominant mutation sites P54, G152 and P241 were screened out. Among them, the specific enzyme activity of P54A was 0.09 U / g, which was 1.78 times higher than that of UGTM2 (0.05 U / g).

[0084] Next, half-saturation mutagenesis was performed on P54, G152, P241, and S272. 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. Relative enzyme activity was measured according to Examples 4 and 5, and the results are as follows: Figure 8 Positive single mutants were obtained: G152T, P241E, and S272H. The specific enzyme activities were 0.13 U / g, 0.08 U / g, and 0.06 U / g, respectively, which were 2.60 times, 1.61 times, and 1.20 times higher than UGTM2 (0.05 U / g).

[0085] (2) UGTM2 combinatorial mutation

[0086] The single mutants P54A, G152T, P241E, and S272H, which exhibited enhanced UGTM2 enzyme activity, were combined and superimposed. The enzyme activity assay results are shown in Table 5. Among them, the four mutants were named UGTM2-4, and the highest specific enzyme activity was measured to be 0.18 U / g, which is 3.60 times the initial enzyme activity.

[0087] Table 5. Relative enzyme activities of UGTM2 combined mutants

[0088]

[0089] 3. Construction of recombinant genetically engineered bacteria

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

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

[0092] 1. Induced expression of glycosyltransferase mutant genes

[0093] The engineered strains containing 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 culture.

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

[0095] 2. Purification of glycosyltransferase mutants

[0096] Step (1) The culture medium was centrifuged at 8000 rpm and 4℃ for 10 min using a high-speed refrigerated centrifuge. The supernatant was discarded, and the bacterial cells were retained. The bacterial cells were then resuspended and washed with ultrapure water, centrifuged at 8000 rpm and 4℃ for 10 min, and the supernatant was discarded. The bacterial cells were collected and weighed. 1 g of wet bacterial cells was weighed and resuspended in 20 mL of 50 mM Tris-HCl (pH 8.0) buffer. The solution was placed on ice and sonicated at 200 W for 30 min with a 2 s working and 4 s resting interval. The cell lysate was then centrifuged at 10000 rpm for 10 min at 4℃. The supernatant was the crude enzyme solution. In order to remove impurities from the crude enzyme solution, the crude enzyme solution was first filtered through a 0.45 μm filter membrane and the filtrate was collected.

[0097] Ni 2+ The affinity chromatography column was cleaned with ultrapure water and binding buffer, and then Ni was installed. 2+The column was flushed again until equilibrium was reached. The collected filtrate was loaded onto the sample at a flow rate of 2 mL / min. First, buffer A was used to elute impurities until the UV absorbance reached baseline equilibrium. Then, buffer B was used to elute the target protein. When the UV absorbance began to rise, the eluent was collected until the UV absorbance returned to baseline; this was the target protein. Finally, the column was flushed with binding buffer. 2+ The affinity chromatography column was brought to baseline equilibration and then maintained with a column containing 20% ​​ethanol.

[0098] The purified target protein was desalted using dialysis. Before dialysis, the dialysis bag (3.5 kDa) was leak-tested to ensure a good seal. The target protein was placed in the dialysis bag in 20 mM pH 8.0 phosphate buffer, and the buffer was changed every 6 hours for 12 hours. The entire desalting process was carried out at low temperature (0°C on ice). The retentate was collected as the pure enzyme solution, yielding pure enzyme solutions UGTM1-3 and UGTM2-4, which were aliquoted into 1.5 mL EP tubes, flash-frozen in liquid nitrogen, and stored at -80°C for later use. Pure enzyme solutions UGTM1 and UGTM2 were prepared using the same method.

[0099] 3. SDS-PAGE analysis

[0100] The SDS-PAGE validation process is as follows: First, mix 30 μL of sample with 10 μL of 4× Protein Loading buffer, boil at 100℃ for 10 min, and then centrifuge at 12000 rpm for 2 min to remove possible impurities and insoluble substances. Finally, add an appropriate amount of sample and protein marker to the wells of a pre-prepared polyacrylamide gel and perform electrophoresis at 160V for 40 min. After electrophoresis, stain with Coomassie Brilliant Blue and then destain with a destaining agent. Finally, place the gel in an imaging system to observe the electrophoresis results.

