Glucosyltransferase and preparation method and application of mutant of glucosyltransferase

Abstract

The invention discloses a glucosyltransferase and a preparation method and application of a mutant of the glucosyltransferase. The amino acid sequence of the glucosyltransferase comprises a sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2. The glucosyltransferase and the mutant thereof can efficiently catalyze the generation of the tocopherol glucoside, the enzyme activity can reach 0.34 U / mg, a new way of synthesizing the tocopherol glucoside by a biological method is developed, the glucosyltransferase and the mutant thereof can be widely applied to the fields of medicines, cosmetics and the like, the industrial production of tocopherol derivatives is promoted, and the production cost is reduced. The requirements of the market on high bioavailability and high stability tocopherol derivatives are met, and the technical progress and industrial upgrading of related industries are promoted.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a preparation method and application of a glucose transferase and a mutant thereof. Background Art

[0002] Since 1922, tocopherols have been considered essential nutrients for reproduction. They act as chain-breaking antioxidants, preventing the propagation of free radical reactions. In recent years, they have been widely studied for their anti-aging, anti-cancer, anti-atherosclerotic, anti-cancer and anti-allergic effects. However, tocopherols are lipophilic compounds and poorly absorbed after oral administration, and these shortcomings have hindered the further development of tocopherol pharmacology.

[0003] Glycosylation is a characteristic reaction that converts water-insoluble, unstable aromatic compounds into corresponding water-soluble, stable compounds to improve their bioavailability and pharmacological properties. Therefore, tocopherol glycosides (tocopherol derivatives) are of great significance in the food, pharmaceutical and cosmetic industries. Tocopherol glycosides have been synthesized by chemical methods to improve the water solubility of tocopherol. Tocopherol glycosides currently on the market are basically produced by chemical methods.

[0004] Enzyme-catalyzed reactions have shown excellent environmental friendliness, high efficiency, safety and other advantages, and have received widespread attention. The glycosylation reaction catalyzed by β-glucosidase and amyloglucosidase follows the reaction mechanism of ordinary glycosidases. Since the transglycosylation reaction catalyzed by them is a reversible reaction, the yield of the synthesized transglycosylation product is not very high. Some researchers used β-glucosidase to synthesize α-tocopherol glycosides to 6-O-β-D-pyranoglucoside-α-tocopherol. The maximum conversion rate was only 23%, and the reaction had to be carried out in an organic solvent diisopropyl ether, which limited its application in industrial production.

[0005] Klebsiella pneumoniae can produce extracellular polysaccharides that are secreted into the culture medium. Kei Shimoda used K. pneumoniae as a biocatalyst to catalyze α-tocopherol to produce tocopherol glycosides, but the conversion rate was only 10% in 48 hours, which limited its industrial application. There is no report on the use of transglycosidase to catalyze tocopherol to produce tocopherol glycosides.

[0006] There are many configurations of tocopherol. As a new active ingredient of tocopherol cosmetics, δ-tocopherol has been shown in literature to have better effects in anti-inflammation, anti-ultraviolet and radiation damage, and anti-skin aging compared with traditional α-tocopherol. As a stable δ-tocopherol prodrug, δ-tocopherol-D-pyranoglucoside can be converted into the active ingredient δ-tocopherol in the skin, while the commonly used α-tocopherol acetate cannot be effectively converted under the same conditions, indicating that δ-tocopherol-D-pyranoglucoside can act as a highly efficient new generation of δ-tocopherol cosmetic active ingredient. Plant oils rich in δ-tocopherol show obvious antifungal, anti-allergic, and wound healing enhancement functions. The above functions are of great significance in skin care and deserve further molecular mechanism research. Therefore, exploring the biological production and application of δ-tocopherol-D-pyranoglucoside will be a new trend in the pharmaceutical and cosmetic industries.

