Glycosyl transferase BsYjic mutant and application of glycosyl transferase BsYjic mutant in synthesis of Isarobrolone C glucoside product

Abstract

The invention relates to a glycosyl transferase BsYjic mutant and an application of the glycosyl transferase BsYjic mutant in the synthesis of an Isarobrolone C glucoside product. According to the invention, two mutants D106M and S128A are obtained by directionally modifying glycosyl transferase BsYjic, and glycosylation modification can be efficiently carried out on Isarobrolone C in a high-specificity manner; according to the present invention, two glucoside products such as 3 '-O-beta-D-Glc-isarobrolone C and 3-O-beta-D-Glc-isarobrolone C are respectively synthesized, the water solubility of the two single glucoside products is significantly improved, the problem of poor water solubility of the isarobrolone C is effectively solved, and the foundation is established for the development of new drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biocatalysis by enzymes, and particularly relates to a glycosyltransferase BsYjic mutant and its application in the synthesis of Isarubrolone C glucoside products. Background Art

[0002] Actinomycetes are widely distributed in nature, especially common in soil and water. Actinomycetes are known for their rich metabolites, including antibiotics, anti-tumor compounds, enzymes, etc. Streptomyces is an important class of actinomycetes that produce antibiotics. Isarubrolone C is a rubrolone compound produced by Streptomyces fermentation. Its molecular structural formula is as follows, and its structural feature is that a tropone ring is included in its pentacyclic skeleton.

[0003]

[0004] Isarubrolone C has low cytotoxicity and has the biological activity of activating autophagy. In addition, there are literature reports that in cells containing HCV-CORE / NS5B, HBx, ZIKV-NS5, and HIV-RT, Isarubrolone C can achieve antiviral effects by activating the autophagy degradation pathway rather than the proteasome pathway. Therefore, it is expected to be used as a drug precursor molecule or an active ingredient in a pharmaceutical composition for the treatment of immune diseases, type II diabetes, neurodegenerative diseases, etc.

[0005] Most natural products have problems such as poor water solubility and low bioavailability. Therefore, it is necessary to modify and transform the structure of natural products to make up for their own defects. Methods for modifying and transforming the structure of natural products include methylation, hydroxylation, sulfonylation, glycosylation, etc. Glycosylation modification is crucial for the formation of the structural and pharmacological activity diversity of natural products and is a common modification mechanism of compounds. According to literature reports, cancer cells can uptake higher levels of glucose than normal cells. Therefore, some anti-cancer compounds have better cancer cell targeting and good water solubility after being glycosylated.

[0006] Currently, it is known that both chemical methods and enzymatic methods can glycosylate natural products. For Isarubrolone C, it has multiple hydroxyl functional groups. Compared with chemical methods for glycosylating it, enzymatic methods can usually catalyze glycosylation reactions with high specificity and selectivity, the product purity is high, and there is no need to synthesize other intermediates. The reaction process is relatively simple, the conditions are mild, and it is environmentally friendly, having broad application prospects.

[0007] Glycosyltransferases (GTs) are one of the largest and most diverse superfamilies of enzymes in nature. They transfer activated glycosyl donors to a wide range of glycosyl acceptors and catalyze the formation of glycosidic bonds, and are key enzymes mediating the formation of regio- and stereospecific glycosidic bonds between glycosyl donors and various compounds. Glycosyltransferase BsYjic (Genbank No. NP_389104.1) belongs to the glycosyltransferase 1 family. Its overall structure is a classical GT-B fold configuration, consisting of two significantly different Rossmann domains connected by a flexible peptide chain. The glycosyl acceptor binding domain is located in the hydrophobic pocket of the N-terminal domain of the glycosyltransferase, while the glycosyl donor binding region is located in a deeper pocket in the C-terminal domain of the glycosyltransferase. Literature reports that BsYjic can catalyze the glycosylation of various types of natural compounds such as flavonoids, stilbenes, sterols, and terpenoids. There is currently no report that BsYjic can catalyze the glycosylation of Isarubrolone C. We found in our research that BsYjic can catalyze the glycosylation of Isarubrolone C, but its catalytic efficiency is low, and there are also isomers of the glucoside product. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the present invention provides a glycosyltransferase BsYjic mutant and its application in the synthesis of Isarubrolone C glucoside products. Through computer-aided rational design, the present invention performs molecular modification on BsYjic to improve the catalytic efficiency of BsYjic for the glycosylation of Isarubrolone C and its ability of specific glycosylation modification, obtaining a tool enzyme for efficiently and specifically preparing Isarubrolone C glucoside, which can change the water solubility of Isarubrolone C compounds and promote the development and application of Isarubrolone C drugs.

