WO2023083359A1 - 糖基转移酶OsUGT91C1突变体及其应用 - Google Patents

糖基转移酶OsUGT91C1突变体及其应用 Download PDF

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WO2023083359A1
WO2023083359A1 PCT/CN2022/131774 CN2022131774W WO2023083359A1 WO 2023083359 A1 WO2023083359 A1 WO 2023083359A1 CN 2022131774 W CN2022131774 W CN 2022131774W WO 2023083359 A1 WO2023083359 A1 WO 2023083359A1
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osugt91c1
enzyme
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phe208met
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朱晓峰
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Sichuan University
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    • C12N9/10—Transferases (2.)
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    • C12N9/1051—Hexosyltransferases (2.4.1)
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Definitions

  • the invention belongs to the field of enzymology, and in particular relates to a glycosyltransferase OsUGT91C1 mutant and application thereof.
  • Stevia is a general term for various steviol glycosides. All steviol glycosides have a similar chemical structure pattern, that is, in the general-purpose steviol (Steviol, CAS 471-80-7) core skeleton C13-hydroxyl (for simplicity, also represented by R1 in the accompanying drawings) and (or) C19- The carboxyl group (for simplicity, also represented by R2 in the figure) has different glucosyl modifications (a in Figure 1).
  • stevioside molecules According to the glycosyl composition of C13-hydroxyl and C19-carboxyl and the combination of glycosidic bonds, stevioside molecules can be named, but stevioside (Stevioside, CAS 57817-89-7) accounts for 57 %, represented by ST) and steviol glycoside A (Rebaudioside A, CAS 58543-16-1, the content can reach 32% of stevioside, represented by Reb A), both of them have the disadvantage of bad taste, which affects the quality of stevia Market acceptance of glycosides.
  • steviol glycoside D (Rebaudioside D, CAS 63279-13-0, represented by Reb D) and M (Rebaudioside M, CAS 1220616-44-3, represented by Reb M) are 200-300 times sweeter than sucrose, and the taste is very similar Sucrose, the sweetness standard product, is considered to be the best quality component of steviol glycosides, and it is also the focus of research and development of stevioside manufacturers.
  • Reb D steviol glycoside D
  • M Rebaudioside M, CAS 1220616-44-3, represented by Reb M
  • Both PepsiCo and Coca-Cola have patents for the use of steviol glycosides D and M in beverages.
  • Glucose group of glycosidic bond No. 1 glucosyl group specifically refers to the glucosyl group directly forming a ⁇ -glycosidic bond with C13-hydroxyl or C19-carboxyl group) (b, c, d in Figure 1). Utilize the ability of glycosyltransferase OsUGT91C1 to add No. 2 glucosyl in both directions of C13-hydroxyl and C19-carboxyl of steviol glycosides, to make up for the lack of ability of stevia itself to add No.
  • Reaction known as normal reaction
  • side reaction adding No. 6 glucose group (or No. 4 glucose group) in the direction of C13-hydroxyl group or C19-carboxyl group, and the two form ⁇ (1 -6) Glycosidic bond (or ⁇ (1-4) glycosidic bond)
  • the side reactions mentioned in this patent all refer to the reaction of adding No. 6 glucosyl (or No. 4 glucosyl)) (b, c, d in Figure 1 ).
  • the technical problem to be solved by the present invention is: how to improve the ability of glycosyltransferase OsUGT91C1 to catalyze the C13-hydroxyl or/and C19-carboxyl direction of steviol glycosides to add No. Eliminate side effects.
  • the original amino acid sequence of the glycosyltransferase OsUGT91C1 was rationally optimized.
  • the inventor changed the 208th phenylalanine to methionine (Phe208Met), and obtained a new Enzyme A.
  • the new enzyme A was purified and prepared by the Escherichia coli protein expression system, and the example verified that the new enzyme A can enhance the ability of the glycosyltransferase OsUGT91C1 to add glucosyl No. 2 at the C13-hydroxyl, especially at the C19-carboxyl position.
  • glycosyltransferase OsUGT91C1 mutant its amino acid sequence is any one of the following a, b, c, d:
  • amino acids 1-14 are arbitrarily deleted or changed.
  • the amino acid sequence shown in SEQ ID No.1 is based on the amino acid sequence of glycosyltransferase OsUGT91C1, and the 208th amino acid is mutated from Phe to Met;
  • the amino acid sequence shown in SEQ ID No.2 is based on the amino acid sequence of glycosyltransferase OsUGT91C1
  • the 208th amino acid was mutated from Phe to Met, and the 379th amino acid was mutated from Phe to Ala.
  • An expression vector containing the above-mentioned expression gene.
  • the enzymatic synthesis or conversion process of the glycosyltransferase OsUGT91C1 mutant in steviol glycoside D or M and other steviol glycoside molecules refers to the addition of No. 2 glucose in the direction of C13-hydroxyl or/and C19-carboxyl
  • the No. 2 glucose group is a glucose group that forms a ⁇ (1-2) glycosidic bond with the No. 1 glucose group
  • the No. 1 glucose group specifically refers to a glucose group that directly forms a ⁇ -glycosidic bond with a C13-hydroxyl or C19-carboxyl group .
  • the substrates that catalyze the normal reaction of adding No. 2 glucose group in the C13-hydroxyl or C-19 carboxyl direction are not unique, including but not limited to, Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O- Monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1) and other steviol glycoside substrates, as long as there is No. 1 glucose group in the C13-hydroxyl or C-19 carboxyl direction, but there is no 3 No. glucosyl, the normal reaction of adding No. 2 glucosyl in the direction of C13-hydroxyl or C-19 carboxyl can occur.
  • OsUGT91C1 or the two new enzymes of the present invention in Escherichia coli, it can also be expressed in other biological systems, non-biological systems, cells, cell-free and other expression systems and preparation; these new enzymes that have eliminated side reactions are introduced into organisms, non-organisms, cells, and non-cells (cell-free) to carry out enzymatic conversion of steviol glycosides through transgenes or other gene introduction means, and in enzymes, In vitro systems such as enzyme immobilization are used for the enzymatic conversion of adding No. 2 glucose group to steviol glycosides, including but not limited to the enzymatic conversion process of steviol glycosides D and M, to achieve the purpose of eliminating side reactions.
  • the side reaction of this patent is defined as adding No. 6 glucosyl (or No. 4 glucosyl) to the C13-hydroxyl and C19-carboxyl groups of steviol or steviol glycosides to form ⁇ (1-6) with No. 1 glucosyl Side reaction of glycosidic bond (or ⁇ (1-4) glycosidic bond); the definition of normal reaction in this patent is to add No. The reaction of glucosyl groups to form ⁇ (1-2) glycosidic bonds.
  • Liquid chromatography-mass spectrometry was used to verify that the new enzyme A (Phe208Met) enhanced the ability to catalyze the addition of No. 2 glucose group, especially the ability to add No. 2 glucose group at the C19-carboxyl position.
  • the new enzyme B (Phe208Met/Phe379Ala) enhances the ability to add No. 2 glucosyl group without side reactions.
  • the Mie kinetic parameters of the two new enzyme-catalyzed reactions were determined by liquid chromatography-mass spectrometry and fluorescence conversion methods, and it was verified that the new enzyme B (Phe208Met/Phe379Ala) not only eliminated the addition of No.
  • 6 glucose group formation of ⁇
  • the side reaction of (1-6) glycosidic bond has an enhancement effect on the normal reaction of adding No. 2 glucose group by about 3-7 times, and can participate in the enzymatic conversion of steviol glycosides more efficiently and more specifically.
  • the new enzyme B catalyzes the enzymatic conversion, and the obtained normal product with added No. 2 glucose group does not contain by-products, and can be used for the synthesis of steviol glycosides D and M.
  • the present invention has the following beneficial effects:
  • the invention solves the enzymatic defect of the rice-derived glycosyltransferase OsUGT91C1 in the conversion of steviol glycosides, and at the same time significantly improves the catalytic ability of the target catalytic reaction.
  • the new enzyme B (Phe208Met/Phe379Ala) can more efficiently and specifically complete the normal enzymatic reaction of adding the No. 2 glucose group to steviol glycosides, and is used for conversion to obtain high-quality, high-purity healthy sweeteners steviol glycosides D and M .
  • Figure 1 The structure of some steviol glycosides related to this patent, the simplified representation of the structure and the schematic diagram of the normal and side reactions during the enzymatic conversion of OsUGT91C1 to steviosides D and M
  • steviol glycosides including the structures of steviol, steviol glycosides D and M, and simplified forms of the structures.
  • Steviol is represented by a complete chemical structure
  • steviol glycosides D and M are represented by a complete chemical structure and a simplified diagram, and the sweetness multiples of the two compared with sucrose are shown in brackets.
  • the glucose group is represented by a hexagon, and the 1-hydroxyl group of each glucose is marked with a black dot.
  • the number in the middle of each glucose represents the number of the glucose group, and also indicates the type of glycosidic bond formed between the glucose group and the No. 1 glucose group , such as 2 for ⁇ (1-2) glycosidic bonds, 3 for ⁇ (1-3) glycosidic bonds, 4 for ⁇ (1-4) glycosidic bonds, and 6 for ⁇ (1-6) glycosidic bonds.
  • OsUGT91C1 catalyzes the normal reaction of adding No. 2 glucose group and adding No. 6 glucose group (or No. Schematic diagram of side reactions.
  • the reaction conditions from top to bottom of the liquid phase diagram are as follows: add 0.75 mg/mL enzyme for 0 hour as a control, 0.15 mg/mL enzyme for 2 hours and 18 hours, and 0.75 mg/mL enzyme for 2 hours and 18 hours. It showed that the side reaction products of natural state OsUGT91C1 increased with time and enzyme amount, but the new enzyme B (Phe208Met/Phe379Ala) had no side reaction products.
  • the bottom of a liquid phase diagram is a schematic diagram of OsUGT91C1 catalyzed STB reaction to generate byproducts
  • the bottom of b liquid phase diagram is a schematic diagram of new enzyme B catalyzed STB reaction, no byproducts are generated, and no side reactions occur.
  • the glucosyl product Reb E is also more than the natural state OsUGT91C1; c,d
  • the natural state OsUGT91C1 and the new enzyme B (Phe208Met/Phe379Ala) catalyze the addition of C13-hydroxyl (R1) and C19-carboxyl (R2) ends of the substrate Rubu, respectively No. 2 Glucosyl, a schematic diagram of the reaction to generate Reb E, the new enzyme B (Phe208Met/Phe379Ala) shows a higher catalytic ability of the target.
  • a, b Liquid phase analysis of the reaction of natural state OsUGT91C1, new enzyme B (Phe208Met/Phe379Ala) and Reb A.
