Glucosyltransferase SlUGT42 and application thereof in catalyzing 2-phenylethanol to generate 2-phenylethyl beta-D-glucoside

Abstract

The invention belongs to the technical field of biological catalysis. The invention provides a glucosyltransferase SlUGT42, and the amino acid sequence of the glucosyltransferase SlUGT42 is as shown in SEQ ID NO. 2. The invention provides a nucleic acid molecule which is a nucleotide sequence for coding the glucosyltransferase SlUGT42, and the nucleotide sequence of the glucosyltransferase SlUGT42 is as shown in SEQ ID NO. 1. The invention also provides an application of the glucosyltransferase SlUGT42 in catalysis of 2-phenylethyl alcohol to generate 2-phenylethyl beta-D-glucoside. The invention further provides a preparation method of the glucosyltransferase SlUGT42. The method for catalyzing the 2-phenylethanol to generate the 2-phenylethyl beta-D-glucoside by using the glucosyltransferase as the biocatalyst is found in tomatoes for the first time, the method is mild in catalysis condition and short in conversion time, the obtained product is easy to separate, and the method is suitable for large-scale separation and purification of the 2-phenylethyl beta-D-glucoside.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalysis, and particularly relates to a glucosyltransferase SlUGT42 and its application in catalyzing the formation of 2-phenylethyl β-D-glucoside from 2-phenylethanol. Background Art

[0002] 2-Phenylethanol has a pleasant rose aroma and is one of the important volatile compounds affecting the flavor of tomato fruits. 2-Phenylethyl β-D-glucoside is a storage and transport form of 2-phenylethanol. 2-Phenylethyl β-D-glucoside can dissolve in water and some organic solvents, and can slowly release the rose flower fragrance under appropriate conditions. It can be used as an aroma releasing agent in foods such as fruit juices and baked goods to improve the taste of the food and release the aroma. Moreover, 2-phenylethyl β-D-glucoside has antioxidant and antibacterial effects and can also be used in the beauty industry, such as as a fixative to slowly release the aroma in perfumes and increase the fragrance retention time. In the past, the acquisition of this glycoside mainly relied on separation from plant metabolites or chemical synthesis. However, metabolite separation has disadvantages such as low yield and difficult separation and purification, while chemical synthesis is relatively expensive and environmentally unfriendly. The enzymatic catalysis method has mild reaction conditions, high yield, and low cost.

[0003] In recent years, the research on the enzymatic glycosylation of volatile substances in plants has received wide attention. The whole-genome analysis of the UDP-glucosyltransferase (CitUGTs) gene family in citrus has been carried out. 136 CitUGTs genes have been identified from the genome of Clementine mandarin and classified into 18 evolutionary groups and 25 gene families. The novel enzyme CitUGT72AZ4 has been discovered and functionally identified. This enzyme can specifically catalyze the glycosylation reaction of the 4'-hydroxy group of flavonoids and shows high catalytic activity towards flavonoids such as quercetin. Its participation in the regulation of the biosynthesis of citrus flavonoid 4'-O-glucoside has been verified by virus-induced gene silencing technology. By analyzing free and glycosylated volatile substances using gas chromatography-mass spectrometry (GC-MS), cloning UGT genes and performing sequence analysis and phylogenetic tree construction, 22 UGT genes related to the content of monoterpene glycosides have been screened out in the grape genome, and it has been found and identified in vitro that UGT85A26 and UGT85A27 are involved in the glycosylation of grape volatile substances.

[0004] Although a variety of UGT enzymes have been identified and their functions studied in citrus and grapes, there is currently no report on the catalysis of 2-phenylethanol to 2-phenylethyl β-D-glucoside by tomato UGT. This indicates that the types of enzymes capable of catalyzing the formation of 2-phenylethyl β-D-glucoside from 2-phenylethanol in tomatoes may be very limited or even undiscovered. The research of this application fills the gap in the catalysis of 2-phenylethanol to 2-phenylethyl β-D-glucoside by tomato UGT, and there is no relevant report in this field. Summary of the Invention

[0005] The present invention aims at the deficiencies of the prior art and provides a glucosyltransferase SlUGT42 and its application in catalyzing the formation of 2-phenylethyl β-D-glucoside from 2-phenylethanol. Based on the function of the tomato glycosyltransferase gene SlUGT42 in catalyzing the formation of 2-phenylethyl β-D-glucoside from 2-phenylethanol, the optimal temperature and optimal pH of this enzyme are explored, providing new ideas for the large-scale production of 2-phenylethyl β-D-glucoside.