[0009] The technical solution of the present invention is as follows:

[0010] A glycosyltransferase BsYjic mutant, wherein the mutant is D106M or S128A. The D106M is obtained by mutating the 106th aspartic acid of the glycosyltransferase BsYjiC with the amino acid sequence shown in SEQ ID NO.1 to methionine, and the S128A is obtained by mutating the 128th serine of the glycosyltransferase BsYjiC with the amino acid sequence shown in SEQ ID NO.1 to alanine.

[0011] A coding gene encoding the above-mentioned glycosyltransferase BsYjic mutant.

[0012] A recombinant plasmid containing the above-mentioned coding gene.

[0013] Preferably according to the present invention, the coding gene is inserted into the pET22b vector to construct a recombinant plasmid.

[0014] A recombinant bacterium contains the above coding gene or the above recombinant plasmid.

[0015] Preferably according to the present invention, the host bacterium of the recombinant bacterium is Escherichia coli BL21(DE3) or Escherichia coli DH5α or Escherichia coli DMT.

[0016] Use of the above recombinant bacterium in the preparation of a glycosyltransferase BsYjic mutant.

[0017] Use of the above glycosyltransferase BsYjic mutant in the synthesis of Isarubrolone C glucoside products.

[0018] Preferably according to the present invention, when synthesizing the Isarubrolone C glucoside product, the glycosyl donor is UDP-Glc, the glycosyl acceptor is Isarubrolone C, and the Isarubrolone C glucoside products are 3’-O-β-D-Glc-isarubrolone C and 3-O-β-D-Glc-isarubrolone C.

[0019] In the present invention, the mutant D106M mainly efficiently synthesizes the glycosylated product 3’-O-β-D-Glc-isarubrolone C and produces a trace amount of the glycosylated product 3-O-β-D-Glc-isarubrolone C; the mutant S128A mainly efficiently synthesizes the glycosylated product 3-O-β-D-Glc-isarubrolone C and produces a trace amount of the glycosylated product 3’-O-β-D-Glc-isarubrolone C.

[0020] In a preferred technical solution of the present invention, a method for synthesizing an Isarubrolone C glucoside product by a glycosyltransferase BsYjic mutant includes the following steps:

[0021] Using Isarubrolone C as the reaction receptor substrate and UDP-Glc as the glycosyl donor, under the catalytic action of the mutant D106M and / or S128A, reacting at a temperature of 35-40°C and a pH of 6.5-8.0 for 24-48 hours to obtain the glucoside products 3’-O-β-D-Glc-isarubrolone C and 3-O-β-D-Glc-isarubrolone C.

[0022] Beneficial effects:

[0023] 1. By directionally modifying the glycosyltransferase BsYjic, the present invention obtained two mutants D106M and S128A, which can efficiently and specifically glycosylate Isarubrolone C, and synthesized two glucoside products 3’-O-β-D-glucopyranosyl isarubrolone C (3’-O-β-D-Glc-isarubrolone C) and 3-O-β-D-glucopyranosyl isarubrolone C (3-O-β-D-Glc-isarubrolone C) respectively. The water solubility of these two monoglucoside products was significantly improved, effectively solving the problem of poor water solubility of Isarubrolone C and laying a foundation for the development of new drugs.

[0024] 2. For the first time, the present invention screened two highly efficient and specific glycosyltransferase mutants D106M and S128A from the directed mutation of glycosyltransferase BsYjic. Mutant D106M can efficiently synthesize 3’-O-β-D-Glc-isarubrolone C using Isarubrolone C as a substrate, and its product conversion rate is 55.9 times higher than that of the wild-type BsYjic; mutant S128A can efficiently synthesize 3-O-β-D-Glc-isarubrolone C using Isarubrolone C as a substrate, and its product conversion rate is 3.6 times higher than that of the wild-type BsYjic. Mutants D106M and S128A successfully changed the product spectrum of glycosyltransferase BsYjic and improved its application flexibility in diversified production.

[0025] 3. The solubilities of the glucoside products 3’-O-β-D-Glc-isarubrolone C and 3-O-β-D-Glc-isarubrolone C at room temperature are 253.6 times and 205.6 times that of Isarubrolone C respectively, indicating that glycosylation modification effectively overcomes the disadvantage of poor water solubility of Isarubrolone C and lays a foundation for the druggability of Isarubrolone C. Brief Description of the Drawings

[0026] Figure 1 It is an SDS-PAGE diagram of the sample solution collected during the protein expression and purification process of glycosyltransferase BsYjic.