[0007] Therefore, there is an urgent need to provide a glucose transferase and a mutant thereof that can overcome the unfriendly effects on production and the environment caused by chemical synthesis and can efficiently catalyze the production of delta-tocopherol-D-pyranoglucoside. Summary of the invention

[0008] In view of the deficiencies in the prior art and actual needs, the present invention provides a method for preparing and applying a glucose transferase and a mutant thereof. A protein capable of catalyzing UGT-glucose and tocopherol to produce glucoside is screened out through AI biological computation, and on this basis, the protein is subjected to codon optimization, protein soluble expression optimization, and high enzyme activity mutation and other modifications to obtain a glucose transferase with a higher expression level, better enzyme activity and more stability, which can efficiently catalyze δ-tocopherol and UGT-glucose to produce δ-tocopherol-D-pyranoglucoside.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a glucose transferase, wherein the amino acid sequence of the glucose transferase comprises the sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0011] SEQ ID NO.1:

[0012] MGSEAHGALHILFFPLMAPGHMLPMVDMAKLFAARGLKPTIFTTPGNVRLIQPSIDNANGSGHQNPIQLFTVPFPSIDGIPDGCENLSSVTQELHVKFFVGLGNLREPFKDSLARLRPDCIVSDMFLPWTADEAAERGMPRLVFHGSNFFTQCAYRSIEHHKVLESLAPGEDTFVIPGLPHRVELRRSTMPDIQTFAPELMDIILQTREAELKSLGTVMNTFYELEPDYADHFCNVMGRRAWNIGPLSLCNKDVADKWRRGNEASITSEFCLNWLDSQEPSSVVYVCFGSLSDFTTAQLREIAAGLEASGRPFVWVVRNHSEEWIPEGLEERVGGKGLIIRGWAPQILILSHVAVGGFLTHCGWNSILEGVSAGLPFVTWPLFADQFYNERLIVDVLKIGVAVGSNEYANKPEERKIIEAAKIEATINELMGDGKDADERREKARALGELAQRAVEKGGSSYRDLDNLISELVEKRSAV。

[0013] SEQ ID NO.2:

[0014] MVVMTTTASRAHIAMFSIAAHGHVNPSLEVIRELVARGHRVSYAVPASFAEKVAATGATPVVYTSTLPTDDDPDAWGTELIDNLEPFLRDAEQALPQLAEAFDRDRPDLVLHDITSYPAPVLAHSWGVPAVSLWPNLVPWEGYEEEVAEPMLAELKASPRGKAYYTRFADWLAEHGIDTDPDRFVARPRRAIVLIPKALQPQADRVDESVYTFVGACQGERAEQGTWQRPADAEKVVLVSLGSAFTKLPGFYRDCVEAFAGLPGWHVVLQIGKFVDPAELGTVPSNVEVRSWVPQLAILRQADGFVTHAGAGGSQEGLATGTPMVAVPQAVDQFGNADMLQSLGVARHLPMDEVTPERLRTAMLALLGDPEVARRAREIQESMAREGGTLRAADLIEAELAGS。

[0015] The present invention can efficiently catalyze the transglycosylation activity of the discovered enzyme by performing specific functional modification on the enzyme, and overcome the unfriendly effects on production and the environment caused by chemical synthesis. The transglycosylation activity of the glucose transferase is increased from 0.24 U / mg to 0.34 U / mg under different rounds of mutation, which is a great improvement, and saves a lot of time and material costs for subsequent scale-up production.

[0016] In a second aspect, the present invention provides a glucose transferase mutant, which undergoes any one or a combination of at least two of the following mutations based on SEQ ID NO.1: H95V, H95L, P126Y, F98L, L291V or L291P; or, the glucose transferase mutant undergoes any one or a combination of at least two of the following mutations based on SEQ ID NO.2: H23F, H23Y, N83F or F87Y.

[0017] In a third aspect, the present invention provides a nucleic acid molecule encoding the glucose transferase described in the first aspect or the glucose transferase mutant described in the second aspect.

[0018] In a fourth aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described in the third aspect.

[0019] In a fifth aspect, the present invention provides a recombinant cell, wherein the recombinant cell contains the nucleic acid molecule described in the third aspect and / or the recombinant vector described in the fourth aspect.