[0101] The crude enzyme solution and pure enzyme solution obtained in step (2) were analyzed by SDS-PAGE, and the results are shown in the figure. Figure 9 The bands in the lanes exhibited uniformity, and the sizes of the protein bands matched the molecular weights of UGTM1 (51.32 kDa) and UGTM2 (50.59 kDa), respectively, which can be used for subsequent experiments.

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

[0103] 1. Method for measuring the specific enzyme activity of glycosyltransferase mutants

[0104] (1) Enzyme activity assay of UGTM1-3

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

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

[0107] After reacting at 40℃ for 10 min, the enzyme was immediately inactivated by heating at 95℃ for 5 min. The mixture was then diluted with an equal volume of methanol, centrifuged at 12000×g for 5 min, and the supernatant was filtered through a 0.22 μm organic filter membrane. The filtrate was analyzed for Mogrol and Mogroside IIE using high-performance liquid chromatography (HPLC). Each reaction was performed in triplicate. Pure enzyme solution UGTM1 was used as a control under the same conditions. The results showed that the enzyme activity of UGTM1-3 was 230 U / mg, while that of UGTM1 was 80 U / mg, representing a 2.88-fold increase in relative enzyme activity.

[0108] HPLC conditions: The processed samples were analyzed using an Aglient 1260 HPLC system. Analytical 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) Enzyme activity assay of UGTM2-4

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

[0111] Reaction system: 0.5mM Mogroside IIE, 10mM UDPG, 10mM MgCl2, UGTM2-4 pure enzyme solution with a protein content of 50mg prepared in Example 3, and 50mM Tris-HCl (pH 8.0) to a final volume of 300μL.

[0112] After reacting at 40℃ for 10 min, the enzyme was immediately inactivated by heating at 95℃ for 5 min. The mixture was then diluted with the same volume of methanol, centrifuged at 12000×g for 5 min, and the supernatant was filtered through a 0.22 μm organic filter membrane. The obtained samples of Mogroside IIE and Mogroside V were analyzed using high-performance liquid chromatography (HPLC). Each reaction was repeated three times. Pure enzyme solution UGTM2 was used as a control under the same conditions. The results showed that the enzyme activity of UGTM2-4 was 180 U / mg, while the enzyme activity of UGTM2 was 50 U / mg, representing a 3.60-fold increase in relative enzyme activity.

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

[0114] 2. Dynamic parameter detection

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

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

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

[0118] The reaction was carried out at 40℃ and pH 8.0 for 10 min. After the reaction, the temperature was increased to 95℃ and maintained for 5 min to effectively terminate the reaction. An equal volume of methanol was added, and the mixture was centrifuged at 12000×g for 5 min. After filtration through a 0.22 μm organic filter membrane, HPLC analysis was performed. The detection method was the same as described in step 1. The initial reaction rates of UGTM1 and UGTM1-3 at different mogroside concentrations were determined. The Km and Vmax values ​​of UGTM1 and UGTM1-3 were obtained by nonlinear fitting using the Michaelis-Menten equation, and the catalytic constant (kcat) and catalytic efficiency (kcat / Km) were calculated using the formula. The results are shown in Table 6. The Km and kcat values ​​of UGTM1 were 96.57 μM and 2.54 min, respectively. -1 The Km and kcat values ​​of UGTM1-3 were 72.98 μM and 5.52 min, respectively. -1 This indicates that the mutant UGTM1-3 has a further enhanced affinity for the substrate. The catalytic rates kcat / Km of UGTM1 and UGTM1-3 are 26.35 min, respectively. -1 ·mM-1 75.68min -1 ·mM -1 These results demonstrate that UGTM1-3 possesses high catalytic activity and substrate affinity.

[0119] Table 6. Dynamic parameters of UGTM1 and UGTM1-3

[0120]

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

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

[0123] The dynamic parameters were analyzed using the method in step (1), and the results are shown in Table 7. The K value of UGTM2 is... m k cat The values ​​were 136.12 μM and 1.33 min, respectively. -1 UGTM2-4's K m k cat The values ​​were 116.01 μM and 3.75 min, respectively. -1 This indicates that the mutant UGTM2-4 has a further enhanced affinity for the substrate. The catalytic rates k of UGTM2 and UGTM2-4 are... cat / K m 9.77 min respectively -1 ·mM -1 32.32min -1 ·mM -1 These results demonstrate that UGTM2-4 possesses high catalytic activity and substrate affinity.

