Glycosyl transferase or mutant thereof and application of glycosyl transferase or mutant thereof in synthesis of raspberry ketone glucoside

Abstract

The invention belongs to the technical field of enzyme engineering, and discloses glycosyltransferase or a mutant thereof and application of the glycosyltransferase in synthesis of raspberry ketone glucoside. The invention provides glycosyl transferase derived from a soil metagenome, and the catalytic efficiency of the glycosyl transferase is remarkably improved through semi-rational design. The substrate conversion rate of the modified mutant to the raspberry ketone glucoside is increased from 75.81% of a wild type to 96.10%, the yield reaches 15.68 g / L, the space time yield reaches 31.36 g / L / d, and the application breakthrough of a biosynthesis technology in industrial whitening and anti-aging product raspberry ketone glucoside is realized. Compared with a traditional extraction process, the method has the advantages that raw material dependence and environmental pollution are effectively reduced, production efficiency and economical efficiency are remarkably improved, and a green and environment-friendly solution is provided for sustainable preparation of raspberry ketone glucoside.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and more specifically, relates to a glycosyltransferase or a mutant thereof and an application thereof in the synthesis of raspberry ketone glucoside. Background Art

[0002] Raspberry Ketone Glucoside (RKG) is an active ingredient naturally found in raspberry fruit. It is widely used in cosmetics such as whitening and anti-aging due to its ability to inhibit melanin synthesis. In recent years, due to its excellent antioxidant and anti-inflammatory biological activities, raspberry ketone glucoside has gradually become a key ingredient in whitening and anti-aging products, and has been included in the "Catalogue of Used Cosmetic Raw Materials" (2015 Edition) and GB / T 35954-2018 "High Performance Liquid Chromatography Method for the Determination of 10 Whitening and Anti-Freckle Agents in Cosmetics".

[0003] The main preparation method of raspberry ketone glucoside currently relies on extraction from raspberry fruit, but this method has several significant problems: 1) High dependence on raw materials: raspberry fruit is in short supply and expensive, which limits the feasibility of large-scale production. 2) Complex extraction process: A large amount of organic solvent is required during the extraction process, which is not only costly but also has adverse effects on the environment. 3) Low efficiency: Traditional extraction processes often have low yields and complex purification processes, making it difficult to meet the needs of industrial production. To solve the above problems, the synthesis of raspberry ketone glucoside based on glycosyltransferases (GT; EC 2.4.xy) has become a green alternative with great potential. Glycosyltransferases can efficiently and specifically catalyze the transfer of sugars from activated glycosyl donors to acceptor molecules, avoiding the use of organic solvents and mild reaction conditions. It is a more environmentally friendly and economical production method. However, there are few reports on glycosyltransferases that can catalyze the synthesis of ketone raspberry ketone glucoside. Shimoda et al. used Phytolacca americana cells containing multiple glycosyl transfers to catalyze raspberry ketone. After 3 days of catalysis, the conversion rate was 23.0%, the yield was 6.0 mg / L, and the space-time yield was 2.0 mg / L / d (Kei Shimoda, et al. Biotransformation of raspberry ketone and zingerone by cultured cells of Phytolacca americana. Phytochemistry, 2007, 68 (4) 487-492); Li et al. used the glycosyltransferase PI-GT1 from Bacillus licheniformis PI15 as a catalyst. After 12 hours of reaction, they successfully achieved the synthesis of raspberry ketone to raspberry ketone glucoside. The conversion rate was 74.1%, the yield was 5.9 g / L, and the space-time yield was 11.8 g / L / d (Li Bingfeng, et al. Biochemical characterization of an organic solvent-tolerant glycosyltransferase from Bacillus licheniformis PI15 with potential application for raspberry ketone glycoside production. Biotechnology and Applied Biochemistry, 2020, 67(2): 249-256). The catalytic efficiency of the glycosyltransferase PI-GT1 is insufficient, and the substrate concentration cannot be further increased. When the substrate concentration increases, the conversion rate decreases significantly.It can be seen that the glycosyltransferase activity currently reported to catalyze raspberry ketone to produce raspberry ketone glucoside is low, resulting in insufficient production and high costs, making it difficult to meet industrial needs. Therefore, the exploration and development of new and efficient glycosyltransferases is the key to achieving the biosynthesis of raspberry ketone glucoside.