[0020] In a sixth aspect, the present invention provides a method for preparing the glucose transferase mutant according to the second aspect, the preparation method comprising the following steps:

[0021] (1) inserting the nucleic acid sequence of glucose transferase SEQ ID NO.3 or SEQ ID NO.4 into a plasmid to obtain a recombinant plasmid, and using the recombinant plasmid as a template, performing site-directed saturation mutagenesis on any one or a combination of at least two of the 95th, 98th, 126th and 291st sites of the glucose transferase;

[0022] (2) transforming the mutant plasmid obtained in step (1) into a host bacterium, culturing and purifying the host bacterium to obtain the glucose transferase mutant.

[0023] SEQ ID NO.3:

[0024]

[0025] SEQ ID NO.4:

[0026]

[0027] Preferably, the plasmid comprises pET28a.

[0028] Preferably, the host bacteria include Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.

[0029] In a seventh aspect, the present invention provides use of the glucose transferase described in the first aspect and / or the glucose transferase mutant described in the second aspect in catalyzing a glycosyl transfer reaction.

[0030] Preferably, the substrates for the transglycosylation reaction include delta-tocopherol and UDP-glucose.

[0031] In an eighth aspect, the present invention provides a method for preparing delta-tocopherol-D-pyranoglucoside, the preparation method comprising:

[0032] The glucose transferase described in the first aspect and / or the glucose transferase mutant described in the second aspect, delta tocopherol and uridine diphosphate glucose are mixed and reacted, and the product is purified after the reaction to obtain the delta tocopherol-D-pyranoglucoside.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The glucose transferase and its mutants of the present invention can efficiently catalyze the production of tocopherol glucoside, and the enzyme activity can reach 0.34 U / mg;

[0035] (2) The present invention has pioneered a new method for the biological synthesis of tocopherol glucoside, which can be widely used in the fields of medicine, cosmetics, etc., promote the industrial production of tocopherol derivatives, meet the market demand for high bioavailability and high stability tocopherol derivatives, and promote technological progress and industrial upgrading in related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Flowchart of AI computation for new enzyme discovery;

[0037] Figure 2 Calculation diagram for virtual mutation of amino acids around AbUGT substrate molecules;

[0038] Figure 3 Calculation diagram for virtual mutation of amino acids around the selected SvUGT substrate molecule;

[0039] Figure 4 This is a graph of HPLC test results;

[0040] Figure 5 This is the result of the hydrogen spectrum;

[0041] Figure 6This is the structural formula result of the enzyme catalyzed substance detected by hydrogen spectrum. DETAILED DESCRIPTION

[0042] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0043] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0044] 1. The culture medium formula involved in the embodiment is as follows:

[0045] LB liquid medium (1 L): 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, dilute to 1 L with pure water, and sterilize with high-pressure steam at 121°C for 15 min.

[0046] LB solid medium (1 L): Add 15 g agar to the LB liquid medium formula.

[0047] 2YT medium (1 L): 16 g tryptone, 10 g yeast extract, 5 g sodium chloride, dilute to 1 L with pure water, and sterilize with high-pressure steam at 121°C for 15 min.

[0048] 2. Glucotransferase activity detection

[0049] In a 2mL centrifuge tube, add substrate 2mmol / Lδ-tocopherol (dissolved in DMSO), 6mmol / LUDP-glucose, 10ug UGT pure enzyme, 5mmol / LpH7.5 Tris-HCl buffer, and the final volume is 200μL. React at 30℃ for 30min, then add 1mL methanol to terminate the reaction. Centrifuge at 12000r / min for 5min, take the supernatant for HPLC liquid phase detection and analysis, and calculate the enzyme activity. The enzyme activity is defined as: at 30℃, pH7.5, the amount of enzyme that converts 1 micromole of δ-tocopherol glycoside per minute is defined as 1 unit (1U).

[0050] Example 1

[0051] AI computational methods for enzyme discovery and construction of strains expressing genes related to delta-tocopherol-D-pyranoglucopyranoside synthesis.

