A glycosyltransferase and its application in preparing anoectochilus glycoside

Abstract

The present invention belongs to the technical field of enzyme engineering, and relates to a glycosyltransferase and its application in the preparation of aucubin. The glycosyltransferase is derived from a soil metagenome, and its amino acid sequence is shown in SEQ ID NO.1. By heterologously expressing the enzyme in Escherichia coli, a recombinant glycosyltransferase was successfully obtained. Using this enzyme, (R)-3-hydroxy-γ-butyrolactone can be efficiently glycosylated to produce aucubin, with a substrate conversion rate exceeding 99%, and the highest space-time yield can reach 12.6 g·L-1·d-1. Compared with the prior art, the present invention has the remarkable advantages of high conversion rate, strong selectivity, mild reaction conditions and few by-products, showing good industrial application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and more specifically, relates to a glycosyltransferase and its application in the preparation of anoectochilus glycoside. Background Art

[0002] Herba Anoectochili is a rare perennial Chinese herbal medicine belonging to the genus Anoectochili of the Orchidaceae family. It is widely distributed in southern my country. Due to its unique medicinal value, Herba Anoectochili is known as "golden grass" and "black ginseng" among the people. Its main active ingredient is kinsenoside, with the chemical name 4-R-β-D-pyranose glucopyranose-butyrolactone (Formula I). ​​It has multiple pharmacological activities such as anti-inflammatory, hypoglycemic, and liver protection, and therefore has broad development prospects in the field of medicine.

[0003]

[0004] Kinsenoside is a natural compound with multiple pharmacological activities, which is widely used in anti-inflammatory, hypoglycemic, liver protection and other therapeutic fields. At present, the main source of kinsenoside is extracted from kinsenoside plants. However, due to the scarcity of kinsenoside resources and the long planting cycle, this extraction method faces a large raw material limitation, resulting in high prices for medicinal materials and difficulty in meeting market demand. In addition, the plant-based extraction method also has the problem of uncertainty in the source, which easily introduces chiral isomer impurities, thereby affecting the purity and efficacy of the product. These factors significantly limit the large-scale application of kinsenoside, and it is urgent to find new production methods to replace traditional plant extraction methods. In order to solve the problems of shortage of raw material sources and high production costs, researchers have tried to prepare kinsenoside through chemical synthesis. However, the chemical synthesis process generally has challenges such as complex reaction routes, numerous steps, frequent protection and deprotection operations, and difficulty in avoiding stereoisomer generation, resulting in high production costs and technical difficulties. Therefore, it is particularly important to find a simpler and more efficient synthesis method. Enzymatic synthesis is expected to overcome the defects of chemical synthesis due to its mild reaction conditions, strong specificity and environmental friendliness. No complicated protective group operations are required during enzymatic synthesis, which can effectively avoid the formation of by-products. There are currently two types of enzymes that can be used for the synthesis of glycosides, namely glycoside hydrolases and glycosyltransferases. Among them, glycoside hydrolases synthesize target glycosides through reverse hydrolysis reactions. Although they are widely available and easy to obtain, their catalytic efficiency is limited and the reaction balance needs to be broken to promote the reaction. Zhang et al. used Sigma's commercial β-D-glucosidase to catalyze the pre-chemically synthesized (R)-3-hydroxy-γ-butyrolactone to synthesize anoectochilus glycoside, but the conversion rate of the enzyme catalysis step was only 22.4%, and the product concentration was only 0.13 g / L (Zhang, et al. Efficient Synthesis of Kinsenoside and Goodyeroside A by a Chemo-Enzymatic Approach. Molecules, 2014, 19: 16950-16958; doi: 10.3390 / molecules191016950). Compared with glycoside hydrolases, glycosyltransferases (GT; EC 2.4.xy) can generate glycoside products by catalyzing the transfer of activated sugar groups on sugar donors to acceptor molecules. However, there is currently a lack of glycosyltransferases that can catalyze the glycosylation reaction of (R)-3-hydroxy-γ-butyrolactone (not reported in the prior art), which greatly limits the development and optimization of the biosynthesis process of anoectochiloside.