[0004] 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

[0005] The purpose of the present invention is to provide a glycosyltransferase or a mutant thereof and application thereof in the synthesis of raspberry ketone glucoside.

[0006] The present invention successfully screened out a microbial glycosyltransferase by deeply mining the soil metagenome, which can efficiently catalyze the glycosylation reaction of raspberry ketone and UDP-glucose to generate raspberry ketone glucoside. In order to further improve the catalytic performance of the enzyme, we conducted a semi-rational design on it and obtained a glycosyltransferase mutant with significantly enhanced enzyme activity. Compared with the wild type, the substrate conversion rate of raspberry ketone by the mutant was increased from 75.81% to 96.10%, the yield reached 15.68g / L, and the space-time yield was as high as 31.36g / L / d, showing excellent prospects for industrial application. The present invention not only provides a new biocatalyst for the green synthesis of raspberry ketone glucoside, but also opens up a new way for its large-scale production, overcoming the problems of resource dependence, low efficiency, environmental pollution, etc. in traditional processes.

[0007] 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.

[0008] The second aspect of the present invention is to provide a glycosyltransferase mutant, the amino acid sequence of the glycosyltransferase mutant is shown in SEQ ID NO.2.

[0009] The third aspect of the present invention discloses a catalyst for catalyzing the synthesis of raspberry ketone glucoside from raspberry ketone, wherein the catalyst comprises or expresses a glycosyltransferase having an amino acid sequence as shown in SEQ ID NO.1 or a glycosyltransferase mutant as shown in SEQ ID NO.2.

[0010] The fourth aspect of the present invention discloses the use of a glycosyltransferase or a mutant thereof in the synthesis of raspberry ketone glucoside, wherein the aforementioned glycosyltransferase or a mutant thereof is used in the glycosylation reaction of raspberry ketone to generate raspberry ketone glucoside.

[0011] The above-mentioned purpose of the present invention is achieved by the following technical means:

[0012] By predicting gene functions based on metagenomic sequencing data of soil from Huoshan County, Lu'an City, Anhui Province, and using the reported glycosyltransferase PI-GT1 sequence from Bacillus licheniformis PI15 (NCBIaccession: AKA94108.1) as a probe, a new glycosyltransferase with an amino acid sequence identity of 31.68% with the probe was obtained from the soil metagenome, named PsUGT, and its amino acid sequence is shown in SEQ ID NO.1. The nucleotide sequence of the gene encoding PsUGT is shown in SEQ ID NO.3.

[0013] Based on the obtained glycosyltransferase PsUGT, the present invention also obtains its mutant M144A through semi-rational design, and the mutant is obtained by site-directed mutation of methionine at position 144 of the glycosyltransferase amino acid sequence shown in SEQ ID NO.1 to alanine, and its amino acid sequence is shown in SEQ ID NO.2. The synthesis of raspberry ketone glucoside using the glycosyltransferase or its mutant overcomes the problems of resource dependence, low efficiency, environmental pollution, etc. existing in the traditional process, and has important industrial application prospects.

[0014] A fifth aspect of the present invention is to provide a method for synthesizing raspberry ketone glucoside, the method comprising the following steps:

[0015] (1) preparing an enzyme solution of the aforementioned glycosyltransferase or a mutant thereof;

[0016] (2) adding the prepared enzyme solution into a mixed reaction system containing raspberry ketone and UDP-glucose regeneration system to carry out glycosylation reaction to obtain raspberry ketone glucoside.

[0017] In a preferred embodiment, the enzyme solution is a crude enzyme solution or a purified enzyme solution obtained by disrupting cells of a genetically engineered bacterium capable of expressing the aforementioned glycosyltransferase or a mutant thereof.