[0052] 1.1 AI computational methods for enzyme discovery

[0053] Search the protein sequence database Uniprot for all enzymes named as flavonoid glycosyltransferase (a total of 2678, of which 969 are labeled as uridine diphosphate glucuronyl transferase, i.e., UGT enzymes). These enzymes generally have the transglycosylation activity of flavonoid analogs, but it is unknown whether they have the transglycosylation activity of delta tocopherol. The 2678 proteins were predicted by the deep learning algorithm FastAF2 (patent publication number: CN115938468A), and the similarity between the structures was calculated using the TM-align method, and then the K-means clustering method was used to obtain 100 representative structures (the central structure of the cluster). These 100 proteins can be used as representatives of the above-mentioned flavonoid transglycosylation enzymes, thereby reducing the structural space for searching for new enzymes.

[0054] Because UGT enzymes or flavone transglycosylases often require uridine diphosphate glucose (UDP-glucoside) as a sugar donor for sugar transfer, during the enzyme reaction, a three-body complex structure of enzyme-substrate-sugar donor will appear. Enzyme discovery and enzyme modification through this structure will increase the success rate. Therefore, the small molecule template modeling method developed by the company (patent publication number: CN117577224A) was first used to model UDP-D-pyranose glucoside in the above 100 representative proteins, and then AutoDock Vina (pocket size = 20 angstroms, search strength parameter exhaustiveness = 32) was used to dock the substrate molecule δ-tocopherol into the protein (20 states will be generated), and the machine learning algorithm OnionNet-SFCT (patent publication number: CN115881243A) was used to score (weight 0.8), and the state with the lowest score among the 20 docking states was used as the score of the protein. The scores of the 100 proteins were sorted, and the top 20 proteins with the lowest scores were selected for the next step of calculation.

[0055] The 20 proteins with potential transglycosylation activity of delta-tocopherol obtained after structural calculation screening were then used based on the deep learning algorithm CataPro (patent application number: 2024112505195) to calculate the kinetic parameter kcat of enzyme catalytic activity using the SMILES chemical representation of protein sequence and substrate molecule, and the five proteins with the highest predicted kcat (GuUGT, AbUGT, FvUGT, SvUGT, TnUGT60) were selected for experimental testing. The AI ​​calculation process for new enzyme discovery is as follows Figure 1 shown.

[0056] 1.2 Construction of strains expressing genes related to the synthesis of δ-tocopherol-D-pyranoglucopyranoside

[0057] The primers in Table 3 were used for PCR amplification to obtain the linear fragment AbUGT (SEQ ID NO. 3) and the linearized pET28a vector. The amplification system is shown in Table 1, and the amplification program is shown in Table 2.

[0058] Table 1

[0059] HSMix 25μL Primer-F 1μL Primer-R 1μL template 0.5ng <![CDATA[ddH2O]]> To50μL total 50μL

[0060] Table 2

[0061]

[0062] Table 3

[0063]

[0064]

[0065] The linearized pET28a vector and the fragment UGT were connected using a recombination cloning kit (CloneExpressⅡOne Step Cloning Kit, Norwegian CAT#: C112-01), and transformed into competent cells E. coli DH5α (Weidi Biotechnology, CAT#: DL1001) using the calcium chloride chemical transformation method. Positive clones were picked, and plasmids were extracted for sequencing. The recombinant plasmid with the correct sequence was recorded as pET28a-UGT. The correct plasmid pET28a-UGT was transformed into E. coli BL21 (DE3) (Weidi Biotechnology, CAT#: EE1002) by the calcium chloride chemical transformation method, and the recombinant bacteria were recorded as BL21-pET28a-UGT, the target gene promoter was T7 promoter, and the screening marker was kanamycin sulfate (Kana).