[0005] It is not easy to find a glycosyltransferase with strong catalytic activity for the substrate (R)-3-hydroxy-γ-butyrolactone. The applicant previously screened glycosyltransferases from various plants and microorganisms in the laboratory preserved enzyme library, and only found that the glycosyltransferase BlYjiC from the microorganism Bacillus licheniformi could successfully catalyze the glycosylation reaction of (R)-3-hydroxy-γ-butyrolactone, but the activity was low. After 10 hours of reaction, the yield of anoectochilus glycoside was only 1.0g / L, which did not reach the level of industrial application. Therefore, exploring and developing new glycosyltransferases that can efficiently catalyze such reactions has become a key step in promoting the biosynthesis of anoectochilus glycosides.

[0006] Traditional enzyme screening methods mainly rely on culturable microorganisms, which limits the discovery of new enzymes. As a rich gene resource library, soil metagenome contains a large amount of uncultivated microbial gene information, which provides the possibility for the mining of new enzymes. Summary of the invention

[0007] The purpose of the present invention is to provide a glycosyltransferase and application thereof in the preparation of anoectochilus glycoside.

[0008] The present invention successfully screened out a new type of glycosyltransferase by deeply mining the soil metagenome. The enzyme can efficiently catalyze the glycosylation reaction of (R)-3-hydroxy-γ-butyrolactone and UDP-glucose to generate anoectin, with a substrate conversion rate exceeding 99% and a space-time yield of up to 12.6 g·L-1·d-1. The method has the advantages of high conversion rate, high selectivity, mild reaction conditions, and few by-products, overcomes the shortcomings of the prior art, and provides a new and feasible approach for the industrial production of anoectin.

[0009] The first aspect of the present invention is to provide a glycosyltransferase, the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO.1.

[0010] The second aspect of the present invention discloses a catalyst for catalyzing the synthesis of anoectochiloside from (R)-3-hydroxy-γ-butyrolactone, wherein the catalyst comprises or expresses a glycosyltransferase having an amino acid sequence as shown in SEQ ID NO.1.

[0011] The third aspect of the present invention is to provide a nucleic acid molecule encoding the aforementioned glycosyltransferase, a host cell expressing the aforementioned glycosyltransferase, or a catalyst comprising the aforementioned glycosyltransferase.

[0012] The fourth aspect of the present invention discloses the use of a glycosyltransferase in the preparation of anoectochiloside, wherein the aforementioned glycosyltransferase is used to generate anoectochiloside in the glycosylation reaction of (R)-3-hydroxy-γ-butyrolactone.

[0013] The fifth aspect of the present invention is to provide a method for preparing anoectochilus glycoside, the method comprising the following steps:

[0014] (1) preparing an enzyme solution of the aforementioned glycosyltransferase;

[0015] (2) adding the prepared enzyme solution to a mixed reaction system containing (R)-3-hydroxy-γ-butyrolactone and a UDP-glucose regeneration system to carry out a glycosylation reaction to obtain anoectochiloside.

[0016] In a preferred embodiment, the enzyme solution is a crude enzyme solution obtained by cell lysis or a purified enzyme solution of a genetically engineered bacterium capable of expressing the aforementioned glycosyltransferase.

[0017] Furthermore, the UDP-glucose regeneration system is a UDP-glucose regeneration system which uses sucrose synthase as the UDP-glucose regeneration enzyme, uses sucrose as a cosubstrate, and contains UDP and UDP-glucose.

[0018] In a preferred embodiment, the concentration of the substrate (R)-3-hydroxy-γ-butyrolactone in the glycosylation reaction is 10 mM to 50 mM.

[0019] Furthermore, the concentration of the crude glycosyltransferase solution in the mixed reaction system is 0.5 to 10 mg / mL (preferably 1 to 5 mg / mL).

[0020] In a preferred embodiment, the temperature of the glycosylation reaction is 15-50°C (preferably 20-45°C, more preferably 25-35°C), and the pH value of the mixed reaction system is 6-10 (preferably 7-8).

[0021] The present invention predicts gene functions based on metagenomic sequencing data of soil in the original forest of Dabie Mountains, and uses the existing conservative sequence of glycosyltransferase PSPG as a probe, thereby obtaining a new glycosyltransferase with an amino acid sequence identity of 45% with the probe from the soil metagenome, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence encoding the glycosyltransferase gene is shown in SEQ ID NO.2.