  • the reaction conditions from top to bottom of the liquid phase diagram are as follows: add 0.25 mg/mL enzyme for 0 hour as a control, 0.05 mg/mL enzyme for 2 hours and 18 hours, and 0.25 mg/mL enzyme for 2 hours and 18 hours. It shows that the natural state of OsUGT91C1 adds No.
  • the reaction conditions from top to bottom in the liquid phase diagram are as follows: add 0.03mg/mL glycosyltransferase UGT76G1 for 0 hours as a control, 0.03mg/mL glycosyltransferase UGT76G1 for 2 hours and 18 hours, 0.15mg/mL mL glycosyltransferase UGT76G1 was reacted for 2 hours and 18 hours.
  • Liquid phase analysis of natural state OsUGT91C1 (a), new enzyme B truncated body (b) after removing amino acids 1-14, and Rubu reaction.
  • the reaction conditions from top to bottom of the liquid phase diagram are as follows: add 0.25 mg/mL enzyme for 0 hour as a control, 0.05 mg/mL enzyme for 2 hours and 18 hours, and 0.25 mg/mL enzyme for 2 hours and 18 hours. It shows that the products of OsUGT91C1 adding No.
  • this patent considers that as long as the amino acid sequence of the translated protein is 50% or more identical to the amino acid sequence of OsUGT91C1, it is considered to belong to OsUGT91C1.
  • the amino acid sequence of OsUGT91C1 (the amino acid in bold is the amino acid site that was changed in this patent, the underlined part is the amino acid sequence added artificially, and LE is for the convenience of connecting into the E. coli expression vector pET21b, HHHHHH (6 histidine tags ) is for the convenience of later purification):
  • OsUGT91C1 codon-optimized nucleotide sequence (the underlined sequence is the coding region of the artificially added amino acid sequence):
  • Amplification program pre-denaturation at 98°C for 2 min, denaturation at 98°C for 30 s, annealing at 69°C for 30 s, extension at 72°C for 8 min, amplification for 20 cycles, extension at 72°C for 10 min, and storage at 4°C.
  • Amplification program pre-denaturation at 98°C for 2 min, denaturation at 98°C for 30 s, annealing at 68°C for 30 s, extension at 72°C for 8 min, amplification for 20 cycles, extension at 72°C for 10 min, and finally storage at 4°C.
  • the eluted protein is further replaced into 20mM HEPES buffer pH 7.2, 50mM NaCl, and can be stored at -80°C for a long time after quick freezing in liquid nitrogen.
  • the above steps can be purified to obtain OsUGT91C1, new enzyme A, and new enzyme B in the natural state, and the purity is detected by SDS-PAGE ( FIG. 2 ).
  • the natural state of OsUGT91C1 can add No. 2 glucose group (with Glucosyl No. 1 forms a ⁇ (1-2) glycosidic bond) (normal reaction) (a in Figure 3), and the new enzyme A (Phe208Met) can also be added in two directions of the C13-hydroxyl and C19-carboxyl of the substrate Rubu No. 2 glucosyl, and can catalyze more products that add No.
  • the reaction system of the embodiment is as follows: at 20°C-40°C, the 200 ⁇ L reaction system includes: 1mM UDP-Glucose, 20mM Tris-HCl buffer pH 7.2, the concentrations are 0.05mg/mL (1 ⁇ ) and 0.25mg/mL ( 5 ⁇ ) enzyme samples (native state or new enzyme A (Phe208Met)), 0.3 mM substrate Rubu.
  • the natural state OsUGT91C1 and the new enzyme A catalyze the addition of No. 2 glucose group in the C13-hydroxyl or C-19 carboxyl direction.
  • the substrates of the normal reaction are not unique, including but not limited to, Rubu (Rubusoside, CAS 64849-39- 4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1) and other steviol glycoside substrates, as long as the C13-hydroxyl or C-19 carboxyl There is No. 1 glucosyl in the direction, but there is no No. 3 glucosyl, and the normal reaction of adding No. 2 glucosyl in the direction of C13-hydroxyl or C-19 carboxyl can occur.
  • the reaction system of the embodiment is as follows: at 20°C-40°C, the 200 ⁇ L reaction system includes: 1mM UDP-Glucose, 20mM Tris-HCl buffer pH 7.2, the concentrations are 0.05mg/mL (1 ⁇ ) and 0.25mg/mL ( 5 ⁇ ) enzyme sample (native state or new enzyme A (Phe208Met)), 0.3mM substrate Reb A.
  • the natural state OsUGT91C1 and the new enzyme A catalyze the addition of No. 2 glucose group in the direction of C13-hydroxyl or C-19 carboxyl.
  • S13G Steviol-13-O-monoglucoside, CAS 60129-60-4
  • Reb A Rebaudioside A, CAS 58543-16-1
  • other steviol glycoside substrates as long as there is 1 in the C13-hydroxyl or C-19 carboxyl direction No. glucosyl, but without the presence of No. 3 glucosyl, the normal reaction of adding No. 2 glucosyl in the direction of C13-hydroxyl or C-19 carboxyl can occur.
  • the natural state of OsUGT91C1 can catalyze the side reaction of adding No. 6 glucosyl (or No. 4 glucosyl) on the substrate STB respectively (A in Fig. 5), the side reaction product of OsUGT91C1 in the natural state increases with time and the increase of enzyme amount, while the new enzyme B (Phe208Met/Phe379Ala) has no side reaction product generation, and the addition of No. 6 glucose group (or 4 No. glucosyl) side reaction ( Figure 5 b).
  • reaction system includes: 1mM UDP-Glucose, 20mM Tris-HCl buffer pH 7.2, the concentrations are 0.15mg/mL (1 ⁇ ) and 0.75mg/mL ( 5 ⁇ ) enzyme sample (native state or new enzyme B (Phe208Met/Phe379Ala)), 0.3mM STB.
  • the natural OsUGT91C1 catalyzes side reactions that are not unique, including but not limited to side reactions to the following steviol glycoside substrates, STB (Steviolbioside, CAS 41093-60-1), Reb E (Rebaudioside E, CAS 63279-14- 1), etc., as long as there are No. 1 and No. 2 glucosyl groups in the direction of C13-hydroxyl or C-19 carboxyl, but there is no No. 3 glucosyl (No. 3 glucosyl refers to the formation of ⁇ (1-3) glycosides with No. 1 glucosyl Glucosyl of the bond), the side reaction of adding No. 6 Glucosyl (or No. 4 Glucosyl) in the corresponding C13-hydroxyl or C-19 carboxyl direction can occur.
  • reaction conditions of the embodiment are as follows: at 20°C-40°C, the 200 ⁇ L reaction system includes: 1mM UDP-Glucose, 20mM Tris-HCl buffer pH 7.2, the concentrations are 0.15mg/mL (1 ⁇ ) and 0.75mg/mL ( 5 ⁇ ) enzyme samples (native state or new enzyme B (Phe208Met/Phe379Ala)), 0.3 mM substrate S13G.
  • the natural state OsUGT91C1 and the new enzyme B catalyze the addition of No. 2 glucose group in the direction of C13-hydroxyl or C-19 carboxyl. 39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1) and other steviol glycoside substrates, as long as the C13-hydroxyl or C- There is No. 1 glucose group in the 19 carboxyl group, but there is no No. 3 glucose group, so the normal reaction of adding No.
  • the natural state of OsUGT91C1 can add No. 2 glucose group (with Glucosyl No. 1 forms ⁇ (1-2) glycosidic bond) (normal reaction) (a in Figure 7), and the new enzyme B (Phe208Met/Phe379Ala) can also be used in the C13-hydroxyl and C19-carboxyl groups of the substrate Rubu Adding No. 2 Glucose in the direction, and can catalyze more products that add No.
  • the reaction system of the embodiment is as follows: at 20°C-40°C, the 200 ⁇ L reaction system includes: 1mM UDP-Glucose, 20mM Tris-HCl buffer pH 7.2, the concentrations are 0.05mg/mL (1 ⁇ ) and 0.25mg/mL ( 5 ⁇ ) enzyme samples (native state or new enzyme B (Phe208Met/Phe379Ala)), 0.3 mM substrate Rubu.
  • the natural state OsUGT91C1 and the new enzyme B catalyze the addition of No. 2 glucose group in the direction of C13-hydroxyl or C-19 carboxyl. 39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1) and other steviol glycoside substrates, as long as the C13-hydroxyl or C- There is No. 1 glucose group in the 19 carboxyl direction, but there is no No. 3 glucose group, and the normal reaction of adding No. 2 glucose group in the C13-hydroxyl or C-19 carboxyl direction can occur.
  • the reaction system of the embodiment is as follows: at 20°C-40°C, the 200 ⁇ L reaction system includes: 1mM UDP-Glucose, 20mM Tris-HCl buffer pH 7.2, the concentrations are 0.05mg/mL (1 ⁇ ) and 0.25mg/mL ( 5 ⁇ ) enzyme samples (native state or new enzyme B (Phe208Met/Phe379Ala)), 0.3mM substrate Reb A.
  • the natural state OsUGT91C1 and the new enzyme B catalyze the addition of No. 2 glucose group in the direction of C13-hydroxyl or C-19 carboxyl. 39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1) and other steviol glycoside substrates, as long as the C13-hydroxyl or C- There is No. 1 glucose group in the 19 carboxyl direction, but there is no No. 3 glucose group, and the normal reaction of adding No. 2 glucose group in the C13-hydroxyl or C-19 carboxyl direction can occur.
  • the new enzyme A (Phe208Met) and the new enzyme B (Phe208Met/Phe379Ala) catalyzed the addition of No. 2 glucose group to the steviol glycoside substrate (formation of ⁇ (1-2) glycosidic bond with No. 1 glucose group) ) the speed of the normal response.
  • the new enzyme A (Phe208Met) enhances the reaction speed of the addition of the 2nd glucose group (with the 1st glucose group to form a ⁇ (1-2) glycosidic bond) on the steviol glycoside substrate required for normal catalysis, which is the natural state OsUGT91C1 active More than 2 times; while the new enzyme B (Phe208Met/Phe379Ala) eliminates the side reaction of adding No. 6 (or No. 4) glucosyl, it enhances the addition of No. 2 glucosyl (with No. 1) to the steviol glycoside substrate required for normal catalysis
  • the reaction speed of glucosyl to form ⁇ (1-2) glycosidic bond) is 3-7 times of the activity of natural state OsUGT91C1 (Table 3).
  • the determination is directly completed using a commercially available glycosyltransferase activity kit.
  • the example uses the UDP-Glo TM Glycosyltransferase Assay kit from Promega Company. Other methods that can be used to detect the reaction speed of glycosyltransferase can obtain the same results.