[0006] To solve the above problems, the present invention provides the following technical solutions: A glucosyltransferase SlUGT42, whose amino acid sequence is shown in SEQ ID NO.2.

[0007] Based on the same inventive concept, the present invention provides a nucleic acid molecule, which is a nucleotide sequence encoding the glucosyltransferase SlUGT42 as described above.

[0008] The nucleic acid molecule as described above, whose nucleotide sequence is shown in SEQ ID NO.1.

[0009] The glucosyltransferase SlUGT42 as described above, the glucosyltransferase SlUGT42 is derived from tomato.

[0010] Based on the same inventive concept, the present invention provides an application of the glucosyltransferase SlUGT42 as described above in catalyzing the formation of 2-phenylethyl β-D-glucoside from 2-phenylethanol.

[0011] The application as described above, the method for catalyzing the formation of 2-phenylethyl β-D-glucoside from 2-phenylethanol includes the following steps: S1. Prepare a purified enzyme solution of the glucosyltransferase SlUGT42 with an amino acid sequence shown in SEQ ID NO.2; S2. Using 2-phenylethanol as a substrate, add uridine-5′-diphosphate glucose disodium salt, and then add the purified enzyme solution prepared in step S1 for catalytic reaction to obtain 2-phenylethyl β-D-glucoside.

[0012] The application as described above, the method for preparing the purified enzyme solution in step S1 includes the following steps: S11. Construct an expression vector carrying the glucosyltransferase SlUGT42 gene; S12. Transform the expression vector into a host cell to obtain a recombinant strain; S13. Induce the recombinant strain to express, collect the cells by centrifugation, take the cell precipitate for disruption to obtain a crude enzyme solution, and then purify it to obtain a purified enzyme solution.

[0013] Preferably, in step S13, the specific steps for inducing the expression of the recombinant strain are as follows: IPTG with a final concentration of 0.3 mM is added for induction, and induction is carried out at 16 °C and 110 rpm for 16 - 20 h. The low-temperature environment of 16 °C can slow down the metabolic reaction rate in the cells, and can also reduce the activity of proteases, reducing the risk of the target protein being degraded, thereby increasing the yield and quality of the target protein. The rotation speed of 110 rpm can avoid the occurrence of over-shearing while ensuring oxygen supply. The induction time of 16 - 20 h balances the growth of the cells and the synthesis of the target protein to a certain extent, enabling the cells to efficiently synthesize the target protein while maintaining a good growth state.

[0014] Preferably, in step S2, using the purified enzyme solution can improve the reaction efficiency, reduce impurity interference; ensure the reaction accuracy and accurately reflect the protein function; improve the product purity and reduce the generation of by-products.

[0015] The application as described above, the expression vector is a pMAL vector. The advantages of using a pMAL vector with a maltose-binding protein (MBP) tag are mainly reflected in aspects such as increasing the protein expression level, enhancing protein solubility, facilitating protein purification, and assisting in the correct folding of the protein.

[0016] Preferably, the host cell is Escherichia coli EC1002. Due to its high protein expression ability, recombinant enzyme deficiency, endonuclease deficiency, restriction enzyme deficiency, low protease activity, folding cofactors, good growth characteristics, and high safety, the BL21 strain is an ideal host strain in the protein expression experiment of the present invention.

[0017] The application as described above, the catalytic reaction temperature condition in step S1 is 30 - 60 °C. Preferably, the catalytic reaction temperature condition in step S1 is 37 - 42 °C. Most preferably, the catalytic reaction temperature condition in step S1 is 42 °C, and the glucosyltransferase SlUGT42 has the highest reaction activity at 42 °C.