[0022] In the presence of UDP-glucose provided by the UDP-glucose regeneration system, the substrate (R)-3-hydroxy-γ-butyrolactone undergoes a glycosylation reaction under the catalysis of glycosyltransferase to produce anoectochilus glycoside. Specifically, the reaction temperature is 15 to 50° C., the pH value of the mixed reaction system is 6 to 10, and the UDP-glucose concentration is 10 to 50 mM.

[0023] Specifically, the UDP-glucose regeneration system is a system in which sucrose is catalyzed by sucrose synthase to generate fructose and UDP-glucose in the presence of UDP, and the generated UDP-glucose is again involved in the synthesis of anoectochilus glycosides. The sucrose concentration is 0 to 500 mM.

[0024] The invention has the following beneficial effects: the glycosyltransferase used in the invention is derived from soil microorganisms, and compared with the glycosyltransferase derived from plants, the glycosyltransferase of the invention is easier to be efficiently and soluble expressed in model strains such as Escherichia coli; the glycosyltransferase obtained in the invention has high catalytic activity for (R)-3-hydroxy-γ-butyrolactone, and the substrate conversion rate is higher than 99%; the glycosyltransferase obtained in the invention has high space-time yield in the process of synthesizing aurocin, and the space-time yield can reach up to 12.6 g·L -1 ·d -1 The invention successfully solves the key problems of low selectivity, low activity, long steps and heavy pollution in the preparation process of roxburghii glycoside, and shows great industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the PCR result of glycosyltransferase gene in soil metagenome, where M is Marker; 1 is glycosyltransferase gene;

[0026] Figure 2 This is the SDS-PAGE analysis of the overexpression effect of glycosyltransferase in Escherichia coli, where M is a marker; 1 is before induction; 2 is the supernatant after induction; 3 is the precipitate after induction;

[0027] Figure 3 It is the HPLC chromatogram of (R)-3-hydroxy-γ-butyrolactone catalyzed by glycosyltransferase;

[0028] Figure 4 Schematic diagram of the preparation of anoectochiloside by glycosyltransferase in the UDP-glucose regeneration system;

[0029] Figure 5 The time curve of glycosyltransferase MmUGT and BlYjiC catalyzing (R)-3-hydroxy-γ-butyrolactone;

[0030] Figure 6 This is the mass spectrum of anoectochiloside, with a [M+Na] peak size of 287;

[0031] Figure 7 This is the 1H NMR spectrum of anoectochiloside;

[0032] Figure 8 This is the 13C nuclear magnetic resonance spectrum of roxburghii glycoside. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] The experimental methods described in the present invention are all conventional methods unless otherwise specified. For details of gene cloning operations, please refer to "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.

[0036] The DNA polymerase (2×Phanta Max Master Mix), Dpn I enzyme, recombination cloning kit and plasmid extraction kit used in the examples of the present invention were purchased from Takara Biotechnology Co., Ltd.; the synthesis of genes and primers and gene sequencing were completed by General Biotechnology (Anhui) Co., Ltd., and the use of the above reagents refers to the product instructions.

[0037] The expression vector involved in the present invention is pET-28a(+), and the host used is Escherichia coli BL21(DE3), all of which are preserved in the laboratory of the inventor.

[0038] All standard products used in the present invention were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; other commonly used reagents were purchased from Anage Chemical Reagent Co., Ltd.

[0039] Example 1: Mining of glycosyltransferase genes and construction of recombinant engineering bacteria