  • the UDP-Glo TM Glycosyltransferase Assay kit from Promega is used to detect the reaction speed of glucosyltransferase with UDP-Glucose as the glycosyl donor, OsUGT91C1 and the two mutant new enzymes involved in the present invention are applicable.
  • the glycosyl donor UDP-Glucose transfers glucose to the substrate to generate UDP.
  • This detection method converts the amount of UDP and other substances (1:1) into ATP, and ATP can make luciferase emit quantitative fluorescence, so the amount of UDP generated by enzymatic glycosyl transfer reaction can be detected by measuring the amount of fluorescence.
  • the fluorescence intensity generated is linearly related to the molar concentration of UDP. According to the standard curve of the corresponding relationship between UDP concentration and fluorescence intensity, the generated amount of UDP can be calculated, which can be converted into the catalytic reaction speed of glycosyltransferase.
  • the substrates of the normal reaction are not unique, including but not limited to, Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1) and other steviol glycoside substrates, as long as There is No. 1 glucose group in the direction of C13-hydroxyl or C-19 carboxyl, but there is no No. 3 glucose group, and the normal reaction of adding No. 2 glucose group in the direction of C13-hydroxyl or C-19 carboxyl can occur.
  • the new enzyme B (Phe208Met/Phe379Ala) has no side reactions and improves the target catalytic ability, which can catalyze the addition of No. 2 glucose group in the direction of the C13-hydroxyl or (and) C-19 carboxyl of the steviol glycoside substrate. reaction to generate a series of steviol glycoside products including Reb E (Rebaudioside E, CAS 63279-14-1). Under these conditions, use glycosyltransferase UGT76G1 to continue to add No.
  • new enzyme B (Phe208Met/Phe379Ala) as an example, remove all amino acids (MDSGYSSSYAAAAG) at positions 1-14 of new enzyme B to obtain a truncated body of new enzyme B, which can still be expressed and purified normally, and shows It shows the activity of the normal reaction of adding No. 2 glucose group in the direction of C13-hydroxyl or C-19 carboxyl of steviol glycoside substrate.
  • Amplification program pre-denaturation at 98°C for 2 min, denaturation at 98°C for 30 s, annealing at 69°C for 30 s, extension at 72°C for 8 min, amplification for 20 cycles, extension at 72°C for 10 min, and storage at 4°C.
  • the eluted protein is further replaced into 20mM HEPES buffer pH 7.2, 50mM NaCl, and can be stored at -80°C for a long time after quick freezing in liquid nitrogen.
  • the above steps can be used to purify the truncated body of Novel Enzyme B, and the purity can be checked by SDS-PAGE ( FIG. 10 ). It can be seen from Figure 10 that when the new enzyme B removes the truncated form of amino acids 1-14, it can be expressed and purified normally. Since the new enzyme A and the new enzyme B have the same 1-14 amino acids, the embodiment of the new enzyme B completely removing the 1-14 amino acids shows that the 1-14 amino acids are redundant. Similarly, amino acids 1-14 of the new enzyme A also have redundancy.
  • the truncation of the new enzyme B does not affect the normal reaction of adding the second glucose group in the two directions of the C13-hydroxyl and C-19 carboxyl groups of the steviol glycoside substrate. Its activity is the same as that of the new enzyme B, and it is better than the natural transformation
  • the catalytic activity of glycosyltransferase OsUGT91C1 was higher, further showing the redundancy of amino acids 1-14 in new enzyme A and new enzyme B.
  • the natural state OsUGT91C1 can add No. 2 glucosyl (with No. 1 glucose group forms ⁇ (1-2) glycosidic bond) (normal reaction) (a in Figure 11), the truncated body of the new enzyme B can also be added in two directions of the C13-hydroxyl and C19-carboxyl of the substrate Rubu Glucose No. 2 (b in Figure 11).
  • reaction system of the example is as follows: at 20°C-40°C, the 200 ⁇ L reaction system includes: 1mM UDP-Glucose, 20mM Tris-HCl buffer pH 7.2, the concentrations are 0.05mg/mL (1 ⁇ ) and 0.25mg /mL (5 ⁇ ) of enzyme sample (native state or new enzyme B truncation), 0.3 mM substrate Rubu.
  • the natural state OsUGT91C1 and the new enzyme B truncated body catalyze the addition of No. 2 glucose group in the C13-hydroxyl or C-19 carboxyl direction.
  • the substrates of the normal reaction are not unique, including but not limited to, Rubu (Rubusoside, CAS 64849 -39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS58543-16-1) and other steviol glycoside substrates, as long as the C13-hydroxyl or C- There is No. 1 glucose group in the 19 carboxyl direction, but there is no No. 3 glucose group, and the normal reaction of adding No. 2 glucose group in the C13-hydroxyl or C-19 carboxyl direction can occur.

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Abstract

提供了糖基转移酶OsUGT91C1突变体及其应用，SEQ ID No.1所示氨基酸序列是在糖基转移酶OsUGT91C1氨基酸序列的基础上将第208位氨基酸由Phe突变为Met；SEQ ID No.2所示氨基酸序列是在糖基转移酶OsUGT91C1氨基酸序列的基础上将第208位氨基酸由Phe突变为Met，且第379位氨基酸由Phe突变为Ala，其中上述两个突变体的第1-14位氨基酸具有冗余性，可以完全去除或改变而不影响正常酶活性。该突变体，提高了催化相同正常反应的能力，进一步去除了OsUGT91C1原始酶所存在的副反应劣势，可催化生成包括Reb E的一系列甜菊糖苷产物。