[0018] For the application described above, the pH of the catalytic reaction in step S1 is 4-8. Preferably, the pH of the catalytic reaction in step S1 is 4.0-8.0. More preferably, the pH of the catalytic reaction in step S1 is 7.0-8.0. Most preferably, the pH of the catalytic reaction in step S1 is 7.5, and the glucosyltransferase SlUGT42 has the highest reaction activity at pH 7.5. Research shows that the enzyme activity is strongly inhibited under acidic conditions and is relatively alkali-tolerant.

[0019] Compared with the existing technology, the effects and advantages of the present invention are as follows: 1. The present invention discovers for the first time a method of using glucosyltransferase as a biocatalyst in tomatoes to catalyze the formation of 2-phenylethyl β-D-glucoside from 2-phenylethanol. This method has mild catalytic conditions, and the obtained 2-phenylethyl β-D-glucoside is easily soluble in water and can be easily separated from the substrate 2-phenylethanol, which is suitable for large-scale separation and purification of 2-phenylethyl β-D-glucoside.

[0020] 2. The present invention explores the optimal temperature and optimal pH for the application of glucosyltransferase SlUGT42, providing new ideas for the large-scale production of 2-phenylethyl β-D-glucoside. Description of the Drawings

[0021] Figure 1 It is the vector map of pMAL- SlUGT42 of the present invention; Figure 2 It is the HPLC chromatogram (A) and mass spectrum (B) of the pMAL empty protein, 2-phenylethyl β-D-glucoside standard product and the recombinant protein SlUGT42 catalyzing the formation of 2-phenylethyl β-D-glucoside from 2-phenylethanol in vitro of the present invention; Figure 3 It is the optimal reaction temperature graph for the application of the glucosyltransferase SlUGT42 of the present invention; Figure 4 It is the optimal reaction pH graph for the application of the glucosyltransferase SlUGT42 of the present invention. Detailed Embodiments

[0022] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the content in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the specification of this invention are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" used in this specification includes any and all combinations of one or more of the related listed items.

[0024] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0025] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0026] The culture media involved in the examples are as follows: LB liquid medium (1L): Add 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl to 950 mL of ddH2O. Make up the volume to 1 L with ddH2O. Autoclave at 121 °C for 20 min, and add ampicillin with a final concentration of 50 μg / L after cooling.

[0027] Example 1 Construction of expression vector 1. Extraction of total RNA from tomato The RNA extraction kit used in this example was purchased from Nanjing Novoprotein Scientific Co., Ltd., and the product number is RC411-01.

[0028] Take an appropriate amount of tomato ripe fruit, cut it into pieces, quickly freeze it in liquid nitrogen, grind it into fine powder at low temperature, add it to a 1.5 mL EP centrifuge tube containing 600 μL of Buffer PSL lysis solution, vortex vigorously for 30 seconds to fully mix the sample and the lysis solution evenly, centrifuge at 12,000 rpm for 5 min, and perform subsequent operations to extract total RNA according to the instructions of the Novoprotein kit. Detect the integrity of the total RNA by agarose gel electrophoresis, and determine and analyze the concentration of the extracted RNA on a ultra-micro ultraviolet spectrophotometer.

[0029] 2. Gene cloning and construction of prokaryotic expression vector Select BamHI and SalI as double digestion sites, design primer sequences (see Table 1), use the cDNA of tomato fruit as a template, and clone it using 2×KeyPo Master Mix high-fidelity enzyme (purchased from Nanjing Novoprotein Scientific Co., Ltd., product number PK511-01) SlUGT42Gene fragment. Among them, the total volume of the cloning system program is 20 μL: 10 μL of 2×KeyPoMaster Mix, 2 μL of template, 0.5 μL of forward primer, 0.5 μL of reverse primer and 7 μL of water. The program is shown in Table 2. The obtained gene fragment was ligated to the pMAL vector (purchased from Beijing Qiyan Biotechnology Co., Ltd., product number QYV0649) using the Novoprotein homologous recombination kit (purchased from Nanjing Novoprotein Biotechnology Co., Ltd., product number C115). The ligation system was directly transformed into DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd., product number DL1001M). Positive clones were selected for sequencing. The total volume of the colony PCR system is 20 μL: 10 μL of 2×Taq PCR Mix, 1 μL of template, 0.5 μL of forward primer, 0.5 μL of reverse primer and 8 μL of water. The program is shown in Table 3 to obtain pMAL- SlUGT42 Expression vector.