[0040] Soil from Mazongling, Jinzhai County, Dabie Mountains was collected and metagenomic sequencing was performed. The online tool MetaGeneMark (http: / / exon.gatech.edu / meta_gmhmmp.cgi) was used to predict gene functions from metagenomic sequencing data of soil from Mazongling, Jinzhai County, Dabie Mountains. The PSPG conservative sequence (RVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQ, SEQ ID NO.8) of glycosyltransferase BlYjiC (NCBI accession NO.: WP_003182014.1) from Bacillus licheniformi was used as a probe. The local BLAST tool was used to search the metagenomic sequencing data of soil from Mazongling, Jinzhai County, Dabie Mountains, and a glycosyltransferase with an amino acid sequence identity of 45% with the probe was obtained. The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO.1, and the gene sequence is shown in SEQ ID NO.2. The amino acid sequence of the glycosyltransferase is consistent with the amino acid sequence of Methylococcus included in the NCBI database. mesophilus had the highest sequence identity, but it was only 48.88%. A group of enzymes similar to this glycosyltransferase in the NCBI database all belonged to microbial sources. Therefore, it can be identified as a new enzyme from a microbial source and named MmUGT.

[0041] The DNA of the soil metagenomics of Mazongling in Jinzhai County, Dabie Mountains was used as a template, MmUGT-F (SEQ ID NO.4) and MmUGT-R (SEQ ID NO.5) were used as primers, and the primer sequences were shown in Table 1 to amplify the glycosyltransferase gene. The results were as follows Figure 1 As shown; At the same time, pET-28a (+) plasmid was used as a template, and pET28a-F (SEQ ID NO.6) and pET28a-R (SEQ ID NO.7) were used as primers to amplify and obtain a linearized vector, and the primer sequence results are shown in Table 1. The amplified glycosyltransferase gene was connected to the pET-28a (+) plasmid by a one-step cloning kit (ClonExpressIIOne Step Cloning Kit) to construct a recombinant plasmid pET28a-UGT. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells, and positive clones were screened and detected to obtain a recombinant engineered bacterium containing a glycosyltransferase gene.

[0042] Table 1 Primer sequences

[0043]

[0044]