[0018] Furthermore, the UDP-glucose regeneration system is a UDP-glucose regeneration system using sucrose synthase as the UDP-glucose regeneration enzyme, sucrose as the cosubstrate, and containing UDP or UDP-glucose.

[0019] In a preferred embodiment, the concentration of the substrate raspberry ketone in the glycosylation reaction is 10-100 mM.

[0020] Furthermore, the concentration of the crude enzyme solution of glycosyltransferase or the crude enzyme solution of glycosyltransferase mutant in the mixed reaction system is 1.0 to 10 mg / mL (preferably 2 to 5 mg / mL).

[0021] 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).

[0022] In the presence of UDP-glucose provided by the UDP-glucose regeneration system, the substrate raspberry ketone undergoes a glycosylation reaction under the catalysis of glycosyltransferase to generate raspberry ketone glucoside. Specifically, the reaction temperature is 15-50° C., the pH value of the mixed reaction system is 6-10, and the UDP concentration is 0.2-2 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 raspberry ketone glucoside. The sucrose concentration is 100-500 mM.

[0024] The present invention has the beneficial effects that the glycosyltransferase and its mutants used in the present invention have high catalytic activity, the substrate conversion rate can reach up to 96.10%, the yield reaches 15.68g / L, and the space-time yield is as high as 31.36g / L / d, which is significantly higher than the reported level. The present invention successfully solves the key problem of low activity in the biosynthesis process of raspberry ketone glucoside, showing great industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure shows the PCR results of the extracted soil metagenome and glycosyltransferase gene, where M is Marker; 1 is the soil metagenome, and 2 is the amplified glycosyltransferase PsUGT gene;

[0026] Figure 2 This is the SDS-PAGE analysis result of pure enzyme of glycosyltransferase PsUGT;

[0027] Figure 3 It is the HPLC chromatogram when the specific enzyme activity of glycosyltransferase PsUGT is measured;

[0028] Figure 4 is the specific enzyme activity of the wild type (WT) and mutant of glycosyltransferase PsUGT;

[0029] Figure 5 represents the amino acid residues near the substrate pocket of the glycosyltransferase PsUGT, UDPG represents UDP-glucose, and RK represents the substrate raspberry ketone;

[0030] Figure 6 Schematic diagram of UDP-glucose regeneration system;

[0031] Figure 7The time course curve of the synthesis of raspberry ketone glucoside catalyzed by wild-type glycosyltransferase and its mutant M144A;

[0032] Figure 8 is the mass spectrum of raspberry ketone glucoside, where [M+Na] + The peak is the target peak with a molecular weight of 349;

[0033] Fig. 9 Raspberry Ketone Glucoside 1 H NMR spectrum;

[0034] Fig.10 Raspberry Ketone Glucoside 13 C NMR spectrum;

[0035] Fig.11 This is the time course curve of the synthesis of raspberry ketone glucoside by glycosyltransferase PI-GT1 at different substrate concentrations. DETAILED DESCRIPTION

[0036] 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.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] In this article, mutant and mutator have the same meaning.

[0043] Example 1: Mining of glycosyltransferase genes and construction of recombinant engineering bacteria

[0044] Soil samples were collected from Huoshan County, Lu'an City, Anhui Province. Soil metagenomes were extracted using a soil genome extraction kit. The results are as follows: Figure 1 As shown, the extracted metagenome was sent to Beijing Qingke Biotechnology Co., Ltd. for metagenomic sequencing. The online tool MetaGeneMark (http: / / exon.gatech.edu / meta_gmhmmp.cgi) was used to predict gene function of metagenomic data. The reported glycosyltransferase PI-GT1 sequence from Bacillus licheniformis PI15 (NCBI accession: AKA94108.1) (Li Bingfeng, et al. Biochemical characterization of an organic solvent-tolerant glycosyltransferase from Bacillus licheniformis PI15 with potential application for raspberry ketoneglycoside production. Biotechnology and Applied Biochemistry, 2020, 67 (2): 249-256) was used as a probe. A new glycosyltransferase with an amino acid sequence identity of 31.68% with the probe was obtained from the soil metagenome. Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence encoding the enzyme is shown in SEQ ID NO.3. The amino acid sequence of this glycosyltransferase has the highest sequence identity of 60.51% with the glycosyltransferase from Phytohabitans suffuscus (NCBI accession: WP_173159979.1) in the NCBI database, so it is named PsUGT. A group of enzymes similar to this glycosyltransferase in the NCBI database are all from microorganisms, so it can be identified as a new enzyme from a microorganism.