[0027] Figure 2 It is an SDS-PAGE diagram of 17 alanine-scanning site mutants.

[0028] Figure 3 It is an SDS-PAGE diagram of the saturation mutants at the D106 site.

[0029] Figure 4 SDS-PAGE diagram of the S128 site saturation mutant

[0030] Figure 5 Bar chart of the conversion rate of the products synthesized by the glycosylation reaction catalyzed by 17 alanine scanning site mutants

[0031] Figure 6 Bar chart of the conversion rate of the products synthesized by the glycosylation reaction catalyzed by the D106 site saturation mutant

[0032] Figure 7 Bar chart of the conversion rate of the products synthesized by the glycosylation reaction catalyzed by the S128 site saturation mutant

[0033] Figure 8 HPLC analysis diagram of the enzymatic reactions of glycosyltransferase BsYjic and its mutants

[0034] Figure 9 Mass spectrometry analysis diagram of the product 3'-O-β-D-Glc-isarubrolone C

[0035] Figure 10 Mass spectrometry analysis diagram of the product 3-O-β-D-Glc-isarubrolone C

[0036] Figure 11 1H NMR spectrum of the product 3'-O-β-D-Glc-isarubrolone C

[0037] Figure 12 13C NMR spectrum of the product 3'-O-β-D-Glc-isarubrolone C

[0038] Figure 13 COSY spectrum of the product 3'-O-β-D-Glc-isarubrolone C

[0039] Figure 14 HSQC spectrum of the product 3'-O-β-D-Glc-isarubrolone C

[0040] Figure 15 HMBC spectrum of the product 3'-O-β-D-Glc-isarubrolone C

[0041] Figure 16 1H NMR spectrum of the product 3-O-β-D-Glc-isarubrolone C

[0042] Figure 17It is the carbon-13 NMR spectrum of the product 3-O-β-D-Glc-isarubrolone C.

[0043] Figure 18 It is the COSY spectrum of the product 3-O-β-D-Glc-isarubrolone C.

[0044] Figure 19 It is the HSQC spectrum of the product 3-O-β-D-Glc-isarubrolone C.

[0045] Figure 20 It is the HMBC spectrum of the product 3-O-β-D-Glc-isarubrolone C.

[0046] Figure 21 It is the enzymatic property diagrams of glycosyltransferase BsYjic and its mutants D106M and S128A. Among them, a is the enzyme activity curve at different reaction temperatures; b is the enzyme activity curve after treatment at different incubation temperatures, c is the enzyme activity curve at different reaction pH values, d is the enzyme activity curve after treatment at different pH values; e is the bar graph of enzyme activity under treatment with different metal ions or compounds.

[0047] Figure 22 It is the kinetic curve diagrams of glycosyltransferase BsYjic and its mutants D106M and S128A.

[0048] Figure 23 It is the standard curve diagram of Isarubrolone C.

[0049] Figure 24 It is the standard curve diagram of 3’-O-β-D-Glc-isarubrolone C.

[0050] Figure 25 It is the standard curve diagram of 3-O-β-D-Glc-isarubrolone C. Detailed implementation manners

[0051] The technical solutions of the present invention will be further described below in conjunction with the embodiments, but the scope protected by the present invention is not limited thereto. The materials and reagents used in the following embodiments are all commercially available biological and chemical experimental materials unless otherwise specified.

[0052] In the following embodiments, the media and their formulations are as follows:

[0053] LB liquid medium: 5 g / L yeast extract, 10 g / L peptone, 7 g / L NaCl (adjust the pH to 7.0).

[0054] LB solid medium: Add 1.8% agar to the LB liquid medium.

[0055] In the following examples, the method for detecting enzyme activity is as follows:

[0056] In a reaction system of 50 μL of 25 mM Tris-HCl buffer (pH 8.0), it contains 2.5 mM UDP-Glc, 0.5 mM Isarubrolone C, and 30 μg of purified glycosyltransferase BsYjiC or its mutant. After reacting at 37 °C for 10 min, the reaction is terminated with 100 μL of pre-cooled methanol, centrifuged at 15000×g for 20 min, the supernatant of the reaction solution is taken, filtered through a filter membrane (0.22 μm), and the glycosylation product is quantitatively analyzed by HPLC.

[0057] Definition of enzyme activity unit: The amount of enzyme required to produce 1 μmol of Isarubrolone C glucoside product per minute is defined as one enzyme activity unit (U).