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

[0125]

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

[0127] Following the reaction system described in step 1 above for enzyme activity determination, the following temperatures were selected for experiments: 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃, to investigate the optimal reaction temperature. The enzyme activity measured at the optimal temperature was set as 100%, and the relative enzyme activities were calculated as a ratio using the highest enzyme activity as a reference value. It was found that the optimal catalytic temperature for UGTM1-3 was 45℃, while for UGTM2-4 it was 40℃. Within the range of 40℃-50℃, both enzymes maintained good catalytic activity, remaining above 80%. Therefore, 40℃ was determined to be the optimal reaction temperature for subsequent experiments.

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

[0129] To investigate the optimal pH for glycosyltransferases, different pH buffers were prepared: 50 mM CH3COOH-CH3COONa buffer (pH 5.0–6.0), 50 mM Tris-HCl buffer (pH 7.0–9.0), and 50 mM NaH2PO4-Na2HPO4 buffer (pH 8.0–10.0). Following the reaction system described in step 1 above for enzyme activity determination, different pH buffers were used as solvents to calculate enzyme activity. The enzyme activity measured at the optimal pH was set as 100%, and the relative enzyme activity was calculated as a ratio with the highest enzyme activity as the reference value. The results showed that the activity trends of UGTM1-3 and UGTM2-4 were basically consistent under different pH conditions. The optimal pH for both was 8.0.

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

[0131] 1. Glycosyltransferase mutant UGTM1-3

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

[0133] After reacting at 40℃ for 10 min, the enzyme was immediately inactivated by heating at 95℃ for 5 min. Then, an equal volume of methanol was added for dilution, and the mixture was centrifuged at 12000×g for 5 min. The supernatant was then filtered through a 0.22 μm organic filter membrane. The obtained sample was analyzed by HPLC under the same conditions as in Example 5. The results are shown below. Figure 10UGTM1 or its mutant UGTM1-3 catalyzes the conversion of Mogrol to Mogroside ⅠE, ultimately leading to Mogroside ⅡE. It was found that UGTM1 catalyzed the incomplete conversion of Mogrol to Mogroside ⅡE, 27.75% to Mogrol, and 42.47% to Mogroside ⅠE. In contrast, UGTM1-3 catalyzed the incomplete conversion of Mogrol to 64.05% to Mogroside ⅡE, 10.27% to Mogrol, and 25.68% to Mogroside ⅠE. This indicates that UGTM1-3 exhibits higher catalytic activity and substrate affinity than UGTM1.

[0134] 2. Glycosyltransferase mutant UGTM2-4

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

[0136] After reacting at 40℃ for 10 min, the enzyme was immediately inactivated by heating at 95℃ for 5 min. Then, an equal volume of methanol was added for dilution, and the mixture was centrifuged at 12000×g for 5 min. The supernatant was then filtered through a 0.22 μm organic filter membrane. The obtained sample was analyzed by HPLC under the same conditions as in Example 5. The results are shown below. Figure 11 UGTM2 or its mutant UGTM2-4 catalyzes the conversion of Mogroside IIE to Mogroside IIIA, further to Mogroside IVA or Siamenoside I, and finally to 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 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 synthesized. This indicates that UGTM2-4 exhibits higher catalytic activity and substrate affinity than UGTM2.

[0137] The above is a detailed implementation method and specific operation process of the present invention, which is implemented under 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 glycosyltransferase mutant was obtained by mutating valine at position 58 of the amino acid sequence shown in SEQ ID NO.4 to leucine, serine at position 181 to phenylalanine, and alanine at position 371 to serine.

2. A recombinant genetically engineered bacterium constructed from the encoding gene of the glycosyltransferase mutant of claim 1.

3. The application of the glycosyltransferase mutant of claim 1 in the catalytic preparation of mogroside IIE from mogroside.

4. The application as described in claim 3, characterized in that, The method of application is as follows: using the pure enzyme solution obtained by ultrasonic extraction of wet bacterial cells from recombinant genetically engineered bacteria containing the glycosyltransferase mutant encoding gene through fermentation culture as a catalyst, using mogroside and uridine diphosphate glucose as substrates, adding MgCl2, and using a buffer solution of pH 6-8 as the reaction medium to construct a reaction system, and reacting at 30-50℃ to obtain mogroside IIE.

5. The application as described in claim 4, 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 monk fruit alcohol is 0.1-1 mM, the final concentration of uridine diphosphate glucose is 5-15 mM, and the final concentration of MgCl2 is 5-15 mM.

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