[0045] The extracted soil metagenome DNA was used as a template, PsUGT-F (SEQ ID NO.5) and PsUGT-R (SEQ ID NO.6) were used as primers, and the primer sequences were shown in Table 1 to amplify the glycosyltransferase gene. Figure 1As shown; At the same time, pET-28a (+) plasmid was used as a template, pET28a-F (SEQ ID NO.7) and pET28a-R (SEQ ID NO.8) were used as primers to amplify and obtain a linearized vector, and the primer sequences are shown in Table 1. The amplified glycosyltransferase gene was connected to the pET-28a (+) plasmid by a one-step cloning kit (ClonExpress II One Step Cloning Kit) to construct a recombinant plasmid pET28a-PsUGT. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells, positive clones were screened and detected, and recombinant engineering bacteria containing glycosyltransferase genes were obtained.

[0046] Table 1 Primer sequences

[0047] Primer name sequence PsUGT-F agcaaatgggtcgcggatccATGCAGTCCTATCACTTGCCTTCA SEQ ID NO.5 PsUGT-R tcgagtgcggccgcaagcttCTATGAGCGCGACTTCAGTCG SEQ ID NO.6 pET28a-F AAGCTTGCGGCCGCACTC SEQ ID NO.7 pET28a-R GGATCCGCGACCCATTTG SEQ ID NO.8