[0030] Table 1 Construction of pMAL- SlUGT42 Primer sequences for expression vector

[0031] Table 2 High-fidelity enzyme PCR reaction program of 2×KeyPo Master Mix

[0032] Table 3 Colony PCR reaction program

[0033] SlUGT42 The gene sequence is shown in SEQ ID NO.1. This sequence contains 1,476 nucleotides, encoding the shown protein, which consists of 491 amino acids, as shown in SEQ ID NO.2.

[0034] Glucosyltransferase SlUGT42 The gene sequence is shown in SEQ ID NO.1:

[0035] Glucosyltransferase SlUGT42 The amino acid sequence is shown in SEQ ID NO.2: MASTGAELDKPHAVCVPYPAQGHINPMLKLAKILNHKGFHITFVNTEYNHRRLLKSRGPHGFKNLPSFRFEAIPDGLPPCDADATQDIIALCKSTDTTCLGPFRELLAKLNNTCSSKVPPVSCIVSDGSNSYTLTAAQELGIPQVHFWTFAACGTLSYMHYCNLVDKGYIPLKDESYLTNGYLEKTTLDWIPGMKDVRLRDLPSFLKTANPDDFMLNFIICKTERSKKYASAIVVNSFEALEKEVLESLQTLVPPVYVIGPLNLLVKHVDDKDLADLGSNLWKEEPKCLEWLDSKKPNSVVYVNFGSITVMTMNHLIEFAWGLANSQLDFLWIIRPDVVLGEEAILPPEFLEETKERGMLASWCQQEKVLNHPAIGGFLTHSGWNSTLESISNGVPMICWPFFAEQPTNCWFCCTKLGIGMEINNNVKRDEVEALVRELMTGEKGKEMKNKATEWKKLAEEAAAKPAGSSYVNIDKLINEILLSPKHSLVK。

[0036] Example 2 In vitro enzyme activity experiment 1. Preparation of crude enzyme solution The pMAL- SlUGT42 ( Figure 1 ) expression vector obtained in Example 1 was transformed into the BL21(DE3) Escherichia coli expression strain (purchased from Shanghai Weidi Biotechnology Co., Ltd., product number EC1002). After overnight culture, 3 mL of the bacterial solution was added to 200 mL of LB liquid medium (containing 50 μg / mL ampicillin), and activated at 37 °C and 200 rpm until the OD 600 value was 0.6 - 0.8. IPTG with a final concentration of 0.3 mM was added for induction, and induction was carried out at 16 °C and 110 rpm for 16 - 20 h. The bacterial cell precipitate was collected by centrifugation for protein disruption to obtain the crude enzyme solution of glycosyltransferase SlUGT42.

[0037] 2. Preparation of purified enzyme solution The crude enzyme solution of glycosyltransferase SlUGT42 obtained by centrifugation was used as the raw material. Take 1 mL of maltose-binding protein-tagged dextrin resin (purchased from Wuhan AmyJet Scientific Co., Ltd., product number BMR2020) and place it in the self-provided gravity column. Wait for the packing liquid to drain completely. First, rinse with deionized water for 3 - 5 column volumes, then elute with 1×PBS (pH 7.2 - 7.4) for 3 - 5 column volumes. Load the crude enzyme solution onto the column and load the sample repeatedly 5 - 6 times. Subsequently, use the impurity washing buffer: 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA-2Na (pH 7.4) to elute the impurity proteins. Take 10 μL of the eluate and observe for impurity proteins in 200 μL of Coomassie Brilliant Blue G-250. Elute until G250 no longer turns blue. Use the elution buffer: 20 mM Tris-HCl, 1 mM EDTA-2Na, 10 mM maltose (pH 7.4) to elute for 3 - 4 column volumes and collect the eluted liquid. Use the same method to detect whether the protein in the eluate is completely eluted with G-250 to obtain the purified protein, that is, the purified enzyme solution.