[0066] The recombinant strain BL21-pET28a-UGT was inoculated into 2YT medium (containing 50 μg / mL Kana) with a volume of 1 L / 5 L and placed in a shaker at 37 °C and 220 rpm until the bacterial concentration OD 600 =0.8, add IPTG at a final concentration of 1mM, and induce expression at 25°C for 16h. Take 10OD Ecoli-UGT culture solution to a centrifuge bottle, centrifuge at 4500rpm for 20min to collect the bacteria, discard the supernatant, add 5mL 50mmol / L phosphate buffer (pH7.5) to the bacterial pellet to resuspend the bacteria. Use an ultrasonic disruptor to disrupt on ice for 10min (70kHz, ultrasound for 3s, pause for 7s) to obtain cell disruption solution, centrifuge at 12000rpm for 30min, and the supernatant is the crude enzyme solution.

[0067] In a 2mL centrifuge tube, 2mmol / L tocopherol (dissolved in DMSO), 6mmol / L UTP-glucose, 10OD crude enzyme solution, 5mmol / L pH7.5 Tris-HCl buffer were added to a final volume of 200μL, and the reaction was carried out at 30°C for 30min, and then 1mL methanol was added to terminate the reaction. After centrifugation at 12000r / min for 5min, the supernatant was taken for HPLC liquid phase detection analysis. The results are shown in Table 4. Two enzymes with glucose transfection activity were found, of which AbUGT and SvUGT had glucose transfection activity, and AbUGT had higher glucose transfection activity.

[0068] Table 4

[0069]

[0070]

[0071] Example 2

[0072] AI calculation method for enzyme activity modification.

[0073] (1) Starting from wild AbUGT and SvUGT, according to the predicted structure of the enzyme-substrate-UDP-glucose complex, 5 amino acids around the substrate were selected, and each mutant was predicted by Rosetta through virtual saturation mutation. Then, a joint calculation method based on molecular docking and CataPro was used, that is, first by molecular docking and OnionNet-SFCT scoring) to rank a total of 95 (5*19, 19 possibilities for each position) mutation points of the 5 amino acids. 50 mutants with the highest ranking were selected, and kcat prediction based on CataPro was performed. No more than 10 mutants with the best scores were selected, and genetic engineering strains were constructed according to the method in Example 1 (AbUGT mutant primers were constructed with reference to Table 7, and SvUGT mutant primers were constructed with reference to Table 8) and crude enzyme solution was prepared.

[0074] The crude enzyme solution was separated and purified by nickel column affinity chromatography. The filler of the nickel column was HispurTM Ni-NTAResin. The target protein with the His tag would specifically bind to the resin. After washing away the impurities with Wash buffer (50mM Tris-HCl, 150mM NaCl, 10mM imidazole; pH7.5), the protein was eluted with Elution buffer (50mM Tris-HCl, 150mM NaCl, 300mM imidazole; pH7.5). The protein eluate obtained above was centrifuged and concentrated with an Amino Ultra-15 (30kDa) ultrafiltration tube, and the imidazole left over during the protein purification process was washed away with Desalination buffer (50mM Tris-HCl, 150mM NaCl; pH7.5). The resulting protein solution was used for enzyme activity detection, and the results are shown in Table 5.

[0075] Table 5

[0076]

[0077]

[0078] (2) Starting from the wild SvUGT, the enzyme activity of the SvUGT mutant was detected by referring to the method steps in step (1). The results are shown in Table 6.

[0079] Table 6

[0080] Amino acid mutation site Vitality (U / mg) Wild-type SvUGT 0.104 H23F 0.121 H23Y 0.157 W76V 0.108 N83F 0.143 F87 0.129 F87L 0.087 F87I 0.055

[0081] Five amino acids within 6 angstroms around the AbUGT substrate molecule, His95, Phe98, Phe99, Phe126 and Leu291, were selected for virtual mutation, the enzyme catalytic kinetic parameters kcat of all mutations were calculated, and the mutant with the highest prediction score was selected for experimental verification. The results are shown in Figure 2 As shown, all five amino acids (purple) interact with the long chain alkane of the substrate molecule (yellow). AbUGT is green and the sugar donor is indigo.