[0048] SEQ ID NO.1

[0049] MQSYHLLFIVCGGNGHLFTTLAVANELTNRGHRVTLVSSAEVKDAVEAVGADFVPYRSAFDGFHLPDAVKAEN

[0050] AEELLHNIYVDDNEAVLRAAEDVAARDQPDAVVYEIFHFIAGKVLAAKLNRPGVRITGLSSNEHYSLWDEMRQA

[0051] VGHGNPEEFESVRQKLTSLLFEAGIGRPIREFWDEVDDLNIVTTSRSYQNAGETFDDKHVFVGPVFTPQQLEAQ

[0052] WQPPAGEPPVLLVSLGSTWNEHPEFFKACVHAFEGTPWHVVLVVGQFVDPAELGALPPNVEARSWVSFVDVL

[0053] KYASAFIMHGTGGSAFPAIYRGCPLLVFNHYATDSAPTAARLLDLGLGHRLGIEQVTPTGLRKAVSDLVADEDVRR

[0054] RVNAMRRDMEDSGGVARAADAIAEHIERLKSRS

[0055] SEQ ID NO.3

[0056] ATGCAGTCCTATCACTTGCTCTTCATCGTGTGCGGAGGCAATGGCCACCTCTTCACTACACTCGCCGTGGCCA

[0057] ACGAACTGACCAATCGCGGGCACCGTGTCACGCTGGTGAGCTCAGCCGAGGTCAAGGACGCGGTCGAGG

[0058] CGGTGGGTGCCGACTTCGTGCCCTATCGGTCAGCGTTCGACGGATTTCACCTACCCGACGCCGTGAAGGCG

[0059] GAAAACGCCGAGGAGCTGTTGCACAACATTTACGTTGACGACAACGAAGCCGTGCTCCGCGCGGCGGAGG

[0060] ACGTCGCGGCCCGGGACCAACCCGATGCCGTGGTGTACGAGATTTTTCACTTCATTGCCGGTAAGGTGCTC

[0061] GCCGCAAAGCTGAACCGACCCGGAGTGAGGATCACCGGCCTCTCCTCGAACGAGCACTACTCGCTCTGGG

[0062] ACGAAATGCGGCAGGCCGTCGGCCACGGGAATCCCGAAGAGTTCGAGAGCGTCCGCCAGAAACTCACCTC

[0063] GCTTCTCTTCGAGGCGGGGATCGGCCGCCCCATCAGAGAGTTCTGGGACGAGGTCGACGATTTAAACATCG

[0064] TTACGACCTCTCGCTCATACCAGAACGCAGGCGAGACCTTTGACGACAAACACGTCTTCGTAGGGCCGGTC

[0065] TTCACCCCGCAGCAGCTCGAGGCACAGTGGCAACCGCCCGCAGGGGAGCCTCCAGTTCTTCTCGTGTCGCT

[0066] CGGTTCGACGTGGAATGAGCATCCCGAGTTCTTCAAGGCGTGCGTCCACGCCTTCGAAGGGACGCCGTGG

[0067] CACGTCGTGCTGGTGGTCGGGCAGTTCGTGGACCCAGCCGAGCTGGGCGCCCTTCCCCCCCAATGTCGAGG

[0068] CGCGGTCCTGGGTCTCGTTCGTCGATGTGTTGAAGTACGCGTCGGCGTTCATCATGCATGGCACCGGCGGCT

[0069] CAGCCTTTCCGGCGATTTACCGTGGTTGCCCGCTACTGGTGTTCAACCACTACGCGACGGACTCTGCACCAA

[0070] CGGCGGCCCGATTGCTCGATCTAGGTCTCGGACATCGCCTCGGCATCGAGCAGGTCACGCCCACGGGGCTG

[0071] CGGAAGGCGGTTTCCGACCTCGTAGCCGATGAGGACGTCCGGCGCCGGGTCAACGCCATGCGCCGCGATA

[0072] TGGAGGATTCCGGAGGCGTCGCCCGAGCCGCCGACGCAATCGCCGAGCACATCGAGCGACTGAAGTCGCG

[0073] CTCATAG

[0074] Example 2 Activity determination of recombinant glycosyltransferase PsUGT

[0075] 1. Preparation of pure enzyme of recombinant glycosyltransferase PsUGT

[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 600When 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 bacteria were collected by centrifugation at 10,000 rpm and 4°C, washed twice with ultrapure water and suspended with 20 mL 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 crude enzyme solution was subjected to Ni column affinity chromatography to obtain the pure enzyme protein. The purified recombinant glycosyltransferase PsUGT was verified by SDS-PAGE. The results are as follows: Figure 2 shown.

[0077] 2. Analysis of the specific activity of recombinant glycosyltransferase PsUGT

[0078] The catalytic activity of pure enzyme on raspberry ketone was determined. The activity detection system was (200 μL): PsUGT pure enzyme (10 μg), 2 mM raspberry ketone, 5 mM UDP-glucose, the reaction medium was 50 mM potassium phosphate buffer (pH 8.0), the reaction temperature was 30 ° C, the reaction was triggered by adding pure enzyme, the reaction lasted for 30 minutes, and the reaction results were determined by high performance liquid chromatography. The results of liquid chromatography analysis are shown in Figure 3 As shown, the substrate peaks at 18.1min and the product peaks at 10.6min. The liquid chromatograph used is Shimadzu LC-2060, and the chromatographic column model used is Elite C18 analytical column, 5μm, 4.6mm×250mm; mobile phase 20% methanol; flow rate 1mL / min; detection wavelength 254nm, injection 10μL. Glycosyltransferase activity unit (U) definition: the amount of enzyme required to generate 1μmol of raspberry ketone glucoside per minute under standard reaction conditions. Enzyme activity test results are shown in Figure 4 As shown, the specific activity of the recombinant glycosyltransferase PsUGT was 2.8 U / mg.