[0038] Reaction system (200 μL): 1 M Tris-HCl 10 μL (pH 7.5), 100 mg / mL uridine-5′-diphosphate glucose disodium salt 1 μL, 100 mg / mL 2-phenylethanol 1 μL, 50 μg of purified enzyme solution. React at 37 °C for 1.5 h, add 200 μL of methanol to stop the reaction, vortex for 1 min, centrifuge at 12000 rpm for 10 min. Take the supernatant and add it to a 2 mL centrifuge tube, store at -20 °C overnight. The next day, dry it at 30 °C, redissolve with 200 μL of methanol, filter through a 0.22 μm filter membrane, load the sample into a vial, and store at -20 °C waiting for on-machine detection.

[0039] Use the products extracted from the detection reaction for qualitative analysis. The detection and analysis conditions are as follows: Injection volume: 3 μL; Chromatographic column: Eclipse Plus C18 chromatographic column (1.8 µm, 2.1 × 50 mm); Column temperature: 35 °C; Mobile phase: (A): water (containing 0.1% formic acid), (B): methanol, flow rate: 0.3 mL / min.

[0040] The elution program is as follows: 0 min, 95% A, 5% B; 0 - 8 min, A linearly decreases to 80%, B linearly increases to 20%; 8 - 10 min, A linearly decreases to 50%, B linearly increases to 50%; 11 - 14 min, A linearly decreases to 0%, B linearly increases to 100%; 14 - 14.5 min, A returns to the initial 95%, B returns to the initial 5%; 14.5 - 17 min, 95% A, 5% B. The above mass percentages are volume fractions.

[0041] The Q-TOF mass spectrometry parameters are as follows: ESI ion source, positive ion mode, 0 - 450 m / z; the drying gas temperature is 300 °C, the flow rate is 6 L / min; the sheath gas temperature is 300 °C, the flow rate is 11.0 L / min; the nebulizer is 30 psi; VCap is 2000 V; metabolites are detected in the Auto-MS / MS mode; ion fragments are obtained using 20 V collision energy.

[0042] In the product peak of the glucosyltransferase SlUGT42 catalyzing 2-phenylethanol, the product peak with the same retention time as the 2-phenylethyl β-D-glucoside standard has a retention time of 9.495 min ( Figure 2 A); using the ESI + mode, the adduct ion is [M+Na] + , and the molecular formula of 2-phenylethyl β-D-glucoside is C 14 H 20 O6, and the mass-to-charge ratio is 307.1152 ( Figure 2 B), and this product also has the same mass spectrometry fragmentation pattern as 2-phenylethyl β-D-glucoside ([M+Na] + :m / z = 307.1152) ( Figure 2 B). Therefore, it is determined that the glucosyltransferase SlUGT42 has the function of glucosyltransferase that catalyzes 2-phenylethanol to produce 2-phenylethyl β-D-glucoside.

[0043] Example 3 Determination of the Optimal Reaction Conditions of Glucosyltransferase SlUGT42 1. The Optimal Reaction Temperature of Glucosyltransferase SlUGT42 The optimal reaction temperature of the purified protein was determined in vitro according to the following reaction system: 10 μL of 1 M Tris-HCl (pH 7.5), 1 μL of 100 mg / mL uridine-5′-diphosphate glucose disodium salt, 1 μL of 100 mg / mL 2-phenylethanol, 50 μg of purified enzyme solution. The total reaction system was 200 μL in total. The reaction was carried out at reaction temperatures of 30 °C, 37 °C, 42 °C, 50 °C and 60 °C for 1.5 h. Then 200 μL of methanol was added to stop the reaction. After vortexing for 1 min and centrifuging at 12,000 rpm for 10 min, the supernatant was taken and added to a 2 mL centrifuge tube, and left overnight at -20 °C. The next day, after drying at 30 °C, it was redissolved with 200 μL of methanol, filtered through a 0.22 μm filter membrane, and then loaded into a vial for detection. The results are as Figure 3 shown. The activity of this enzyme was strongly inhibited at high temperatures of 50 °C and 60 °C, indicating its characteristic of intolerance to high temperatures. The activity of this enzyme was relatively low at 30 °C and 37 °C, and it had the highest reaction activity at 42 °C.