[0045] SEQ ID NO.1

[0046] MSKIILFNIPATGHVNPSLPIVTELVKRGEEILYVNSEEYRQKVEATGASFMAYPDLGDFQR

[0047] LMEEDAAEGNLPRNMRDLLRLTAQITPFAKDLIKKEKPDLIIFDTLARWGYLAAKQSNLPH

[0048] ISFCSTFVVNPTDMPPLPASALLDMVAKMMRVIPSYVSDSWKMFRQQGIFPGFLLDAVMS

[0049] TGNITLVFTSREFQPNSEKFDESYKFIGTSLAPRPTDSDFPLSELNQAPITYISLGTLAQNPDF

[0050] LKKCYQAFADFEGQFVLSAGKKTHIADLGTIPENFIVRNFVSQLEILERASAFITHGGLNSI

[0051] HEGLVYGVPMIAVPHQIEQAIVALQLQTQGAGVALKTSPPYGDVSIVELQNSLREILSNPTY

[0052] AQNAKRLGESLKNAGGYQRAADEIQRFIGKI

[0053] SEQ ID NO.2

[0054] ATGTCTAAGATAATCCTATTCAATATTCCAGCTACAGGGCATGTAAATCCATCCTTGCCG

[0055] ATTGTCACTGAATTGGTGAAGCGTGGCGAGGAAATCCTCTATGTGAATAGTGAAGAATA

[0056] TCGTCAGAAGGTTGAAGCCACTGGCGCAAGTTTTATGGCTTACCCTGATTTGGGCGAC

[0057] TTCCAACGCCTAATGGAAGAGGATGCGGCAGAAGGCAATTTGCCCCGAAATATGCGTG

[0058] ACCTTTTGCGATTAACTGCTCAAATAACACCATTTGCGAAAGACCTCATTAAAAAAGAA

[0059] AAACCTGATCTCATTATTTTTGACACATTAGCACGCTGGGGATACCTTGCCGCCAAACA

[0060] GAGCAACTTACCACATATTTCATTCTGCTCTACCTTTGTTGTGAATCCCACTGATATGCC

[0061] TCCTTTACCTGCCAGTGCTTTGCTGGATATGGTCGCAAAAATGATGCGGGTCATACCTT

[0062] CGTATGTGTCAGATTCATGGAAAATGTTTCGTCAGCAGGGTATCTTTCCCGGATTTTTAT

[0063] TGGATGCTGTCATGTCTACGGGGAATATTACACTTGTTTTCACATCGCGTGAGTTCCAG

[0064] CCCAACAGTGAAAAATTTGATGAGAGCTACAAATTTATCGGCACATCTCTCGCCCCACG

[0065] TCCAACAGACAGCGACTTTCCCTTATCTGAATTAAATCAAGCACCAATAACCTACATTT

[0066] CATTGGGGACACTCGCTCAAAATCCCGACTTCCTGAAAAAATGTTACCAAGCCTTTGC

[0067] CGACTTTGAGGGGCAATTTGTTTTATCCGCAGGGAAAAAGACTCATATAGCGGATTTAG

[0068] GTACTATCCCTGAAAACTTCATCGTCAGGAACTTTGTGTCTCAGCTTGAGATTTTAGAA

[0069] CGCGCCTCGGCTTTTATTACACATGGTGGATTGAATAGCATTCATGAAGGTTTGGTCTAT

[0070] GGTGTACCGATGATTGCCGTACCGCATCAAATAGAACAAGCGATTGTTGCCTTACAATT

[0071] GCAAACACAAGGTGCAGGGGTTGCCCTCAAAACCAGCCCACCTTATGGGGATGTTTCT

[0072] ATAGTAGAGCTACAAAATAGTCTGCGTGAAATTTTGTCGAATCCCACTTACGCGCAGAA

[0073] TGCCAAGCGATTAGGGGAATCACTCAAGAACGCAGGGGGGTATCAGCGTGCTGCCGAT

[0074] GAGATTCAGAGATTTATAGGCAAGATCTAA

[0075] Example 2 Inducible expression and solubility analysis of recombinant glycosyltransferase MmUGT

[0076] The recombinant engineering bacteria constructed above were streaked and activated on LB solid medium containing 50 μg / mL Kan; a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL Kan, and cultured at 37°C, 200 rpm, and shaken overnight; 1% of the inoculum was transferred to 250 mL of fresh LB liquid medium also containing 50 μg / mL Kan, and cultured at 37°C, 200 rpm, until OD 600 When the pH value reached 0.8, IPTG with a final concentration of 0.1 mM was added, and the culture was induced at 18°C ​​and 200 rpm for 20 hours. After the culture was completed, the cells were collected by centrifugation at 10,000 rpm and 4°C, washed twice with ultrapure water and suspended in 20 mL of 50 mM phosphate buffer (pH 7.5). The cells were disrupted by ultrasonic disruptor, and the disrupted liquid was centrifuged at 12,000 rpm for 20 minutes. The supernatant was the crude enzyme solution. The supernatant and precipitate were analyzed by SDS-PAGE, and the results were as follows: Figure 2 As shown, it can be seen that the target protein content in the supernatant is equivalent to the total target protein content, while there is almost no target protein in the precipitate, proving that glycosyltransferase can be efficiently and soluble expressed in Escherichia coli.

[0077] The crude enzyme solution hereinafter refers to the above-mentioned cell lysis supernatant.

[0078] Example 3 Analysis of glycosylation activity of glycosyltransferase MmUGT

[0079] The crude enzyme of glycosyltransferase was prepared according to the expression and culture method described in Example 2, and the catalytic activity of the crude enzyme on the (R)-3-hydroxy-γ-butyrolactone substrate was determined. The activity detection system is (1 mL): crude enzyme solution (50 μg), 2 mM (R)-3-hydroxy-γ-butyrolactone, 5 mM UDP-glucose, the reaction medium is 50 mM potassium phosphate buffer (pH 7.5), the reaction temperature is 30°C, the reaction is triggered by adding the enzyme solution, the reaction is carried out for 30 minutes, and the reaction results are determined by high performance liquid chromatography. The liquid chromatograph used is Shimadzu LC-2060 equipped with an ELSD detector, and the chromatographic column model used is: Ultimate AQ-C18 analytical column, 5 μm, 4.6 mm×250 mm; the mobile phase is 100% ultrapure water; the flow rate is 0.6 mL / min; the drift tube temperature of the evaporative light scattering detector is 115°C; the N2 flow rate is 3 L / min, and 20 μL is injected each time. The results are as follows Figure 3 As shown, the substrate peaks at 9.5 min and the product peaks at 7.5 min.