[0079] Example 3 Semi-rational design of glycosyltransferase PsUGT

[0080] 1. Site-directed mutagenesis of glycosyltransferase PsUGT

[0081] The structure of glycosyltransferase PsUGT was modeled using AlphaFold3 (https: / / golgi.sandbox.google.com / about) online software. UDP-glucose and substrate raspberry ketone were docked into the protein model using AutoDock Vina software. The docking results were analyzed using Pymol to find out the amino acid composition near the substrate. The results are as follows: Figure 5As shown. By designing specific primers (see Table 2), the gene sequence of Sequence NO.1 was introduced with mutations using the Quickchange technology (An efficient onestep site-directed and site-saturation mutagenesis protocol [J]. Nucleic Acids Research, 2004, 32 (14): e115), and 11 site-directed mutants were constructed. Specifically, the PCR reaction system was: ddH2O 2μL, DNA polymerase 5μL, upstream primer 1μL, downstream primer 1μL, template 1μL. PCR amplification conditions: 1) pre-denaturation at 94℃ for 2min; 2) denaturation at 98℃ for 10s, annealing at 60℃ for 15s, extension at 72℃ for 40s (this step was cycled 30 times); post-extension at 72℃ for 5min. The PCR product was digested with Dpn I restriction enzyme at 37℃ for 20 minutes. The digested product was then transformed into E. coli BL21 (DE3) competent cells, spread on LB / Kan solid plates and cultured at 37°C overnight. The clones were selected and the mutation of each site was confirmed by gene sequencing.

[0082] Table 2 Primers for site-directed mutagenesis of glycosyltransferase PsUGT

[0083]

[0084]

[0085] 2. Specific activity analysis of glycosyltransferase PsUGT mutants

[0086] The obtained mutant single colonies were streaked and activated on LB solid medium containing 50 μg / mL Kan; according to the method described in Example 2, the recombinant PsUGT mutants were cultured and collected and the protein was purified by nickel column affinity chromatography to obtain the mutant pure enzyme. The specific enzyme activity of the mutant was determined by the method described in Example 2. The results are as follows: Figure 4 As shown. Figure 4 The results show that different mutants of PsUGT exhibit different specific enzyme activities from the wild type, among which the mutant M144A has the highest specific enzyme activity, reaching 10.1 U / mg, which is 3.6 times that of the wild type, indicating that the mutant significantly improves the catalytic activity of PsUGT. The amino acid sequence of the mutant M144A is shown in SEQ ID NO.2.

[0087] SEQ ID NO.2

[0088] MQSYHLLFIVCGGNGHLFTTLAVANELTNRGHRVTLVSSAEVKDAVEAVGADFVPYRSAFDGFHLPDAVKAEN

[0089] AEELLHNIYVDDNEAVLRAAEDVAARDQPDAVVYEIFHFIAGKVLAAKLNRPGVRITGLSSNEHYSLWDEARQA

[0090] VGHGNPEEFESVRQKLTSLLFEAGIGRPIREFWDEVDDLNIVTTSRSYQNAGETFDDKHVFVGPVFTPQQLEAQ

[0091] WQPPAGEPPVLLVSLGSTWNEHPEFFKACVHAFEGTPWHVVLVVGQFVDPAELGALPPNVEARSWVSFVDVL

[0092] KYASAFIMHGTGGSAFPAIYRGCPLLVFNHYATDSAPTAARLLDLGLGHRLGIEQVTPTGLRKAVSDLVADEDVRR

[0093] RVNAMRRDMEDSGGVARAADAIAEHIERLKSRS

[0094] Example 4 Biocatalytic Preparation Method of Raspberry Ketone Glucoside

[0095] 1. Preparation of crude enzyme solution of recombinant sucrose synthase

[0096] The biocatalytic preparation method of raspberry ketone glucoside in the present invention adopts a double enzyme coupling system, such as Figure 6As shown, the sucrose synthase used to construct the UDP-glucose regeneration system is derived from Micractinium conductrix. The sucrose synthase used is 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. 4. According to the method of Example 1, the sucrose synthase mutant S31D gene is cloned into the pET-28a (+) plasmid. Subsequently, the plasmid is 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 is 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 (ie, cell lysis supernatant) was prepared.