[0058] Example 1: Construction, heterologous expression and purification of recombinant expression plasmids of glycosyltransferase BsYjic and its mutants

[0059] (1) Construction of BsYjic recombinant expression plasmid

[0060] The required gene fragment was synthesized according to the amino acid sequence of glycosyltransferase BsYjic from Bacillus subtilis (GenBank accession number: NP_389104.1, SEQ ID NO.1), and the nucleotide sequence of the gene fragment is shown in SEQ ID NO.2. The above gene fragment was ligated into the pET22b vector by homologous recombination, and the ligation product was transformed into E. coli DH5α for sequencing screening to obtain the parental plasmid pET22b-BsYjic.

[0061] (2) Construction of mutants and their recombinant expression plasmids

[0062] Using the parental plasmid pET22b-BsYjic as a template, PCR amplification was carried out. The PCR amplification reaction system is shown in Table 1. Primers were designed according to the parental gene sequence (SEQ ID NO.2) (as shown in Table 2). Through PCR amplification technology, site-directed alanine scanning mutations were first carried out at the 11th, 16th, 80th, 106th, 107th, 108th, 110th, 128th, 129th, 132nd, 138th, 139th, 140th, 144th, 171st, 317th, and 318th positions of the wild-type glycosyltransferase BsYjic to screen for mutant sites with increased conversion rate of glycosylation products. Subsequently, saturation mutations were carried out at the 106th and 128th amino acid sites screened. The saturation mutation primers are shown in Tables 3-4 to screen for mutants with increased conversion rate of glycosylation products.

[0063] Table 1. PCR amplification reaction system

[0064]

[0065] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 20 s; annealing at Tm - 5°C for 20 s; extension at 72°C for 5 min; 32 cycles; and finally, compensatory extension at 72°C for 5 min.

[0066] Table 2. Site-directed alanine scanning mutation primers

[0067]

[0068] Table 3. Saturation mutation primers at the D106 site

[0069]

[0070]

[0071] Table 4. Saturation mutation primers at the S128 site

[0072]

[0073]

[0074] The PCR amplification products were digested with 1 μL of DpnI enzyme for 30 min to obtain mutant plasmids, which were then transformed into E. coli DMT competent cells. After screening on an LB solid medium plate containing Amp, single colonies were picked for sequencing to screen for mutant plasmids with correct amino acid site mutations. The parental plasmid of glycosyltransferase BsYjic and the mutant plasmids were transferred into Escherichia coli BL21(DE3) for the expression of related proteins.

[0075] (3) Heterologous Expression and Purification of Glycosyltransferase

[0076] Inoculate the overnight-cultured recombinant Escherichia coli BL21(DE3) bacterial solution into 300 mL of LB liquid medium at an inoculation amount of 1%, and culture it at 37 °C and 220 rpm for 1.5 h (OD 600 is 0.6 - 0.8), add the inducer IPTG to a final concentration of 0.1 mM, and culture it at 16 °C and 120 rpm for 18 h. Collect the bacterial cells, resuspend the cells with 20 mM disodium hydrogen phosphate - sodium dihydrogen phosphate (NaPB) buffer (pH 7.4), and disrupt the cells with an ultrasonic disruptor (the power is 45%, run for 3 s, interval 9 s, disrupt the cells for about 10 min, and the cell suspension becomes clear). After disruption, centrifuge at 4 °C, 13000×g for 20 min, and the finally obtained supernatant is the crude enzyme solution.

[0077] Equilibrate the nickel column with 20 mM NaPB buffer (pH 7.4) containing 20 mM imidazole, then load the crude enzyme solution, and wash it with 20 mM NaPB buffer (pH 7.4) containing 20 mM imidazole for 8 - 10 column volumes; elute with 20 mM NaPB buffer (pH 7.4) containing 40 mM imidazole, collect and combine 15 mL of the eluate with enzyme activity, transfer it to an ultrafiltration tube (10 kD), and centrifuge at 3700×g for 30 min. Repeat the above centrifugation conditions three times, and collect the retained enzyme solution, which is the pure enzyme solution. Finally, the wild-type glycosyltransferase BsYjic (WT) and its mutants I11A, H16A, L80A, D106A, F107A, V108A, L110A, S128A, S129A, Q132A, L138A, G139A, N140A, L144A, L171A, E317A, Q318A, S128C, S128D, S128E, S128F, S128G, S128H, S128I, S128K, S128L, S128M, S128N, S128P, S128Q, S128R, S128T, S128V, S128W, S128Y, D106C, D106E, D106F, D106G, D106H, D106I, D106K, D106L, D106M, D106N, D106P, D106Q, D106R, D106S, D106T, D106V, D106W, D106Y are obtained.