Description

糖基转移酶OsUGT91C1突变体及其应用 技术领域
本发明属于酶学领域，具体涉及一种糖基转移酶OsUGT91C1突变体及其应用。
背景技术
近年来高热量糖的危害逐步得到重视，但由于甜味可刺激产生内源性阿片、多巴胺等奖励性神经递质，使人难于戒除对甜味的依赖。几种量产的人造甜味剂虽可满足热量低的需求，但口感、安全性和心理接受度仍存在争议，难以有效替代传统高热量甜味剂。原产于南美的甜叶菊在当地作为天然甜味来源已有超过百年的历史。2006年世界卫生组织(WHO)和联合国粮农组织(FAO)联合食品添加剂专用委员会(JECFA)通过了甜菊糖健康无害的安全评价。2008年美国FDA正式批准允许甜菊糖在食品中使用。2011年甜菊糖取得了欧洲的食品安全局安全性检验证书。随着消费者对低糖、低热量的食品和饮料的需求持续增长，甜菊糖已成为全球使用量增长最快的天然甜味剂之一。
甜菊糖是多种甜菊糖苷的总称。所有甜菊糖苷具有相似的化学结构模式，即在通用的甜菊醇(Steviol，CAS 471-80-7)核心骨架C13-羟基(为了简化，在附图中也用R1代表)和(或)C19-羧基(为了简化，在附图中也用R2代表)存在不同的葡萄糖基修饰(图1中a)。根据C13-羟基和C19-羧基的糖基组成和糖苷键的组合情况，可对甜菊糖苷分子进行命名，但目前市场上的甜菊糖产品以甜菊苷(Stevioside，CAS 57817-89-7占比57％，用ST表示)和甜菊糖苷A(Rebaudioside A，CAS 58543-16-1，含量可达甜菊糖苷的32％，用Reb A表示)为代表，二者存在口感不佳的劣势，影响了甜菊糖苷的市场接受度。研究发现甜菊糖苷D(Rebaudioside D，CAS 63279-13-0，Reb D代表)和M(Rebaudioside M，CAS 1220616-44-3，Reb M代表)甜度是蔗糖的200-300倍，口感非常接近于甜味标准品蔗糖，被认为是甜菊糖苷品质最好的组分，也是甜菊糖苷生产商研发的重点。百事可乐和可口可乐都获得了在饮料中使用甜菊糖苷D和M的专利。
然而，甜菊糖苷D和M在天然甜叶菊叶片中的含量低于1％，难于直接从天然种植的甜叶菊提取。由于含量低，从甜叶菊提取过程繁琐，成本高，限制了口感最好的甜菊糖苷D和M的市场化应用。研究表明甜叶菊自身缺少在甜菊糖苷的C19-羧基方向添加2号葡萄糖基的催化能力，不能有效合成Reb D和Reb M，导致了二者天然含量低的现象；同时Reb D和Reb M在C19-羧基方向的2号葡萄糖基，使二者同其他甜菊糖苷相比，口感得以显著提升的原因。因此，利用异源酶促转化的方法弥补甜叶菊催化在甜菊糖苷的C19-羧基方向添 加2号葡萄糖基的能力不足，是规模化制备甜菊糖苷D和M的解决手段。一种水稻来源的糖基转移酶OsUGT91C1(也可称为EUGT11)，可催化在甜菊糖苷的C13-羟基和C19-羧基方向添加2号葡萄糖基(2号葡萄糖基是与1号葡萄糖基形成β(1-2)糖苷键的葡萄糖基，1号葡萄糖基特指直接与C13-羟基或C19-羧基形成β-糖苷键的葡萄糖基)(图1中b，c，d)。利用糖基转移酶OsUGT91C1在甜菊糖苷的C13-羟基和C19-羧基两个方向都可添加2号葡萄糖基的能力，弥补甜叶菊自身在甜菊糖苷的C19-羧基方向添加2号葡萄糖基能力的不足，是能否规模化生产甜菊糖苷D和M的决定因素。通过包含糖基转移酶OsUGT91C1的一系列酶促反应，在生物体内(不一定是甜叶菊)或生物体外利用前体物质进行甜菊糖苷的全合成转化，或通过酶促反应对甜叶菊天然提取物，特别是在自然含量较为丰富的甜菊苷(ST)、甜菊糖苷A(Reb A)或其他可利用的甜菊糖苷分子的特定位置添加葡萄糖基，转化生成甜菊糖苷D和M(Reb D和Reb M)。
由于在甜菊糖苷的C19-羧基方向添加2号葡萄糖基是生成Reb D和Reb M的关键，发明人希望能进一步提高糖基转移酶OsUGT91C1在甜菊糖苷C19-羧基方向催化添加2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)的能力，用于更加高效地转化生成甜菊糖苷D和M。同时，发明人发现糖基转移酶OsUGT91C1在催化甜菊糖苷D、M酶促转化过程中的特异性不好，除了添加2号葡萄糖基外(这是酶促转化生成甜菊糖苷D、M所需的反应，称为正常反应)，还存在明显的副反应，即在C13-羟基方向或C19-羧基方向添加6号葡萄糖基(或4号葡萄糖基)，二者与1号葡萄糖基形成β(1-6)糖苷键(或β(1-4)糖苷键)(本专利提及的副反应均指添加6号葡萄糖基(或4号葡萄糖基)的反应)(图1中b，c，d)。副反应将浪费酶催化资源和底物，形成明显的副产物，既影响了甜菊糖苷D和M的生成效率，还带来杂质，给获得高品质甜菊糖苷D、M纯品带来很大困难。
发明内容
本发明所要解决的技术问题为：如何提高糖基转移酶OsUGT91C1在催化甜菊糖苷的C13-羟基或/和C19-羧基方向添加2号葡萄糖基的能力，另一方面，在提高催化能力的前提下消除副反应。
基于发明人独立发现的糖基转移酶OsUGT91C1三维空间结构，理性优化糖基转移酶OsUGT91C1原有氨基酸序列，发明人将第208位的苯丙氨酸改为甲硫氨酸(Phe208Met)，得到新酶A。通过大肠杆菌蛋白表达系统，纯化制备新酶A，实施例验证新酶A可增强糖基转移酶OsUGT91C1在C13-羟基，特别是在C19-羧基位添加2号葡萄糖基的能力。
经前期研究发现，通过改变糖基转移酶OsUGT91C1第379位的苯丙氨酸为丙氨酸 (Phe379Ala)，可完全消除副反应。发明人综合新酶A(Phe208Met)催化能力得到增强的特点，组合Phe208Met和Phe379Ala两个位点的氨基酸改变，成功构建了一个既消除副反应又能增强目标催化能力(添加2号葡萄糖基)的新酶B(Phe208Met/Phe379Ala)。验证了新酶B(Phe208Met/Phe379Ala)既消除了添加6号葡萄糖基(形成β(1-6)糖苷键)的副反应，又对添加2号葡萄糖基的正常反应有约3-7倍的增强作用，可更高效和更加专一地参与甜菊糖苷的酶促转化。
进一步地研究发现，在上述突变的基础上，删除或改变糖基转移酶OsUGT91C1前端1-14个氨基酸(MDSGYSSSYAAAAG)均不影响突变后的酶性质。说明前端的14个氨基酸具有冗余性，可以完全去除或改变。
本发明的技术方案为：糖基转移酶OsUGT91C1突变体，其氨基酸序列为以下a、b、c、d中的任一种：
a、SEQ ID No.1所示；
b、SEQ ID No.2所示；
c、SEQ ID No.1所示氨基酸序列的基础上任意删除或改变第1-14位氨基酸；
d、SEQ ID No.2所示氨基酸序列的基础上任意删除或改变第1-14位氨基酸。
SEQ ID No.1所示氨基酸序列是在糖基转移酶OsUGT91C1氨基酸序列的基础上将第208位氨基酸由Phe突变为Met；SEQ ID No.2所示氨基酸序列是在糖基转移酶OsUGT91C1氨基酸序列的基础上将第208位氨基酸由Phe突变为Met，且第379位氨基酸由Phe突变为Ala。
一种表达基因，编码上述所述的糖基转移酶OsUGT91C1突变体。
一种表达载体，含有上述所述表达基因。
携带上述所述表达载体的重组菌或细胞。
上述所述的糖基转移酶OsUGT91C1突变体在甜菊糖苷D或M及其他甜菊糖苷分子的酶促合成或转化过程的用途。
进一步地，所述糖基转移酶OsUGT91C1突变体在甜菊糖苷D或M及其他甜菊糖苷分子的酶促合成或转化过程是指在甜菊糖苷的C13-羟基或/和C19-羧基方向添加2号葡萄糖基，所述2号葡萄糖基是与1号葡萄糖基形成β(1-2)糖苷键的葡萄糖基，1号葡萄糖基特指直接与C13-羟基或C19-羧基形成β-糖苷键的葡萄糖基。
催化在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应的底物不唯一，包括但不限于，Rubu(Rubusoside，CAS 64849-39-4)、S13G(Steviol-13-O-monoglucoside，CAS  60129-60-4)及Reb A(Rebaudioside A,CAS 58543-16-1)等甜菊糖苷底物，只要在C13-羟基或C-19羧基方向存在1号葡萄糖基，但不存在3号葡萄糖基，就可发生在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应。
除了可在大肠杆菌中表达、纯化、制备天然状态OsUGT91C1或本发明的两个新酶，还可在其他生物系统、非生物系统、细胞、非细胞(Cell-free)等其他表达体系中进行表达和制备；将这些消除了副反应的新酶，通过转基因或其他基因导入手段，引入生物体、非生物体、细胞、非细胞(cell-free)进行甜菊糖苷的酶促转化，以及在酶、酶固定化等体外体系，用于在甜菊糖苷添加2号葡萄糖基的酶促转化，包括制备但不限于甜菊糖苷D和M的酶促转化过程，达到消除副反应的目的。
本专利的副反应定义为，在甜菊醇或甜菊糖苷的C13-羟基，C19-羧基两个方向添加6号葡萄糖基(或4号葡萄糖基)，与1号葡萄糖基形成β(1-6)糖苷键(或β(1-4)糖苷键)的副反应；本专利的正常反应的定义是在甜菊醇或甜菊糖苷的C13-羟基，C19-羧基两个方向添加2号葡萄糖基，与1号葡萄糖基形成β(1-2)糖苷键的反应。
利用了液相色谱-质谱联用验证了新酶A(Phe208Met)增强了催化添加2号葡萄糖基，特别是在C19-羧基位添加2号葡萄糖基的能力。新酶B(Phe208Met/Phe379Ala)在增强添加2号葡萄糖基能力的同时，无副反应的出现。通过液相色谱-质谱联用、荧光转化法等方法测定了两个新酶催化反应的米氏动力学参数，验证了新酶B(Phe208Met/Phe379Ala)既消除了添加6号葡萄糖基(形成β(1-6)糖苷键)的副反应，又对添加2号葡萄糖基的正常反应有约3-7倍的增强作用，可更高效和更加专一地参与甜菊糖苷的酶促转化。新酶B催化酶促转化，得到的添加了2号葡萄糖基的正常产物不含副产物，可用于合成甜菊糖苷D和M。
与现有技术相比，本发明具有以下有益效果：
本发明解决了水稻来源的糖基转移酶OsUGT91C1在甜菊糖苷转化中的酶学缺陷，同时显著提升了对目标催化反应的催化能力。新酶B(Phe208Met/Phe379Ala)可更为高效和专一地完成对甜菊糖苷添加2号葡萄糖基的正常酶促反应，用于转化获得高品质、高纯度的健康甜味剂甜菊糖苷D和M。
附图说明
图1与本专利相关的部分甜菊糖苷结构，结构的简化表示及OsUGT91C1酶促转化生成甜菊糖苷D和M过程中正常反应和副反应的示意图
a部分甜菊糖苷的结构，包括甜菊醇、甜菊糖苷D和M的结构及结构的简化形式。甜 菊醇用完整的化学结构表示，甜菊糖苷D和M用完整的化学结构和简化图两种方法表示，括号内为二者相比于蔗糖的甜度倍数。简化图中C13-羟基(用R1简化表示)，C19-羧基(用R2简化表示)。葡萄糖基用六边形表示，每个葡萄糖的1-羟基用黑点标注，每个葡萄糖中间的数字代表是几号葡萄糖基，同时也表示该葡萄糖基与1号葡萄糖基形成的糖苷键的类型，如2代表β(1-2)糖苷键，3代表β(1-3)糖苷键，4代表β(1-4)糖苷键，6代表β(1-6)糖苷键。
b OsUGT91C1在甜菊糖苷底物C13-羟基(用R1简化表示)或(和)C19-羧基(用R2简化表示)两端催化添加2号葡萄糖基正常反应及添加6号葡萄糖基(或4号葡萄糖基)副反应的示意图。
c OsUGT91C1在甜菊糖苷底物C13-羟基(用R1简化表示)催化添加2号葡萄糖基正常反应及添加6号葡萄糖基(或4号葡萄糖基)副反应的示意图。
d OsUGT91C1在甜菊糖苷底物C19-羧基(用R2简化表示)催化添加2号葡萄糖基正常反应及添加6号葡萄糖基(或4号葡萄糖基)副反应的示意图。
图2新酶A(Phe208Met)、天然状态OsUGT91C1、新酶B(Phe208Met/Phe379Ala)SDS-PAGE分析