[0097] SEQ ID NO.4

[0098] MSAGADSPSSQPFLASPRGVITPRTFTRSLDFAGGTPSEILKAGLVHSRNELVLLFSRCMAK

[0099] SKADKPILLPHIIMDELCAVCDECNNPMLKSGEIAAILKTVQEAVVIAPRIAFALRPTMGEW

[0100] YYVRVSVEDMRVEEMTAAHYLAFKEKLVPLDQDRHGYDPFVLELDLKPFGAHQPKISLQ

[0101] SHIGNGVSFLNRTLSAKMFSQNANAEGSQLMLDFLREFKHGGEKLLLSPRVNSVQKLRHS

[0102] LLRADRLLEKHEDEDPLSVVQGIDELGFLPGWGNTVGRVRESFQLLLDIIQAPDADTLEKF

[0103] LARLPLMVKVVILSPHGYFGQTNVLGMPDTGGQVVYILDQVRAMEREMQQRLDEAGLQ

[0104] NVKADVVVLTRLIPDAHGTSCNERLEPISGCQNARILRVPFRDSEGRILNHWVSRFDLWPY

[0105] LERFTIDATKEILAEMGGKPDFIIGNYSDGNLVATLMSHRMNVTQCNIAHALEKTKYDDA

[0106] DIYWQKLEDKYHFSCQFTADLIAMNSADFIVTSTYQEIAGHEEMVGQYESYKSFTMPQLY

[0107] RVVEGIDIYNPKFNIVSPGADLDIYFPYQEKERRLTGLHKDIEALLFDPDFKGTVGQLEDRD

[0108] KPILFSMARLDKVKNLTGLAEWYAGNQRLRGLVNLVIVGGVIDPAATMDREEAAECEHM

[0109] HELVEKYKMHGTFRWIVAQKNRVRNGELYRYIADTRGAFAQPALYEAFGLTVIEAMTCGL

[0110] PTFATNHGGPSEIIKHKKSGFHIDPYHGAEAADLMADFFERSQKEPSHWTKISEAAQERIFS

[0111] RYTWSIYAKRLVTLSHVYTFWKHVTSLESRETKRYLEMFYILQMRKLVAKMSEETVEKEK

[0112] AAAEAGPAGPPKVGFGAM

[0113] 2. Double enzyme coupling preparation method of raspberry ketone glucoside

[0114] The total system for the biocatalytic preparation of raspberry ketone glucoside is 50 mL: raspberry ketone 50 mM, UDP 0.5 mM, sucrose 500 mM, phosphate buffer (pH 8.0) 50 mM, DMSO 1% (v / v), 100 mg of crude enzyme solution of PsUGT glycosyltransferase or its mutant M144A, and 100 mg of crude enzyme solution of sucrose synthase. The reaction device is a magnetic stirrer, and the reaction temperature is controlled at 30°C by a water bath. Samples are taken at regular intervals during the reaction, and the reaction progress is detected by the detection method described in Example 2. The results are as follows: Figure 7 As shown. Figure 7 It can be seen that the 12h conversion rates of the wild-type glycosyltransferase and the mutant M144A provided by the present invention are 75.81% and 96.10%, respectively, and the yields are 12.37 g / L and 15.68 g / L, respectively. Calculation shows that the space-time yields of the wild-type glycosyltransferase and the mutant M144A are 24.74 g / L / d and 31.36 g / L / d, respectively.

[0115] The reaction solution after the reaction is collected, and the catalytic product is purified by semi-preparative liquid phase, and the obtained pure product is detected by liquid chromatography-mass spectrometry, such as Figure 8 As shown, the results are consistent with the theoretical molecular weight of raspberry ketone glucoside; in addition, the purified raspberry ketone glucoside was dissolved in deuterated methanol for hydrogen and carbon spectrum analysis, and the results are as follows Fig. 9 , Fig.10 As shown, the result is consistent with the literature report (Li Bingfeng, et al. Biochemical characterization of an organic solvent-tolerant glycosyltransferase from Bacillus licheniformis PI15 with potential application for raspberry ketone glycoside production. Biotechnology and Applied Biochemistry, 2020, 67(2): 249-256).