[0078] Measure the protein concentration of the pure enzyme solution with a Nanodrop 2000 Spectrophotometer (Thermo Scientific), dilute the pure enzyme solution with an equal volume of 40% glycerol, aliquot it into 1.5 mL EP tubes, and store it in an -80 °C refrigerator for subsequent experiments.

[0079] The above various pure enzyme solutions were analyzed by SDS-PAGE respectively, and the results are as Figure 1 , Figure 2 , Figure 3 and Figure 4 shown. The theoretical molecular weight of the wild-type glycosyltransferase BsYjic is 44 kDa. SDS-PAGE shows that the sizes of the recombinant glycosyltransferase BsYjic-His6-tag and its mutants are about 44 kDa, which is consistent with their theoretical molecular weights, indicating that the glycosyltransferase BsYjiC and its mutants were successfully heterologously expressed.

[0080] Example 2: HPLC and mass spectrometry analysis and detection of enzyme reaction system and products

[0081] In a 100 μL reaction system of 25 mM Tris-HCl buffer (pH 8.0), it contained 2.5 mM UDP-Glc, 0.5 mM Isarubrolone C, and 200 μg of purified glycosyltransferase BsYjiC or its mutants. After reacting at 37 °C and 200 rpm for 24 h, the reaction was terminated with 200 μL of pre-cooled methanol, centrifuged at 15000×g for 20 min, and the supernatant of the reaction solution was taken. After filtering through a filter membrane (0.22 μm), the glucosylated products were quantitatively analyzed by HPLC.

[0082] HPLC quantitative analysis: An Agilent 1260 high-performance liquid chromatograph (Agilent, US) was used, and an Agilent ZORBAX SB-Aq (4.6 mm × 250 mm, chromatographic column part number 880975-914, Agilent Technologies Co., Ltd.) chromatographic column was used for the analysis and detection of glucoside products. The HPLC gradient elution conditions were as follows: Phase A was 0.1% acetic acid, Phase B was acetonitrile, the mobile phase B increased from 20% to 50% within 12 min, the flow rate was 1.0 mL / min, and the ultraviolet detection wavelength was 280 nm.

[0083] The calculation formula for the conversion rate (yield, %) of Isarubrolone C glucoside:

[0084]

[0085] where S represents the peak area of the compound analyzed by HPLC quantitative analysis.

[0086] The conversion rate results are as Figure 5 , Figure 6 and Figure 7. In the above reaction system, the conversion rates of the wild-type glycosyltransferase BsYjic catalyzing the substrate Isarubrolone C to produce the glucoside product 3'-O-β-D-Glc-isarubrolone C and the glucoside product 3-O-β-D-Glc-isarubrolone C were 1.19% and 24.29% respectively, with relatively low conversion rates. The alanine scanning site-directed mutants could basically produce the above two glucoside products. Among them, the mutant D106A had the highest conversion rate of producing the glucoside product 3'-O-β-D-Glc-isarubrolone C, and the mutant S128A had the highest conversion rate of producing the glucoside product 3-O-β-D-Glc-isarubrolone C. Based on this, saturation mutagenesis was carried out on the amino acid sites at positions 106 and 128. The results showed that the conversion rate of the mutant D106M catalyzing the substrate Isarubrolone C to produce the glucoside product 3'-O-β-D-Glc-isarubrolone C was 66.58%, and the conversion rate of producing the glucoside product 3-O-β-D-Glc-isarubrolone C was 0.92%; the conversion rate of the mutant S128A catalyzing the substrate Isarubrolone C to produce the glucoside product 3'-O-β-D-Glc-isarubrolone C was 1.39%, and the conversion rate of producing the glucoside product 3-O-β-D-Glc-isarubrolone C was 86.03%. The HPLC analysis results are as shown in Figure 8 .

[0087] Among them, the above mutant D106M was obtained by mutating the aspartic acid at position 106 of the glycosyltransferase BsYjiC with the amino acid sequence shown in SEQ ID NO.1 to methionine, and mutating the codon GAC at positions 316 - 318 of the glycosyltransferase BsYjiC encoding gene with the nucleotide sequence shown in SEQ ID NO.2 to ATG; the above mutant S128A was obtained by mutating the serine at position 128 of the glycosyltransferase BsYjiC with the amino acid sequence shown in SEQ ID NO.1 to alanine, and mutating the codon TCG at positions 382 - 384 of the glycosyltransferase BsYjiC encoding gene with the nucleotide sequence shown in SEQ ID NO.2 to GCA.