a新酶A(Phe208Met)的SDS-PAGE分析。M,Marker；1，诱导后样品；2，菌体裂解液；3，菌体裂解液上清；4，菌体裂解液沉淀；5，Ni柱流穿样品；6-12，Ni柱洗脱蛋白样品。
b天然状态OsUGT91C1和新酶B(Phe208Met/Phe379Ala)的SDS-PAGE分析。M,Marker；WT，天然状态OsUGT91C1；2，Q柱流穿样品；3-7，Q柱洗脱蛋白样品。两个图右侧箭头表示目的蛋白条带的位置。
图3天然状态OsUGT91C1和新酶A(Phe208Met)催化在底物Rubu的C13-羟基(R1)和C19-羧基(R2)两端添加2号葡萄糖基，形成β(1-2)糖苷键正常酶促反应的对比
a,b天然状态OsUGT91C1、新酶A(Phe208Met)与Rubu反应的液相分析。液相图从上至下的反应条件依次为：加0.25mg/mL酶反应0小时作为对照，0.05mg/mL酶反应2小时和18小时，0.25mg/mL酶反应2小时和18小时。显示天然状态的OsUGT91C1在Rubu的C13-羟基(R1)和C19-羧基(R2)端分别添加2号葡萄糖基的产物随时间和酶量的增加而增多，新酶A(Phe208Met)同样可在底物Rubu的C13-羟基、C19-羧基两个方向添加2号葡萄糖基，而且可催化得到更多的在C19-羧基方向添加2号葡萄糖基的产物，具有增强的在C19-羧基方向的目标催化能力，最终得到在C13-羟基和C19-羧基两个方向都加有2号 葡萄糖基的产物Reb E也多于天然状态的OsUGT91C1；c,d天然状态OsUGT91C1和新酶A(Phe208Met)催化底物Rubu的C13-羟基(R1)和C19-羧基(R2)端分别添加2号葡萄糖基，生成Reb E的反应示意图，新酶A(Phe208Met)显示具有在C19-羧基方向增强的目标催化能力。
图4天然状态OsUGT91C1和新酶A(Phe208Met)催化在底物Reb A的C19-羧基(R2)端添加2号葡萄糖基，形成β(1-2)糖苷键正常酶促反应的对比
a,b天然状态OsUGT91C1、新酶A(Phe208Met)与Reb A反应的液相分析。液相图从上至下的反应条件依次为：加0.25mg/mL酶反应0小时作为对照，0.05mg/mL酶反应2小时和18小时，0.25mg/mL酶反应2小时和18小时。显示天然状态的OsUGT91C1在Reb A的C19-羧基(R2)端添加2号葡萄糖基，生成产物Reb D随时间和酶量的增加而增多，而新酶A(Phe208Met)可明显率先将底物Reb A完全消耗并生成目标产物Reb D，具有增强的在C19-羧基方向的目标催化能力；c,d天然状态OsUGT91C1和新酶A(Phe208Met)催化底物Reb A的C19-羧基(R2)端添加2号葡萄糖基，生成Reb D的反应示意图，新酶A(Phe208Met)该反应得到增强。
图5天然状态OsUGT91C1和新酶B(Phe208Met/Phe379Ala)在底物STB的C13-羟基(R1)端添加6号(或4号)葡萄糖基副反应的情况对比
天然状态的OsUGT91C1(a)、新酶B(Phe208Met/Phe379Ala)(b)与底物STB反应的液相分析。液相图从上至下的反应条件依次为：加0.75mg/mL酶反应0小时作为对照，0.15mg/mL酶反应2小时和18小时，0.75mg/mL酶反应2小时和18小时。显示天然状态的OsUGT91C1副反应产物随时间和酶量的增加而增多，而新酶B(Phe208Met/Phe379Ala)无副反应产物的生成。a液相图下方为OsUGT91C1催化STB反应生成副产物的示意图，b液相图下方为新酶B催化STB反应示意图，无副产物生成，无副反应的发生。
图6天然状态OsUGT91C1和新酶B(Phe208Met/Phe379Ala)催化在底物S13G的C13-羟基(R1)端添加2号葡萄糖基，形成β(1-2)糖苷键正常酶促反应，以及随后发生添加6号(或4号)葡萄糖基副反应的情况对比
天然状态的OsUGT91C1(a)、新酶B(Phe208Met/Phe379Ala)(b)与底物S13G反应的液相分析。液相图从上至下的反应条件依次为：加0.75mg/mL酶反应0小时作为对照，0.15mg/mL酶反应2小时和18小时，0.75mg/mL酶反应2小时和18小时。显示天然状态的OsUGT91C1先催化在底物S13G的C13-羟基(R1)端添加2号葡萄糖基的正常酶促反应生成STB，然后在STB发生副反应，添加6号(或4号)葡萄糖基，生成副产物；与之相 反，新酶B(Phe208Met/Phe379Ala)只催化在底物S13G的C13-羟基(R1)端添加2号葡萄糖基的正常反应生成STB，不催化副反应，无副产物生成；a液相图下方为天然状态OsUGT91C1催化S13G先催化正常反应生成STB，然后催化副反应产生副产物的示意图，b液相图下方为新酶B(Phe208Met/Phe379Ala)只催化S13G正常反应生成STB，不生成副产物的示意图。
图7天然状态OsUGT91C1和新酶B(Phe208Met/Phe379Ala)催化在底物Rubu的C13-羟基(R1)端和C19-羧基(R2)端添加2号葡萄糖基，形成β(1-2)糖苷键正常酶促反应的对比
a,b天然状态OsUGT91C1、新酶B(Phe208Met/Phe379Ala)与Rubu反应的液相分析。液相图从上至下的反应条件依次为：加0.25mg/mL酶反应0小时作为对照，0.05mg/mL酶反应2小时和18小时，0.25mg/mL酶反应2小时和18小时。显示天然状态的OsUGT91C1在Rubu的C13-羟基(R1)和C19-羧基(R2)端分别添加2号葡萄糖基的产物随时间和酶量的增加而增多，而新酶B(Phe208Met/Phe379Ala)可催化得到更多的在C19-羧基方向添加2号葡萄糖基的产物，具有增强的在C19-羧基方向的目标催化能力，而且最终得到在C13-羟基和C19-羧基两个方向都加有2号葡萄糖基的产物Reb E也多于天然状态的OsUGT91C1；c,d天然状态OsUGT91C1和新酶B(Phe208Met/Phe379Ala)催化底物Rubu的C13-羟基(R1)和C19-羧基(R2)端分别添加2号葡萄糖基，生成Reb E的反应示意图，新酶B(Phe208Met/Phe379Ala)显示具有更高的目标催化能力。
图8天然状态OsUGT91C1和新酶B(Phe208Met/Phe379Ala)催化在底物Reb A的C19-羧基(R2)端添加2号葡萄糖基，形成β(1-2)糖苷键，生成Reb D的正常酶促反应的对比
a,b天然状态OsUGT91C1、新酶B(Phe208Met/Phe379Ala)与Reb A反应的液相分析。液相图从上至下的反应条件依次为：加0.25mg/mL酶反应0小时作为对照，0.05mg/mL酶反应2小时和18小时，0.25mg/mL酶反应2小时和18小时。显示天然状态的OsUGT91C1在Reb A的C19-羧基(R2)端添加2号葡萄糖基，生成产物Reb D随时间和酶量的增加而增多，而新酶B(Phe208Met/Phe379Ala)可明显率先将底物反应完全，具有增强的在C19-羧基方向的目标催化能力；c,d天然状态OsUGT91C1和新酶B(Phe208Met/Phe379Ala)催化底物Reb A的C19-羧基(R2)端添加2号葡萄糖基，生成Reb D的反应示意图，其中新酶B(Phe208Met/Phe379Ala)的该反应得到增强。
图9利用新酶B(Phe208Met/Phe379Ala)催化获得的甜菊糖苷底物Reb E(不产生副 产物)，通过糖基转移酶UGT76G1在C13-羟基或(和)C-19羧基方向继续添加3号葡萄糖基，获得甜菊糖苷D和M
a,以新酶B(Phe208Met/Phe379Ala)催化获得的甜菊糖苷底物Reb E(不产生副产物)的基础上，再利用糖基转移酶UGT76G1转化形成Reb D和Reb M的反应示意图。方框显示两个新酶不发生副反应，只在C13-羟基或(和)C-19羧基方向正常添加2号葡萄糖基，形成甜菊糖苷E(Reb E)；然后糖基转移酶UGT76G1接力，继续催化在C13-羟基或(和)C-19羧基方向添加3号葡萄糖基，形成甜菊糖苷D和M(Reb D和Reb M)的过程。b,利用新酶B(Phe208Met/Phe379Ala)催化获得的甜菊糖苷底物Reb E(不产生副产物)，进一步利用糖基转移酶UGT76G1催化从Reb E到Reb D和Reb M的转化过程。在液相图从上至下的反应条件依次为：加0.03mg/mL的糖基转移酶UGT76G1反应0小时作为对照，0.03mg/mL糖基转移酶UGT76G1反应2小时和18小时，0.15mg/mL糖基转移酶UGT76G1反应2小时和18小时。
图10新酶B(Phe208Met/Phe379Ala)去除第1-14位氨基酸后的截短体的SDS-PAGE分析；
新酶B(Phe208Met/Phe379Ala)和新酶B去除第1-14位氨基酸后的截短体的SDS-PAGE比较分析。M,Marker；1，新酶B；2，新酶B的截短体从Q柱流穿的样品；3-8，新酶B的截短体从Q柱洗脱的样品
图11天然状态OsUGT91C1、新酶B去除第1-14位氨基酸后的截短体催化在底物Rubu的C13-羟基(R1)和(或)C19-羧基(R2)端分别添加2号葡萄糖基，形成β(1-2)糖苷键正常酶促反应的对比；
天然状态的OsUGT91C1(a)、新酶B去除第1-14位氨基酸后的截短体(b)与Rubu反应的液相分析比较。液相图从上至下的反应条件依次为：加0.25mg/mL酶反应0小时作为对照，0.05mg/mL酶反应2小时和18小时，0.25mg/mL酶反应2小时和18小时。显示天然状态的OsUGT91C1在Rubu的C13-羟基(R1)和C19-羧基(R2)端分别添加2号葡萄糖基的产物随时间和酶量的增加而增多，而新酶B截短体具有同样的酶促催化反应，且同新酶B一样，具有比天然状态OsUGT91C1更好的催化能力。c,天然状态OsUGT91C1和新酶B截短体可进行同样催化在底物Rubu的C13-羟基(R1)和C19-羧基(R2)端分别添加2号葡萄糖基的正常目标反应，生成Reb E的反应示意图，进一步验证第1-14位氨基酸的冗余性，可以完全去除或改变。
具体实施方式
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为从商业渠道购买得到的。
一、天然状态OsUGT91C1蛋白表达载体的构建
(1)获取OsUGT91C1的氨基酸序列(NCBI Reference Sequence:XP_015629141.1)，根据蛋白表达系统的要求对OsUGT91C1编码密码子进行优化和合成。发明人根据大肠杆菌的表达体系优化后的编码密码子如下。由于密码子的兼并性，密码子优化的选择可有多种。
为了防止通过替换OsUGT91C1的非重要氨基酸序列，达到规避本专利权利要求的目的，本专利认为只要翻译后的蛋白氨基酸序列和OsUGT91C1的氨基酸序列相同度在50％及以上，则认为属于OsUGT91C1。
不管是在OsUGT91C1的氨基酸序列还是空间结构上，通过改变同208和379位氨基酸等效的位点或其他位点(由于氨基酸冗余性的原因，通过去除第1-14位的一个或任意个氨基酸，导致第208和379位氨基酸不一定一直是第208和379位的编号)，达到提升OsUGT91C1添加2号葡萄糖基的正常反应或(和)消除OsUGT91C1副反应的目的，都属于本专利的权利要求。
OsUGT91C1的氨基酸序列(加粗的氨基酸是本专利进行改动的氨基酸位点，划线部分是人为添加的氨基酸序列，其中LE是为了方便连接进入大肠杆菌表达载体pET21b，HHHHHH(6个组氨酸标签)是为了方便后期的纯化)：
Figure PCTCN2022131774-appb-000001
OsUGT91C1密码子优化后的核苷酸序列(划线序列为上述人为添加氨基酸序列的编码区)：
Figure PCTCN2022131774-appb-000002
Figure PCTCN2022131774-appb-000003