[0082] From the above mutation screening results, it can be seen that the effects of mutants H95V, H95L, P126Y, F98L, L291V and L291P are better than the wild type, and more products can be generated under the same conditions per unit time.

[0083] We selected four amino acids within 6 angstroms around the SvUGT substrate molecule: His23, Trp76, Asn83, and Phe87, calculated the enzyme catalytic kinetic parameter kcat for all mutations, and selected the mutant with the highest prediction score for experimental verification. Figure 3As shown, all four amino acids (purple) interact with the long chain alkane of the substrate molecule (yellow). SvUGT is green and the sugar donor is indigo.

[0084] From the above mutation screening results, it can be seen that the effects of mutants H23F, H23Y, W76V, N83F and F87 are better than those of wild-type SvUGT, and more products can be generated under the same conditions per unit time.

[0085] Table 7

[0086]

[0087]

[0088] Table 8

[0089]

[0090]

[0091] Example 3

[0092] Isolation, purification and structure determination of delta-tocopherol-D-glucopyranoside.

[0093] 1. Isolation and purification of δ-tocopherol-D-pyranoglucopyranoside

[0094] The mutant reaction solution obtained in Example 2 was centrifuged at 14000 rpm, the supernatant was discarded, 3 volumes of dichloromethane and 3 volumes of water were added, stirred, and allowed to stand for stratification, and the upper layer was discarded. An equal volume of water was added to the lower organic layer for washing, and the washing was repeated three times. The lower organic layer was taken, dried with anhydrous sodium sulfate, and filtered to obtain a crude reaction product.

[0095] The crude reaction product was dissolved with 1 volume of methanol, and an equal amount of silica gel (400 mesh) was added. After stirring evenly, the solvent methanol was removed by rotary evaporation under reduced pressure at 50°C until a fine powder solid with good fluidity was obtained by rotary evaporation. The solid was then evenly added to the column bed. During the process, the dichloromethane layer at the top of the column was kept slightly above the sample layer at the top of the column.

[0096] First, the column was eluted with dichloromethane to wash away the residual substrate in the sample, and the content of δ-tocopherol in the eluent was monitored until the content was low. Then, the column was rinsed with 2 volumes of ethyl acetate-methanol (1:1, V / V) mixed solvent, and when the product was eluted out of the column, the eluent containing the product was collected and combined.

[0097] The above-mentioned eluent was concentrated by rotary evaporation at 50°C under reduced pressure until no solvent was evaporated and the sample was a foamy solid. 1 volume of ethanol and 5 volumes of water were added, and the mixture was heated at 50°C for 1 h. The mixture was filtered and the operation was repeated twice to obtain a white jelly-like substance. The substance was placed in a freeze dryer until it became a white powder.

[0098] 2. Detection and structure determination of delta-tocopherol-D-pyranoglucoside

[0099] 2.1HPLC detection

[0100] The detection method of δ-tocopherol-D-pyranoglucoside was established: δ-tocopherol and δ-tocopherol-D-pyranoglucoside standard samples were dissolved in 100% methanol solution, and the contents of δ-tocopherol and δ-tocopherol-D-pyranoglucoside were detected by high performance liquid chromatography (HPLC).

[0101] The HPLC detector was a 1260 Infinity II liquid chromatography system; the chromatographic column was Hypersil BDS C18 (4.6 mm × 150 mm; 5 μm); the mobile phase was methanol, the time was 12 min; the flow rate was 1.2 mL / min; the detection wavelength was 280 nm; the column temperature was set to 30°C; and the injection volume was 10 μL.

[0102] Through trial and error, a detection method for δ-tocopherol-D-pyranoglucoside was successfully established, in which the absorption peaks of δ-tocopherol are at 6.372 min, and that of δ-tocopherol-D-pyranoglucoside are at 4.459 min, both of which are single strong absorption peaks. The peak position of δ-tocopherol-D-pyranoglucoside is consistent with that of the standard. By making a standard curve for the standard, the content of δ-tocopherol-D-pyranoglucoside can be calculated.