[0088] Before mass spectrometry detection of the glucoside product, a small amount of the glucoside product was separated by HPLC, the product sample was dissolved in methanol, and after filtration through a membrane (0.22 μm), it was detected using a cation mode on the test instrument Shimadzu LCMS-IT-TOF. The results are as shown in Figure 9 and Figure 10As shown, the product showed an ion peak with a mass-to-charge ratio of 634.2156 under the ESI-MS detection in the positive ion mode, which corresponded to the theoretical molecular weight of Isarubrolone C monoglucoside, further confirming that the product in the reaction solution was Isarubrolone C monoglucoside. + The ion peak corresponded to the theoretical molecular weight of Isarubrolone C monoglucoside, further confirming that the product in the reaction solution was Isarubrolone C monoglucoside.

[0089] Example 3: Preparation and Structural Analysis of Isarubrolone C Glucoside

[0090] The reaction system was enlarged according to the enzyme activity detection method. 40 mL of the reaction solutions of mutants D106M and S128A were inactivated with twice the volume of cold methanol, centrifuged at 15000×g for 20 min, and the supernatant was freeze-dried, dissolved in 10 mL of methanol, filtered through a membrane (0.22 μm), and then the glucoside pure product was prepared by HPLC. The glucoside pure product was separated and prepared using a 5 μm Supersil AQ-C18 liquid chromatography column (10 mm×250 mm, column part number 31113128, Dalian Elite Analytical Instruments Co., Ltd.). The HPLC gradient elution conditions were as follows: Phase A was pure water (0.05% trifluoroacetic acid), Phase B was acetonitrile (0.05% trifluoroacetic acid), the proportion of mobile phase B increased from 20% to 50% within 25 min, the flow rate was 1.4 mL / min, and the UV detection wavelength was 280 nm. The glucoside product peak was collected and the pure glucoside product was obtained after freeze-drying.

[0091] NMR analysis of the glycosylation product structure: 7 mg of the freeze-dried glucoside product pure product was weighed and dissolved in a 5 mm standard NMR tube containing 500 μL of Methanol-d4. The 1H (600 MHz), 13C (150 MHz), COSY, HMBC, and HSQC NMR data of the sample were collected at 25 °C using a 600 MHz nuclear magnetic resonance spectrometer from Bruker Corporation, and the data was processed by MestReNova software. Combining the one-dimensional and two-dimensional NMR detection results ( 1 1H (600 MHz), 13 13C (150 MHz), COSY, HMBC, and HSQC NMR data, through MestReNova software for data processing. Combining the one-dimensional and two-dimensional NMR detection results ( Figure 11 、 Figure 12 、 Figure 13 、 Figure 14 、 Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 、 Figure 19 、 Figure 20 ), it can be seen that the structures of the glucoside products are all of the β type, and glucose is connected to the C skeletons C3’ and C3 of Isarubrolone C respectively. Therefore, it can be determined that the structures of Isarubrolone C and its glucoside products are as follows:

[0092]

[0093] Example 4: Biochemical Property Studies of Glycosyltransferase BsYjic, Mutants D106M and S128A

[0094] (1) Determination of the Optimal Reaction Temperature:

[0095] In a 50 μL reaction system of 25 mM Tris-HCl buffer (pH 8.0), containing 2.5 mM UDP-Glc, 0.5 mM Isarubrolone C, and 30 μg of glycosyltransferase (BsYjiC, mutant D106M, or mutant S128A), the prepared reaction solutions were respectively detected at temperatures of 25, 30, 35, 37, 40, 45, 50, 55, and 60 °C according to the enzyme activity detection method. Taking the maximum enzyme activity in each group of reactions as 100% relative activity, and the relative activity under other conditions relative to the maximum enzyme activity as the ordinate, a reaction temperature curve was plotted. The results are as shown in Figure 21 Figure a in it. The optimal enzymatic reaction temperature of glycosyltransferase BsYjic is 45 °C; the optimal enzymatic reaction temperature of mutant D106M is 35 °C; the optimal enzymatic reaction temperature of mutant S128A is 40 °C.

[0096] (2) Determination of Temperature Stability:

[0097] After the enzymes were incubated at 4, 10, 20, 30, 40, 50, and 60 °C for 2 h respectively, in a 50 μL reaction system of 25 mM Tris-HCl buffer (pH 8.0), containing 2.5 mM UDP-Glc, 0.5 mM Isarubrolone C, and 30 μg of the incubated glycosyltransferase (BsYjiC, mutant D106M, or mutant S128A), the enzyme activity was measured by the enzyme activity detection method. Taking the maximum enzyme activity in each group of reactions as 100% relative activity, and the relative activity under other conditions relative to the maximum enzyme activity as the ordinate, a temperature stability curve was plotted. The results are as shown in Figure 21 Figure b in it. Glycosyltransferase BsYjic and mutant S128A are relatively stable at temperatures below 40 °C, and when the temperature is higher than 40 °C, the relative enzyme activity decreases significantly; mutant D106M is relatively stable at temperatures below 30 °C, and when the temperature is higher than 30 °C, the relative enzyme activity decreases significantly.