[0080] Example 4 Preparation of crude enzyme solution of recombinant sucrose synthase

[0081] The sucrose synthase derived from Micractinium conductrix was selected for constructing a UDP-glucose regeneration system. The sucrose synthase used was its highly active mutant S31D (Chen et al. Identification of sucrose synthase from Micractinium conductrix to favor biocatalytic glycosylation. Front. Microbiol. 2023, 14: 1220208. doi: 10.3389 / fmicb. 2023.1220208), and the mutant amino acid sequence is shown in SEQ ID NO. 3. According to the method of Example 1, the sucrose synthase mutant S31D gene was cloned into the pET-28a (+) plasmid. Subsequently, the plasmid was transformed into E. coli BL21 (DE3) competent cells, and after screening and detection of positive clones, a recombinant engineered bacterium containing a sucrose synthase mutant gene was obtained. According to the method of Example 2, the obtained recombinant engineered bacteria were expressed and cultured, and a crude enzyme solution of recombinant sucrose synthase was prepared.

[0082] SEQ ID NO.3

[0083] MSAGADSPSSQPFLASPRGVITPRTFTRSLDFAGGTPSEILKAGLVHSRNELVLLFSRCMAK

[0084] SKADKPILLPHIIMDELCAVCDECNNPMLKSGEIAAILKTVQEAVVIAPRIAFALRPTMGEW

[0085] YYVRVSVEDMRVEEMTAAHYLAFKEKLVPLDQDRHGYDPFVLELDLKPFGAHQPKISLQ

[0086] SHIGNGVSFLNRTLSAKMFSQNANAEGSQLMLDFLREFKHGGEKLLLSPRVNSVQKLRHS

[0087] LLRADRLLEKHEDEDPLSVVQGIDELGFLPGWGNTVGRVRESFQLLLDIIQAPDADTLEKF

[0088] LARLPLMVKVVILSPHGYFGQTNVLGMPDTGGQVVYILDQVRAMEREMQQRLDEAGLQ

[0089] NVKADVVVLTRLIPDAHGTSCNERLEPISGCQNARILRVPFRDSEGRILNHWVSRFDLWPY

[0090] LERFTIDATKEILAEMGGKPDFIIGNYSDGNLVATLMSHRMNVTQCNIAHALEKTKYDDA

[0091] DIYWQKLEDKYHFSCQFTADLIAMNSADFIVTSTYQEIAGHEEMVGQYESYKSFTMPQLY

[0092] RVVEGIDIYNPKFNIVSPGADLDIYFPYQEKERRLTGLHKDIEALLFDPDFKGTVGQLEDRD

[0093] KPILFSMARLDKVKNLTGLAEWYAGNQRLRGLVNLVIVGGVIDPAATMDREEAAECEHM

[0094] HELVEKYKMHGTFRWIVAQKNRVRNGELYRYIADTRGAFAQPALYEAFGLTVIEAMTCGL

[0095] PTFATNHGGPSEIIKHKKSGFHIDPYHGAEAADLMADFFERSQKEPSHWTKISEAAQERIFS

[0096] RYTWSIYAKRLVTLSHVYTFWKHVTSLESRETKRYLEMFYILQMRKLVAKMSEETVEKEK

[0097] AAAEAGPAGPPKVGFGAM

[0098] Example 5 Biocatalytic Preparation Method of Anoectochilus glycoside

[0099] The biocatalytic preparation method of anoectochilus glycosides uses a dual enzyme coupling system, such as Figure 4 As shown, the total reaction system is 50mL: (R)-3-hydroxy-γ-butyrolactone 20mM, UDP 0.5mM, sucrose 500mM, phosphate buffer (pH 7.5) 50mM, 50mg glycosyltransferase crude enzyme solution and 100mg sucrose synthase crude enzyme solution. The reaction device is a magnetic stirrer, and the reaction temperature is controlled at 30°C by a water bath. Samples are taken regularly during the reaction, and the reaction progress is detected by the detection method described in Example 3. The results are shown in FIG. Figure 5 As shown. Figure 5 It can be seen that the conversion rate of the substrate is greater than 99.0% at 10 h, and the concentration of the product anoectochiloside is 5.26 g / L. The calculated space-time yield is 12.6 g L -1 d -1 .