(2)对合成得到的OsUGT91C1编码区用表1所列的引物进行扩增，扩增片段的5’和3’端将分别带上Nde I和Xho I的限制酶位点。
表1 pET21b-OsUGT91C1的引物序列
Figure PCTCN2022131774-appb-000004
(3)用Nde I和Xho I对pET21b表达载体和步骤2的OsUGT91C1编码区扩增片段进行双酶切，将OsUGT91C1编码区用T4连接酶连接进pET21b载体的Nde I和Xho I酶切位点之间，组成在大肠杆菌可表达糖基转移酶OsUGT91C1的表达载体pET21b-OsUGT91C1。
(4)将连接产物全部转入100μL E.coli DH5α感受态细胞中，挑取平板上的阳性单克隆菌落接种于10mL的LB培养基中，200rpm 37℃培养12-16小时后提取质粒，测序验证 表达质粒的正确性，完成天然状态OsUGT91C1糖基转移酶表达载体的构建。
二、为了增强添加2号葡萄糖基的正常反应，构建新酶A(Phe208Met)的表达载体；为了既提升酶促添加2号葡萄糖基的目标催化活性，又消除副反应，构建新酶B(Phe208Met/Phe379Ala)的表达载体
1、新酶A(Phe208Met)表达载体的构建
(1)以天然状态pET21b-OsUGT91C1为模板，设计新酶A(Phe208Met)和新酶B(Phe208Met/Phe379Ala)所使用的定点突变引物如表2。
表2突变体引物序列如下：(下划线部分表示突变位点)
Figure PCTCN2022131774-appb-000005
(2)用ddH 2O溶解表2的引物，稀释引物浓度为10μM。用Phe208Met的一对突变引物以pET21b-OsUGT91C1为模板进行PCR扩增，体系同为：dNTP 4μL，5×PS buffer 10μL，上下游引物各2μL，模板1μL(约10ng)，PCR扩增酶Primer star 0.5μL，剩余用ddH2O补齐至50μL。混匀后用PCR扩增，扩增程序：98℃预变性2min，98℃变性30s，69℃退火30s，72℃延伸8min，扩增20个循环，72℃再延伸10min，最后4℃保存。
(3)从上述扩增产物取出10μL进行琼脂糖胶验证PCR的扩增效果，剩余的40μL体系里各加1μL DpnI酶，37℃孵育1-2小时后取10μL分别转入100μL E.coli DH5α感受态细胞中，冰浴30min，42℃热激2min，再冰浴3min后加入新鲜LB 300μL，200rpm 37℃摇床孵育1小时后，分别取150μL均匀涂布于Amp抗性的固体LB平板上，37℃静置培养过夜。
(4)挑取两个平板上的单克隆菌落接种于10mL的LB培养基中，200rpm 37℃培养12-16小时后提取质粒，DNA测序验证突变位点Phe208Met的正确性，编码的氨基酸序列如SEQ ID No.1所示。
2、新酶B(Phe208Met/Phe379Ala)表达载体的构建
(1)获得上述2.2.2.1第4步经过测序验证的新酶A(Phe208Met)的表达载体为模板，进行新酶B(Phe208Met/Phe379Ala)表达载体的构建；
(2)用ddH 2O溶解表2的引物，稀释引物浓度为10μM。用Phe379Ala的一对突变引物以新酶A(Phe208Met)的表达载体为模板进行PCR扩增，体系同上：dNTP 4μL，5×PS  buffer 10μL，上下游引物各2μL，模板1μL(约10ng)，PCR扩增酶Primer star 0.5μL，剩余用ddH 2O补齐至50μL。混匀后用PCR扩增，扩增程序：98℃预变性2min，98℃变性30s，68℃退火30s，72℃延伸8min，扩增20个循环，72℃再延伸10min，最后4℃保存。
(3)从上述扩增产物取出10μL进行琼脂糖胶验证PCR的扩增效果，剩余的40μL体系里各加1μL DpnI酶，37℃孵育1-2小时后取10μL分别转入100μL E.coli DH5α感受态细胞中，冰浴30min，42℃热激2min，再冰浴3min后加入新鲜LB 300μL，200rpm 37℃摇床孵育1小时后，分别取150μL均匀涂布于Amp抗性的固体LB平板上，37℃静置培养过夜。
(4)挑取两个平板上的单克隆菌落接种于10mL的LB培养基中，200rpm 37℃培养12-16小时后提取质粒，DNA测序验证两个突变位点Phe208Met和Phe379Ala的正确性，编码的氨基酸序列如SEQ ID No.2所示。
三、天然状态OsUGT91C1和新酶A，新酶B的诱导表达和纯化
(1)将上述三种酶对应的表达质粒转化至E.coli BL21(DE3)表达菌株，第二天挑取单克隆接种于10mL新鲜的LB培养基中，200rpm 37℃培养过夜后加入终浓度为8％的甘油保种，该菌种可在-80℃长期保存。
(2)挑取上一步保存的E.coli BL21(DE3)甘油菌接种于含Amp 50μg/mL的100mL新鲜LB培养基中，200rpm 37℃过夜培养。第二天以1％的接种比例接种于含Amp 50μg/mL的1L新鲜LB培养基，180rpm 37℃培养至OD600＝1.0后，菌液降温至16℃，添加终浓度为0.5mM的IPTG，160rpm 16℃–20℃诱导表达18小时。
(3)完成诱导表达后，4000rpm离心15min弃上清，收集菌体。菌体用重悬缓冲液(20mM Tris-HCl buffer pH 7.8,0.5M NaCl,30mM imidazole)重悬后，用高压破碎仪在1000bar压力下反复破碎3次。13500rpm 4℃离心60min，离心后的上清上样到NTA-Ni柱，用上述重悬缓冲液洗去非特异性结合的杂蛋白，带组氨酸标签的目的蛋白可被洗脱缓冲液(20mM Tris-HCl buffer pH 7.8,0.5M NaCl,250mM imidazole)洗脱下来。洗脱下来的蛋白进一步置换到20mM HEPES buffer pH 7.2,50mM NaCl里，经液氮速冻后，可长期保存于-80℃。上述步骤可纯化得到天然状态的OsUGT91C1、新酶A、新酶B，并通过SDS-PAGE检测纯度(图2)。
四、新酶A(Phe208Met)和新酶B(Phe208Met/Phe379Ala)的酶活性测定
1、酶促产物分析，表明新酶A(Phe208Met)提升了在C13-羟基，特别是C19-羧基方 向添加2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)的催化能力
(1)以OsUGT91C1的底物Rubu(Rubusoside,CAS 64849-39-4)为实施例，天然状态的OsUGT91C1可在底物Rubu的C13-羟基、C19-羧基两个方向添加2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)(正常反应)(图3中a)，而新酶A(Phe208Met)同样可在底物Rubu的C13-羟基、C19-羧基两个方向添加2号葡萄糖基，而且可催化得到更多的在C19-羧基方向添加2号葡萄糖基的产物，具有增强的在C19-羧基方向的目标催化能力，最终得到在C13-羟基和C19-羧基两个方向都加有2号葡萄糖基的产物Reb E(Rebaudioside E,CAS 63279-14-1)也多于天然状态的OsUGT91C1(图3中b)。
实施例的反应体系如下：在20℃-40℃，200μL反应体系包括：1mM UDP-Glucose,20mM Tris-HCl缓冲液pH 7.2，浓度分别为0.05mg/mL(1×)和0.25mg/mL(5×)的酶样品(天然状态或者新酶A(Phe208Met))，0.3mM底物Rubu。通过加入酶样品启动反应，分别于0，2，18小时取样60μL，与等体积的正丁醇混合涡旋振荡，终止反应并萃取对应的酶促反应产物，室温17000rpm离心10min，室温静置1min，取上层正丁醇的萃取相50μL真空干燥后，用等体积的25％乙腈重悬后，利用HPLC检测酶促产物。本实施例所列的反应条件不唯一，只要能使OsUGT91C1发生酶促反应，都可得到同样的结果。检测手段也不唯一，只要能区分每个酶促产物，可得到相同的检测结果。
天然状态的OsUGT91C1和新酶A(Phe208Met)催化在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应的底物不唯一，包括但不限于，Rubu(Rubusoside，CAS 64849-39-4)、S13G(Steviol-13-O-monoglucoside，CAS 60129-60-4)及Reb A(Rebaudioside A,CAS 58543-16-1)等甜菊糖苷底物，只要在C13-羟基或C-19羧基方向存在1号葡萄糖基，但不存在3号葡萄糖基，就可发生在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应。
(2)以OsUGT91C1的底物Reb A(Rebaudioside A,CAS 58543-16-1)为实施例，天然状态的OsUGT91C1可在底物Reb A的C19-羧基方向添加2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)(正常反应)(图4中a)，而新酶A(Phe208Met)可明显率先将底物Reb A完全消耗并生成目标产物Reb D(Rebaudioside D,CAS 63279-13-0)，具有增强的在C19-羧基方向的目标催化能力，提高在C19-羧基方向添加2号葡萄糖基形成对应产物的能力(图4中b)。
实施例的反应体系如下：在20℃-40℃，200μL反应体系包括：1mM UDP-Glucose,20mM Tris-HCl缓冲液pH 7.2，浓度分别为0.05mg/mL(1×)和0.25mg/mL(5×)的酶样品 (天然状态或者新酶A(Phe208Met))，0.3mM底物Reb A。通过加入酶样品启动反应，分别于0，2，18小时取样60μL，与等体积的正丁醇混合涡旋振荡，终止反应并萃取对应的酶促反应产物，室温17000rpm离心10min，室温静置1min，取上层正丁醇的萃取相50μL真空干燥后，用等体积的25％乙腈重悬后，利用HPLC检测酶促产物。本实施例所列的反应条件不唯一，只要能使OsUGT91C1发生酶促反应，都可得到同样的结果。检测手段也不唯一，只要能区分每个酶促产物，可得到相同的检测结果。
天然状态的OsUGT91C1和新酶A催化在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应的底物不唯一，包括但不限于，Rubu(Rubusoside，CAS 64849-39-4)、S13G(Steviol-13-O-monoglucoside，CAS 60129-60-4)及Reb A(Rebaudioside A,CAS 58543-16-1)等甜菊糖苷底物，只要在C13-羟基或C-19羧基方向存在1号葡萄糖基，但不存在3号葡萄糖基，就可发生在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应。
2、酶促产物分析，表明新酶B(Phe208Met/Phe379Ala)消除了添加6号葡萄糖基(或4号葡萄糖基)的副反应，但不影响在C13-羟基和C19-羧基方向添加2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)的催化能力
(1)以OsUGT91C1的底物STB(Steviolbioside，CAS 41093-60-1)为实施例，天然状态的OsUGT91C1可分别在底物STB上催化添加6号葡萄糖基(或4号葡萄糖基)的副反应(图5中a)，天然状态的OsUGT91C1副反应产物随时间和酶量的增加而增多，而新酶B(Phe208Met/Phe379Ala)无副反应产物的生成，已经消除添加6号葡萄糖基(或4号葡萄糖基)的副反应(图5中b)。