[0098] (3) Determination of the Optimal pH:

[0099] In reaction systems of different pH buffer solutions with a volume of 50 μL, there are 2.5 mM UDP-Glc, 0.5 mM Isarubrolone C, and 30 μg of glycosyltransferase (BsYjiC, mutant D106M, or mutant S128A). Among them, the citrate-sodium citrate buffer system is used for the pH 3.5 - 6.5 buffer solution, the dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer system is used for the pH 6.5 - 8.0 buffer solution, and the Tris-HCl buffer system is used for the pH 8.0 - 9.0 buffer solution. According to the enzyme activity detection method, with the maximum enzyme activity in each group of reactions as 100% relative activity, and the relative activity under other conditions relative to the maximum enzyme activity as the ordinate, a reaction pH curve is plotted to measure the optimal reaction pH of the enzyme. The results are as Figure 21 shown in Figure c of [reference]. The optimal enzymatic reaction pH of glycosyltransferase BsYjic is 8.0 (dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer); the optimal enzymatic reaction pH of mutant D106M is 6.5 (citrate-sodium citrate buffer); the optimal enzymatic reaction pH of mutant S128A is 8.0 (Tris-HCl buffer).

[0100] (4) pH stability determination:

[0101] The enzyme is stored at 4 °C for 2 h in buffer solutions with a pH range of 4.0 - 9.0 (at intervals of 0.5). In a 50-μL reaction system of 25 mM Tris-HCl buffer (pH 8.0), there are 2.5 mM UDP-Glc, 0.5 mM Isarubrolone C, and 30 μg of glycosyltransferase (BsYjiC, mutant D106M, or mutant S128A) treated with different pH buffer solutions. The enzyme activity is measured using the enzyme activity detection method. With the maximum enzyme activity in each group of reactions as 100% relative activity, and the relative activity under other conditions relative to the maximum enzyme activity as the ordinate, a pH stability curve is plotted. The results are as Figure 21 shown in Figure d of [reference]. Glycosyltransferase BsYjic is stable between pH 5.5 and 8.0, and the relative enzyme activity reaches over 70%; mutant D106M is stable between pH 6.5 and 7.5, and the relative enzyme activity reaches over 70%; mutant S128A is stable between pH 5.5 and 8.5, and the relative enzyme activity reaches over 70%.

[0102] (5) Effect of metal ions on enzyme activity:

[0103] In a 50 μL reaction system of 25 mM Tris-HCl buffer (pH 8.0), it contains 2.5 mM UDP-Glc, 0.5 mM Isarubrolone C, 30 μg of glycosyltransferase (BsYjiC, mutant D106M or mutant S128A), and KCl, NaCl, AgNO3, CaCl2, ZnCl2, CuCl2, CoCl2, MnCl2, NiSO4, MgCl2, MgSO4, FeSO4, FeCl3 or EDTA with a final concentration of 2 mM. In the control group, no compounds and metal ions are added. The enzyme activity is measured by the enzyme activity detection method. Taking the maximum enzyme activity in each group of reactions as 100% relative activity, the relative activity relative to the maximum enzyme activity under other conditions is used as the ordinate to draw a relative enzyme activity bar chart.

[0104] The results are as Figure 21 shown in Figure e in. Metal ions affect the enzyme activities of glycosyltransferase BsYjic, mutant D106M and S128A. Among them, MgCl2 has a significant activating effect on them, increasing the catalytic activities of BsYjic, S128A and D106M by 108%, 59% and 184% respectively; AgNO3, ZnCl2 and GuCl2 can strongly inhibit the catalytic activities of BsYjic, D106M and S128A, reducing the enzyme activity to less than 30% of the control group. Interestingly, NiSO4 has a strong inhibitory effect on the enzyme activities of BsYjic and S128A, but has a certain promoting effect on the enzyme activity of D106M.