[0044] 2. Optimal reaction pH of glycosyltransferase SlUGT42 The optimal reaction temperature of the purified protein was determined in vitro according to the following reaction system: 10 μL of 1 M Tris-HCl (pH 4.0, 5.5, 7.0, 7.5, 8.0), 1 μL of 100 mg / mL uridine-5′-diphosphate glucose disodium salt, 1 μL of 1 mg / mL 2-phenylethanol, 50 μg of purified enzyme solution. The total reaction system was 200 μL in total. The reaction was carried out at a reaction temperature of 37 °C for 1.5 h. Then 200 μL of methanol was added to stop the reaction. After vortexing for 1 min and centrifuging at 12,000 rpm for 10 min, the supernatant was taken and added to a 2 mL centrifuge tube, and left overnight at -20 °C. The next day, after drying at 30 °C, it was redissolved with 200 μL of methanol, filtered through a 0.22 μm filter membrane, and then loaded into a vial for detection. As Figure 4 shown, the activity of this enzyme was relatively low at acidic pH 4.0. When transitioning from acidic to neutral pH, the activity of this enzyme increased. This enzyme had the highest reaction activity at pH 7.5. When transitioning to alkaline, the activity of this enzyme decreased.

[0045] It should be noted that the specific embodiments are only relatively representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and there can be many variations. Those of ordinary skill in the art, which are clearly disclosed by the present invention or obtained without any objection according to the written description of the document, should be regarded as the scope protected by this patent.

Claims

1. A glucosyltransferase SlUGT42, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that, It is a nucleotide sequence encoding the glucosyltransferase SlUGT42 as described in claim 1.

3. The nucleic acid molecule according to claim 2, wherein Its nucleotide sequence is shown in SEQ ID NO.

1.

4. The glucosyltransferase SlUGT42 according to claim 1, wherein The glucosyltransferase SlUGT42 is derived from tomato.

5. Use of the glucosyltransferase SlUGT42 as described in claim 1 in catalyzing the production of 2-phenylethyl β-D-glucoside from 2-phenylethanol.

6. The application according to claim 5, wherein The method for catalyzing the production of 2-phenylethyl β-D-glucoside from 2-phenylethanol comprises the following steps: S1. Prepare a purified enzyme solution of the glucosyltransferase SlUGT42 with an amino acid sequence shown in SEQ ID NO.2; S2. Using 2-phenylethanol as a substrate, add uridine-5′-diphosphate glucose disodium salt, and then add the purified enzyme solution prepared in step S1, and carry out a catalytic reaction to obtain 2-phenylethyl β-D-glucoside.

7. The application according to claim 6, characterized in that, The method for preparing the purified enzyme solution in step S1 comprises the following steps: S11. Construct an expression vector carrying the glucosyltransferase SlUGT42 gene; S12. Transform the expression vector into a host cell to obtain a recombinant strain; S13. Induce the expression of the recombinant strain, collect the bacterial cells by centrifugation, take the bacterial cell precipitate for disruption to obtain a crude enzyme solution, and then purify it to obtain a purified enzyme solution.

8. The application according to claim 7, wherein The expression vector is a pMAL vector, and the host cell is Escherichia coli.

9. The application according to claim 6, characterized in that, The catalytic reaction temperature condition in step S1 is 30-60 °C.

10. The application according to claim 6, characterized in that, The catalytic reaction pH in step S1 is 4-8.

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