副反应的实施例如下：在20℃-40℃，200μL反应体系包括：1mM UDP-Glucose,20mM Tris-HCl缓冲液pH 7.2，浓度分别为0.15mg/mL(1×)和0.75mg/mL(5×)的酶样品(天然状态或者新酶B(Phe208Met/Phe379Ala))，0.3mM STB。通过加入酶样品启动反应，分别于0，2，18小时取样60μL，与等体积的正丁醇混合涡旋振荡，终止反应并萃取对应的酶促反应产物，室温17000rpm离心10min，室温静置1min，取上层正丁醇的萃取相50μL真空干燥后，用等体积的25％乙腈重悬后，利用HPLC检测酶促产物。本实施例所列的反应条件不唯一，只要能使OsUGT91C1发生酶促反应，都可得到同样的结果。检测手段也不唯一，只要能区分每个酶促产物，可得到相同的检测结果。
天然状态的OsUGT91C1催化副反应的底物不唯一，包括但不限于对以下甜菊糖苷底物发生副反应，STB(Steviolbioside，CAS 41093-60-1)，Reb E(Rebaudioside E，CAS 63279-14-1)等，只要在C13-羟基或C-19羧基方向存在1号和2号葡萄糖基，但不存在3号葡萄 糖基(3号葡萄糖基指与1号葡萄糖基形成β(1-3)糖苷键的葡萄糖基)，就可发生在对应的C13-羟基或C-19羧基方向添加6号葡萄糖基(或4号葡萄糖基)的副反应。
(2)以OsUGT91C1的底物S13G(Steviol-13-O-monoglucoside，CAS 60129-60-4)为实施例，天然状态的OsUGT91C1先催化在底物S13G的C13-羟基(R1)端添加2号葡萄糖基的正常酶促反应生成STB，然后对STB发生副反应，添加6号(或4号)葡萄糖基，生成副产物(图6中a)；与之相反，新酶B(Phe208Met/Phe379Ala)只催化在底物S13G的C13-羟基(R1)端添加2号葡萄糖基的正常反应生成STB，不催化副反应，无副产物生成(图6中b)。
实施例的反应条件如下：在20℃-40℃，200μL反应体系包括：1mM UDP-Glucose,20mM Tris-HCl缓冲液pH 7.2，浓度分别为0.15mg/mL(1×)和0.75mg/mL(5×)的酶样品(天然状态或者新酶B(Phe208Met/Phe379Ala))，0.3mM底物S13G。通过加入酶样品启动反应，分别于0，2，18小时取样60μL，与等体积的正丁醇混合涡旋振荡，终止反应并萃取对应的酶促反应产物，室温17000rpm离心10min，室温静置1min，取上层正丁醇的萃取相50μL真空干燥后，用等体积的25％乙腈重悬后，利用HPLC检测酶促产物。本实施例所列的反应条件不唯一，只要能使OsUGT91C1发生酶促反应，都可得到同样的结果。检测手段也不唯一，只要能区分每个酶促产物，可得到相同的检测结果。
天然状态的OsUGT91C1和新酶B(Phe208Met/Phe379Ala)催化在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应的底物不唯一，包括但不限于，Rubu(Rubusoside，CAS 64849-39-4)、S13G(Steviol-13-O-monoglucoside，CAS 60129-60-4)及Reb A(Rebaudioside A,CAS 58543-16-1)等甜菊糖苷底物，只要在C13-羟基或C-19羧基方向存在1号葡萄糖基，但不存在3号葡萄糖基，就可发生在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应；天然状态的OsUGT91C1催化副反应的底物不唯一，包括但不限于对以下甜菊糖苷底物发生副反应，STB(Steviolbioside，CAS 41093-60-1)，Reb E(Rebaudioside E，CAS 63279-14-1)等，只要在C13-羟基或C-19羧基方向存在1号和2号葡萄糖基，但不存在3号葡萄糖基(3号葡萄糖基指与1号葡萄糖基形成β(1-3)糖苷键的葡萄糖基)，就可发生在对应的C13-羟基或C-19羧基方向添加6号葡萄糖基(或4号葡萄糖基)的副反应。
3、酶促产物分析，表明新酶B(Phe208Met/Phe379Ala)提升了在C13-羟基，特别是C19-羧基方向添加2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)的催化能力
(1)以OsUGT91C1的底物Rubu(Rubusoside,CAS 64849-39-4)为实施例，天然状态 的OsUGT91C1可在底物Rubu的C13-羟基、C19-羧基两个方向添加2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)(正常反应)(图7中a)，而新酶B(Phe208Met/Phe379Ala)同样可在底物Rubu的C13-羟基、C19-羧基两个方向添加2号葡萄糖基，而且可催化得到更多的在C19-羧基方向添加2号葡萄糖基的产物，具有增强的在C19-羧基方向的目标催化能力，最终得到在C13-羟基和C19-羧基两个方向都加有2号葡萄糖基的产物Reb E(Rebaudioside E,CAS 63279-14-1)也明显多于天然状态的OsUGT91C1(图7中b)。
实施例的反应体系如下：在20℃-40℃，200μL反应体系包括：1mM UDP-Glucose,20mM Tris-HCl缓冲液pH 7.2，浓度分别为0.05mg/mL(1×)和0.25mg/mL(5×)的酶样品(天然状态或者新酶B(Phe208Met/Phe379Ala))，0.3mM底物Rubu。通过加入酶样品启动反应，分别于0，2，18小时取样60μL，与等体积的正丁醇混合涡旋振荡，终止反应并萃取对应的酶促反应产物，室温17000rpm离心10min，室温静置1min，取上层正丁醇的萃取相50μL真空干燥后，用等体积的25％乙腈重悬后，利用HPLC检测酶促产物。本实施例所列的反应条件不唯一，只要能使OsUGT91C1发生酶促反应，都可得到同样的结果。检测手段也不唯一，只要能区分每个酶促产物，可得到相同的检测结果。
天然状态的OsUGT91C1和新酶B(Phe208Met/Phe379Ala)催化在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应的底物不唯一，包括但不限于，Rubu(Rubusoside，CAS 64849-39-4)、S13G(Steviol-13-O-monoglucoside，CAS 60129-60-4)及Reb A(Rebaudioside A,CAS 58543-16-1)等甜菊糖苷底物，只要在C13-羟基或C-19羧基方向存在1号葡萄糖基，但不存在3号葡萄糖基，就可发生在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应。
(2)以OsUGT91C1的底物Reb A(Rebaudioside A,CAS 58543-16-1)为实施例，天然状态的OsUGT91C1可在底物Reb A的C19-羧基方向添加2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)(正常反应)(图8中a)，而新酶B(Phe208Met/Phe379Ala)可明显率先将底物Reb A完全消耗并生成目标产物Reb D(Rebaudioside D,CAS 63279-13-0)，具有增强的在C19-羧基方向的目标催化能力，提高在C19-羧基方向添加2号葡萄糖基形成对应产物的能力(图8中b)。
实施例的反应体系如下：在20℃-40℃，200μL反应体系包括：1mM UDP-Glucose,20mM Tris-HCl缓冲液pH 7.2，浓度分别为0.05mg/mL(1×)和0.25mg/mL(5×)的酶样品(天然状态或者新酶B(Phe208Met/Phe379Ala))，0.3mM底物Reb A。通过加入酶样品启动反应，分别于0，2，18小时取样60μL，与等体积的正丁醇混合涡旋振荡，终止反应并 萃取对应的酶促反应产物，室温17000rpm离心10min，室温静置1min，取上层正丁醇的萃取相50μL真空干燥后，用等体积的25％乙腈重悬后，利用HPLC检测酶促产物。本实施例所列的反应条件不唯一，只要能使OsUGT91C1发生酶促反应，都可得到同样的结果。检测手段也不唯一，只要能区分每个酶促产物，可得到相同的检测结果。
天然状态的OsUGT91C1和新酶B(Phe208Met/Phe379Ala)催化在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应的底物不唯一，包括但不限于，Rubu(Rubusoside，CAS 64849-39-4)、S13G(Steviol-13-O-monoglucoside，CAS 60129-60-4)及Reb A(Rebaudioside A,CAS 58543-16-1)等甜菊糖苷底物，只要在C13-羟基或C-19羧基方向存在1号葡萄糖基，但不存在3号葡萄糖基，就可发生在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应。
4、通过荧光转化的方法，测定新酶A(Phe208Met)和新酶B(Phe208Met/Phe379Ala)催化在甜菊糖苷底物添加2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)正常反应的速度。
新酶A(Phe208Met)增强了催化正常所需的在甜菊糖苷底物添加第2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)的反应速度，是天然状态OsUGT91C1活性的2倍以上；新酶B(Phe208Met/Phe379Ala)消除添加6号(或4号)葡萄糖基副反应的同时，增强了催化正常所需的在甜菊糖苷底物添加第2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)的反应速度，是天然状态OsUGT91C1活性的3-7倍(表3)。
表3 天然状态OsUGT91C1、新酶A(Phe208Met)、新酶B(Phe208Met/Phe379Ala)催化在底物C13-羟基(R1)和C19-羧基(R2)端分别添加2号葡萄糖基(正常所需的催化反应)的动力学参数
Figure PCTCN2022131774-appb-000006
(1)该测定直接利用商品化的糖基转移酶活性试剂盒完成，实施例使用Promega公司的UDP-Glo TM Glycosyltransferase Assay试剂盒，其他可用于糖基转移酶反应速度检测方法可得到同样结果。
Promega公司的UDP-Glo TM Glycosyltransferase Assay试剂盒用于检测以UDP-Glucose为糖基供体的葡萄糖基转移酶反应速度，OsUGT91C1及本发明涉及的两个突变新酶均适用。
在葡萄糖基转移酶的作用下，糖基供体UDP-Glucose将葡萄糖转移给底物后生成UDP。该检测方法将生成UDP等物质的量(1:1)转化为ATP，ATP可使荧光素酶发出定量的荧光，因此通过测定荧光量可检测酶促糖基转移反应生成UDP的量。当生成UDP在0-25μM浓度范围内，产生的荧光强度和UDP的摩尔浓度成线性关系。根据UDP浓度和荧光强度对应关系的标准曲线，计算UDP的生成量，可转化为糖基转移酶的催化反应速度。