[0100] The catalytic product was purified by semi-preparative liquid phase, and the obtained pure product was detected by liquid phase mass spectrometry, such as Figure 6 As shown, the results are consistent with the theoretical molecular weight of anoectin; in addition, the purified anoectin product was dissolved in deuterated pyridine for hydrogen and carbon spectrum analysis, and the results are as follows Figure 7 , Figure 8 As shown, the results are consistent with those reported in the literature (Zhang, et al. Efficient Synthesis of Kinsenoside and Goodyeroside Aby a Chemo-Enzymatic Approach. Molecules, 2014, 19: 16950-16958;

[0101] doi:10.3390 / molecules191016950).

[0102] Comparative Example

[0103] According to the method shown in Example 1, the gene encoding the glycosyltransferase YjiC (NCBI accession NO.: WP_003182014.1) from Bacillus licheniformi was connected to the pET-28a (+) plasmid, and the ligation product was transformed into E. coli BL21 (DE3) competent cells, and the positive clones were screened and detected to obtain the recombinant engineered bacteria containing the glycosyltransferase BlYjiC gene. According to Example 2, the obtained recombinant engineered bacteria were expressed and cultured to obtain the recombinant glycosyltransferase BlYjiC crude enzyme solution. According to the method of Examples 4-5, the recombinant glycosyltransferase BlYjiC and the prepared sucrose synthase enzyme solution were coupled, and the reaction progress was detected by the detection method described in Example 3. The results are as shown in FIG. Figure 5 As shown. Figure 5 It can be seen that the conversion rate of the substrate was only 19.1% at 10 h, and the concentration of the product aurocin was 1.0 g / L. The calculated space-time yield was 2.4 g L -1 d -1 This level is much lower than the yield catalyzed by the glycosyltransferase provided by the present invention, which proves the high efficiency of the glycosyltransferase provided by the present invention.

[0104] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A glycosyltransferase, characterized in that The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO.

1.

2. A catalyst for catalyzing the synthesis of anoectochiloside from (R)-3-hydroxy-γ-butyrolactone, characterized in that: The catalyst comprises or expresses a glycosyltransferase having an amino acid sequence as shown in SEQ ID NO.

1.

3. A nucleic acid molecule encoding the glycosyltransferase according to claim 1, a host cell expressing the glycosyltransferase according to claim 1, or a catalyst comprising the glycosyltransferase according to claim 1.

4. Use of a glycosyltransferase in the preparation of anoectochilus glycoside, characterized in that: The glycosyltransferase according to claim 1 is used to produce anoectochiloside in the glycosylation reaction of (R)-3-hydroxy-γ-butyrolactone.

5. A method for preparing anoectochiloside, characterized in that: The method comprises the following steps: (1) preparing an enzyme solution of the glycosyltransferase according to claim 1; (2) adding the prepared enzyme solution to a mixed reaction system containing (R)-3-hydroxy-γ-butyrolactone and a UDP-glucose regeneration system to carry out a glycosylation reaction to obtain anoectochiloside.

6. The method for preparing anoectochiloside according to claim 5, characterized in that: The enzyme solution is a crude enzyme solution or a purified enzyme solution of a genetically engineered bacterium capable of expressing the glycosyltransferase according to claim 1.

7. The method for preparing roxiliglycoside according to claim 5, characterized in that: The UDP-glucose regeneration system is a UDP-glucose regeneration system which uses sucrose synthase as a UDP-glucose regeneration enzyme, uses sucrose as a cosubstrate, and contains UDP and UDP-glucose.

8. The method for producing anoectochiloside according to claim 5, characterized in that: The concentration of the substrate (R)-3-hydroxy-γ-butyrolactone in the glycosylation reaction is 10 mM to 50 mM.

9. The method for preparing roxiliposide according to claim 5, characterized in that: The concentration of the crude glycosyltransferase solution in the mixed reaction system is 0.5-10 mg / mL.

10. The method for preparing anoectochiloside according to claim 5, characterized in that: The temperature of the glycosylation reaction is 15-50° C., and the pH value of the mixed reaction system is 6-10.

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