[0103] The results are as follows Figure 4 The glucose transferase mutant of the present invention can successfully catalyze the production of delta-tocopherol-D-glucopyranoside. The results of the content of delta-tocopherol-D-glucopyranoside are shown in Table 9.

[0104] Table 9

[0105]

[0106]

[0107] 2.2 Hydrogen spectrum results

[0108] Detection steps: NMR spectrometer, 500Hz, Bruker brand, Germany. 0.5 ml MeOD was added to the solvent sample, and the sample was scanned 16 times. The scanning time was 90 seconds, the sampling point number Td was 64k, the inspection temperature was 298K, and the NMR spectrum was obtained using topspin 4.0.9 version.

[0109] The results are as follows Figure 5 and Figure 6 As shown, 1H NMR (MeOD, 500 MHz, 300 K): δ6.73 (dd, J = 30.0, 2.9 Hz, 2H), δ4.77 (d, J = 7.6 Hz, 1H), δ3.89 (dd, J = 12.2, 2.2 Hz, 1H), δ3.75 (d dd, J = 12.1, 4.9 Hz, 1H), δ3.55-3.36 (m, 4.9 Hz, 4H), δ2.72 (q, J = 6.2 Hz, 1H), δ2.12 (s, 3H), δ1.77 (ddt, J = 18.1, 13.6, 6.7 Hz, 1H), δ1.63-1.07 (m, 15H), δ0.9 (t, J = 6.7 Hz, 10H), which is consistent with the structure of δ-tocopherol-D-pyranoglucoside.

[0110] Through structural identification, the substance purified by enzymatic reaction is delta-tocopherol glucoside, which can be used as a candidate mutant for industrial application.

[0111] In summary, the glucose transferase and its mutants of the present invention can efficiently catalyze the production of tocopherol glucoside, and the enzyme activity can reach 0.34 U / mg.

[0112] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A glucose transferase, characterized in that The amino acid sequence of the glucose transferase includes the sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A glucose transferase mutant, characterized in that: The glucose transferase mutant has any one or a combination of at least two of the following mutations based on SEQ ID NO.1: H95V, H95L, P126Y, F98L, L291V or L291P; or, The glucose transferase mutant undergoes any one or a combination of at least two of the following mutations based on SEQ ID NO.2: H23F, H23Y, N83F, W76V or F87Y.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the glucose transferase according to claim 1 or the glucose transferase mutant according to claim 2.

4. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule according to claim 3.

5. A recombinant cell, characterized in that The recombinant cell contains the nucleic acid molecule according to claim 3 and / or the recombinant vector according to claim 4.

6. A method for preparing the glucose transferase mutant according to claim 2, characterized in that: The preparation method comprises the following steps: (1) inserting the nucleic acid sequence of glucose transferase SEQ ID NO.3 or SEQ ID NO.4 into a plasmid to obtain a recombinant plasmid, and using the recombinant plasmid as a template, performing site-directed saturation mutagenesis on any one or a combination of at least two of the 95th, 98th, 126th and 291st sites of the glucose transferase; (2) transforming the mutant plasmid obtained in step (1) into a host bacterium, culturing and purifying the host bacterium to obtain the glucose transferase mutant.

7. The method for preparing a glucose transferase mutant according to claim 6, characterized in that: The plasmid includes pET28a; Preferably, the host bacteria include Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.

8. Use of the glucose transferase according to claim 1 and / or the glucose transferase mutant according to claim 2 in catalyzing a glycosyl transfer reaction.

9. The use according to claim 8, characterized in that: The substrates for the transglycosylation reaction include delta-tocopherol and UDP-glucose.

10. A method for preparing delta-tocopherol-D-pyranoglucoside, characterized in that: The preparation method comprises: The glucose transferase according to claim 1 and / or the glucose transferase mutant according to claim 2, delta tocopherol and uridine diphosphate glucose are mixed and reacted, and the product is purified after the reaction to obtain the delta tocopherol-D-pyranoglucoside.

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