[0105] (6) Determination of the donor-acceptor kinetic parameters of glycosyltransferase BsYjic, mutant D106M and S128A:

[0106] It is measured by the enzyme activity detection method. Among them, when determining the kinetic parameters of glycosyltransferase for Isarubrolone C, first fix the final concentration of the glycosyl donor UDP-Glc in the 50 μL reaction solution at 2.5 mM, and then measure the initial velocity of the enzyme reaction corresponding to the concentration range of 0 - 1.5 mM Isarubrolone C. When determining the kinetic parameters of glycosyltransferase for UDP-Glc, first fix the concentration of the glycosyl acceptor Isarubrolone C in the 50 μL reaction solution at 0.5 mM, and then measure the initial velocity of the enzyme reaction corresponding to the concentration range of 0 - 5.0 mM UDP-Glc. Vmax (the maximum reaction rate of the enzyme-catalyzed reaction) and Km (the substrate concentration when reaching half of the maximum reaction rate) are calculated using GraphPad Prism 10. The enzyme kinetic curve is as Figure 22 , and the kinetic parameters are shown in Table 5.

[0107] Table 5. Enzyme reaction kinetic parameters

[0108]

[0109] Example 5: Water solubility detection of Isarubrolone C and its glucoside products

[0110] Excess amounts of Isarubrolone C, 3’-O-β-D-Glc-isarubrolone C, and 3-O-β-D-Glc-isarubrolone C were separately placed in EP tubes containing 50 μL of ddH2O and sonicated at room temperature for 5 min until the solid no longer decreased, to obtain saturated solutions of Isarubrolone C, 3’-O-β-D-Glc-isarubrolone C, and 3-O-β-D-Glc-isarubrolone C. Standard curves were plotted using the HPLC peak areas of the above three samples at 0 - 0.4 mg / mL. The results are as Figure 23 、 Figure 24 and Figure 25 shown. The concentration of the saturated solution was diluted to within the linear range of the standard curve using the serial dilution method, substituted into the standard equation, and the concentration of the diluted solution and the saturated solution were calculated.

[0111] The solubilities of Isarubrolone C, 3’-O-β-D-Glc-isarubrolone C, and 3-O-β-D-Glc-isarubrolone C were measured under the conditions of room temperature and sonication-assisted dissolution. The results showed that the solubility of Isarubrolone C was 0.45 mg / mL, the solubility of 3’-O-β-D-Glc-isarubrolone C was 114.10 mg / mL, which was 253.6 times that of Isarubrolone C, and the solubility of 3-O-β-D-Glc-isarubrolone C was 92.51 mg / mL, which was 205.6 times that of Isarubrolone C.

Claims

1. Glycosyltransferase BsYjic mutant, characterized in that, The mutant is D106M or S128A. The D106M is obtained by mutating the 106th aspartic acid of the glycosyltransferase BsYjiC with an amino acid sequence as shown in SEQ ID NO.1 into methionine, and the S128A is obtained by mutating the 128th serine of the glycosyltransferase BsYjiC with an amino acid sequence as shown in SEQ ID NO.1 into alanine.

2. A coding gene, characterized in that, Encoding the glycosyltransferase BsYjic mutant described in claim 1.

3. A recombinant plasmid, characterized in that, Containing the encoding gene described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The encoding gene is inserted into the pET22b vector to construct a recombinant plasmid.

5. A recombinant bacterium, characterized in that, Containing the encoding gene described in claim 2 or the recombinant plasmid described in claim 3.

6. The recombinant bacterium according to claim 5, characterized in that, The host bacterium of the recombinant bacterium is Escherichia coli BL21(DE3) or Escherichia coli DH5α or Escherichia coli DMT.

7. Use of the recombinant bacterium described in claim 5 in the preparation of the glycosyltransferase BsYjic mutant.

8. Use of the glycosyltransferase BsYjic mutant described in claim 1 in the synthesis of Isarubrolone C glucoside product.

9. The application according to claim 8, wherein The glycosyl donor in the synthesis of the Isarubrolone C glucoside product is UDP-Glc, the glycosyl acceptor is Isarubrolone C, and the Isarubrolone C glucoside product is 3’-O-β-D-Glc-isarubrolone C and 3-O-β-D-Glc-isarubrolone C.

10. A method for synthesizing Isarubrolone C glucoside product by using the glycosyltransferase BsYjic mutant described in claim 1, characterized in that, Including the following steps: Using Isarubrolone C as the reaction acceptor substrate, UDP-Glc as the glycosyl donor, and under the catalytic action of the mutant D106M and / or S128A, reacting at a temperature of 35-40 °C and a pH of 6.5-8.0 for 24-48 hours to obtain the glucoside products 3’-O-β-D-Glc-isarubrolone C and 3-O-β-D-Glc-isarubrolone C.

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