[0116] 3. Comparative ratio

[0117] The present invention synthesized the reported glycosyltransferase PI-GT1 sequence from Bacillus licheniformis PI15 (NCBI accession: AKA94108.1) (Li Bingfeng, et al. Biochemical characterization of an organic solvent-tolerant glycosyltransferase from Bacilluslicheniformis PI15 with potential application for raspberry ketone glycoside production. Biotechnology and Applied Biochemistry, 2020, 67 (2): 249-256), cloned it into the pET-28 (a) vector using the method described in Example 1, and transformed it into E. coli BL21 (DE3) competent cells to obtain a recombinant engineered bacterium containing the glycosyltransferase PI-GT1 gene. According to the method of Example 2, the obtained recombinant engineered bacteria were expressed and cultured, and a recombinant glycosyltransferase PI-GT1 crude enzyme solution was prepared. Raspberry ketone glucoside was synthesized according to the dual enzyme coupling preparation method described in Example 4, and the results are as follows. Fig.11 As shown. Fig.11 It can be seen that when the recombinant glycosyltransferase PI-GT1 catalyzes 30mM substrate, the 12h conversion rate can reach 74.60%, the corresponding yield is 7.30g / L, and the calculated time-space yield is 14.60g / L / d; however, when the substrate concentration is increased to the substrate concentration consistent with the present invention (50mM), the conversion rate is only 54.31%, the corresponding yield is 8.86g / L, and the calculated time-space yield is 17.72g / L / d. From the above results, it can be seen that the catalytic efficiency of the glycosyltransferase PI-GT1 is limited, the substrate concentration cannot be further increased, and the conversion rate is significantly reduced when the substrate concentration increases. Compared with the present invention, the yield and time-space yield are significantly lower. In summary, the conversion rate, yield and space-time yield of the glycosyltransferase PsUGT and its mutants provided by the present invention in the preparation of raspberry ketone glucoside are higher than the highest level reported, which proves that the glycosyltransferase and its mutants provided by the present invention have good industrial application potential in the preparation of raspberry ketone glucoside.

[0118] 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 glycosyltransferase mutant, characterized in that: The amino acid sequence of the glycosyltransferase mutant is shown in SEQ ID NO.

2.

3. A catalyst for catalyzing the synthesis of raspberry ketone glucoside from raspberry ketone, characterized in that: The catalyst comprises or expresses the glycosyltransferase according to claim 1 or the glycosyltransferase mutant according to claim 2.

4. Use of a glycosyltransferase or a mutant thereof in the synthesis of raspberry ketone glucoside, characterized in that: The glycosyltransferase according to claim 1 or the glycosyltransferase mutant according to claim 2 is used in the glycosylation reaction of raspberry ketone to produce raspberry ketone glucoside.

5. A method for synthesizing raspberry ketone glucoside, characterized in that: The method comprises the following steps: (1) preparing an enzyme solution of the glycosyltransferase according to claim 1 or the glycosyltransferase mutant according to claim 2; and (2) adding the prepared enzyme solution to a mixed reaction system containing raspberry ketone and UDP-glucose regeneration system to carry out a glycosylation reaction to obtain raspberry ketone glucoside.

6. The method for synthesizing raspberry ketone glucoside 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 that is capable of expressing the glycosyltransferase according to claim 1 or the glycosyltransferase mutant according to claim 2 after cell lysis.

7. The method for synthesizing raspberry ketone glucoside according to claim 5, characterized in that: The UDP-glucose regeneration system is a UDP-glucose regeneration system that uses sucrose synthase as a UDP-glucose regeneration enzyme, uses sucrose as a cosubstrate, and contains UDP or UDP-glucose.

8. The method for synthesizing raspberry ketone glucoside according to claim 5, characterized in that: The concentration of the substrate raspberry ketone in the glycosylation reaction is 10-100 mM.

9. The method for synthesizing raspberry ketone glucoside according to claim 6, characterized in that: The concentration of the crude enzyme solution of glycosyltransferase or the crude enzyme solution of glycosyltransferase mutant in the mixed reaction system is 1-10 mg / mL.

10. The method for synthesizing raspberry ketone glucoside 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.

Images