(2)配制UDP不同浓度的一系列溶液，根据上述转化的反应，测定对应的荧光强度。天然状态OsUGT91C1及本发明涉及的两个新酶，以不同底物检测各种催化反应的速度及酶动力学常数等信息，用于评价本发明的新酶A(Phe208Met)和新酶B(Phe208Met/Phe379Ala)在促进添加2号葡萄糖基正常反应上的效果。利用Rubu(Rubusoside，CAS 64849-39-4)、S13G(Steviol-13-O-monoglucoside，CAS 60129-60-4)及Reb A(Rebaudioside A,CAS 58543-16-1)为底物分别检测新酶A(Phe208Met)和新酶B(Phe208Met/Phe379Ala)在C13-羟基，C19-羧基添加2号葡萄糖基的催化能力，并和天然状态OsUGT91C1的催化能力进行比较。天然状态的OsUGT91C1及新酶A(Phe208Met)、新酶B(Phe208Met/Phe379Ala)催化在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应的底物不唯一，包括但不限于，Rubu(Rubusoside，CAS 64849-39-4)、S13G(Steviol-13-O-monoglucoside，CAS 60129-60-4)及Reb A(Rebaudioside A,CAS 58543-16-1)等甜菊糖苷底物，只要在C13-羟基或C-19羧基方向存在1号葡萄糖基，但不存在3号葡萄糖基，就可发生在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应。
(3)天然状态OsUGT91C1与新酶A(Phe208Met)和新酶B(Phe208Met/Phe379Ala)针对不同底物的酶动力学常数表3所示，新酶A(Phe208Met)增强了催化正常所需的在甜菊糖苷底物添加第2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)的反应速度，是天然状态OsUGT91C1活性的2倍以上；新酶B(Phe208Met/Phe379Ala)消除添加6号(或4号)葡萄糖基副反应的同时，增强了催化正常所需的在甜菊糖苷底物添加第2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)的反应速度，是天然状态OsUGT91C1活性的3-7倍(表3)。
五、新酶B(Phe208Met/Phe379Ala)既无副反应的发生又提升了目标催化能力，可催 化完成在甜菊糖苷底物C13-羟基或(和)C-19羧基方向添加2号葡萄糖基的正常反应，生成包括Reb E(Rebaudioside E，CAS 63279-14-1)的一系列甜菊糖苷产物。在此条件下，利用糖基转移酶UGT76G1在C13-羟基或(和)C-19羧基方向继续添加3号葡萄糖基，则可获得甜菊糖苷D和M(Rebaudioside D，CAS 63279-13-0和Rebaudioside M，CAS 1220616-44-3)(图9)。
六、新酶A(Phe208Met)、新酶B(Phe208Met/Phe379Ala)的氨基酸序列具有冗余性，可以去除或改变第1-14位的任一氨基酸。
以新酶B(Phe208Met/Phe379Ala)为实施例，对新酶B去除第1-14位的全部氨基酸(MDSGYSSSYAAAAG)得到新酶B截短体，该截短体仍能正常表达、纯化，并显示出在甜菊糖苷底物C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应的活性。
1、为了验证第1-14位氨基酸(MDSGYSSSYAAAAG)的冗余性，在新酶B(Phe208Met/Phe379Ala)的表达载体上构建去除第1-14位的全部氨基酸(MDSGYSSSYAAAAG)(新酶B截短体)的表达载体
(1)以新酶B(Phe208Met/Phe379Ala)的表达质粒为模板，设计去除第1-14位的全部
氨基酸的截短体的引物如表4。
表4 突变体引物序列如下：
Figure PCTCN2022131774-appb-000007
(2)用ddH 2O溶解表4的引物，稀释引物浓度为10μM。用去除1-14-F和去除1-14-R的一对引物分别以新酶B(Phe208Met/Phe379Ala)的表达质粒为模板进行PCR扩增，体系同为：dNTP 4μL，5×PS buffer 10μL，上下游引物各2μL，模板1μL(约10ng)，PCR扩增酶Primer star 0.5μL，剩余用ddH 2O补齐至50μL。混匀后用PCR扩增，扩增程序：98℃预变性2min，98℃变性30s，69℃退火30s，72℃延伸8min，扩增20个循环，72℃再延伸10min，最后4℃保存。
(3)从上述扩增产物各取出10μL进行琼脂糖胶验证PCR的扩增效果，剩余的40μL体系里各加1μL DpnI酶，37℃孵育1-2小时后取10μL分别转入100μL E.coli DH5α感受态细胞中，冰浴30min，42℃热激2min，再冰浴3min后加入新鲜LB 300μL，200rpm37℃摇床孵育1小时后，分别取150μL均匀涂布于Amp抗性的固体LB平板上，37℃静 置培养过夜。
(4)挑取两个平板上的单克隆菌落接种于10mL的LB培养基中，200rpm 37℃培养12-16小时后提取质粒，DNA测序验证新酶B截短体的表达质粒，成功去除了第1-14位的全部氨基酸，除了第1-14位氨基酸被去除，其余位点氨基酸序列同SEQ ID No.2维持一致。
2、新酶B(Phe208Met/Phe379Ala)去除第1-14位的全部氨基酸后截短体的诱导表达和纯化
(1)将对应的表达质粒转化至E.coli BL21(DE3)表达菌株，第二天挑取单克隆接种于10mL新鲜的LB培养基中，200rpm 37℃培养过夜后加入终浓度为8％的甘油保种，该菌种可在-80℃长期保存。
(2)挑取上一步保存的E.coli BL21(DE3)甘油菌接种于含Amp 50μg/mL的100mL新鲜LB培养基中，200rpm 37℃过夜培养。第二天以1％的接种比例接种于含Amp 50μg/mL的1L新鲜LB培养基，180rpm 37℃培养至OD600＝1.0后，菌液降温至16℃，添加终浓度为0.5mM的IPTG，160rpm 16℃–20℃诱导表达18小时。
(3)完成诱导表达后，4000rpm离心15min弃上清，收集菌体。菌体用重悬缓冲液(20mM Tris-HCl buffer pH 7.8,0.5M NaCl,30mM imidazole)重悬后，用高压破碎仪在1000bar压力下反复破碎3次。13500rpm 4℃离心60min，离心后的上清上样到NTA-Ni柱，用上述重悬缓冲液洗去非特异性结合的杂蛋白，带组氨酸标签的目的蛋白可被洗脱缓冲液(20mM Tris-HCl buffer pH 7.8,0.5M NaCl,250mM imidazole)洗脱下来。洗脱下来的蛋白进一步置换到20mM HEPES buffer pH 7.2,50mM NaCl里，经液氮速冻后，可长期保存于-80℃。上述步骤可纯化得到新酶B的截短体，并通过SDS-PAGE检测纯度(图10)。由图10可见，当新酶B去除第1-14位氨基酸的截短体可以进行正常的表达和纯化。由于新酶A和新酶B具有同样的第1-14位氨基酸，对新酶B完全去除第1-14位氨基酸的实施例，显示第1-14位氨基酸具有冗余性。同理，新酶A的第1-14位氨基酸也具有冗余性。
3、新酶B的截短体不影响在甜菊糖苷底物的C13-羟基和C-19羧基两个方向添加第2号葡萄糖基的正常反应，其活性和新酶B一样，比天然转态糖基转移酶OsUGT91C1的催化活性更高，进一步显示第1-14位氨基酸在新酶A和新酶B中的冗余性。
(1)以甜菊糖苷底物Rubu(Rubusoside,CAS 64849-39-4)为实施例，天然状态的OsUGT91C1可在底物Rubu的C13-羟基、C19-羧基两个方向添加2号葡萄糖基(与1号葡萄糖基形成β(1-2)糖苷键)(正常反应)(图11中a)，新酶B的截短体同样可在底物Rubu的C13-羟基、C19-羧基两个方向添加2号葡萄糖基(图11中b)。
(2)实施例的反应体系如下：在20℃-40℃，200μL反应体系包括：1mM UDP-Glucose,20mM Tris-HCl缓冲液pH 7.2，浓度分别为0.05mg/mL(1×)和0.25mg/mL(5×)的酶样品(天然状态或者新酶B截短体)，0.3mM底物Rubu。通过加入酶样品启动反应，分别于0，2，18小时取样60μL，与等体积的正丁醇混合涡旋振荡，终止反应并萃取对应的酶促反应产物，室温17000rpm离心10min，室温静置1min，取上层正丁醇的萃取相50μL真空干燥后，用等体积的25％乙腈重悬后，利用HPLC检测酶促产物。本实施例所列的反应条件不唯一，只要能使OsUGT91C1发生酶促反应，都可得到同样的结果。检测手段也不唯一，只要能区分每个酶促产物，可得到相同的检测结果。
(3)天然状态的OsUGT91C1和新酶B截短体催化在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应的底物不唯一，包括但不限于，Rubu(Rubusoside，CAS 64849-39-4)、S13G(Steviol-13-O-monoglucoside，CAS 60129-60-4)及Reb A(Rebaudioside A,CAS58543-16-1)等甜菊糖苷底物，只要在C13-羟基或C-19羧基方向存在1号葡萄糖基，但不存在3号葡萄糖基，就可发生在C13-羟基或C-19羧基方向添加2号葡萄糖基的正常反应。

Claims (6)

  1. 糖基转移酶OsUGT91C1突变体，其氨基酸序列为以下a、b、c、d中的任一种：
    a、如SEQ ID No.1所示氨基酸序列；
    b、如SEQ ID No.2所示氨基酸序列；
    c、删除第1-14位氨基酸的SEQ ID No.1所示氨基酸序列；
    d、删除第1-14位氨基酸的SEQ ID No.2所示氨基酸序列。
  2. 一种表达基因，编码权利要求1所述的糖基转移酶OsUGT91C1突变体。
  3. 一种表达载体，含有权利要求2所述表达基因。
  4. 携带权利要求3所述表达载体的重组菌。
  5. 权利要求1所述的糖基转移酶OsUGT91C1突变体在甜菊糖苷D或M的酶促合成或转化过程的用途。
  6. 根据权利要求5所述的用途，其特征在于，所述糖基转移酶OsUGT91C1突变体在甜菊糖苷D或M的酶促合成或转化过程是指在甜菊糖苷的C13-羟基或/和C19-羧基方向添加2号葡萄糖基，所述2号葡萄糖基是与1号葡萄糖基形成β(1-2)糖苷键的葡萄糖基，1号葡萄糖基特指直接与C13-羟基或C19-羧基形成β-糖苷键的葡萄糖基。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150315623A1 (en) * 2014-05-05 2015-11-05 Conagen Inc. Non-caloric sweetener
CN113584016A (zh) * 2021-08-17 2021-11-02 安徽金禾实业股份有限公司 一种通过易错pcr技术及高通量筛选提高葡萄糖基转移酶eugt11酶活方法
CN114045273A (zh) * 2021-11-15 2022-02-15 四川大学 糖基转移酶OsUGT91C1突变体及其应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG11201505597SA (en) * 2013-02-06 2015-08-28 Evolva Sa Methods for improved production of rebaudioside d and rebaudioside m
AU2017308143B2 (en) * 2016-08-12 2022-04-07 Amyris, Inc. UDP-dependent glycosyltransferase for high efficiency production of rebaudiosides
WO2019178541A1 (en) * 2018-03-16 2019-09-19 Purecircle Usa Inc. High-purity steviol glycosides
CN112080480B (zh) * 2019-06-14 2023-01-03 中国科学院分子植物科学卓越创新中心 糖基转移酶突变体及其应用
CN110656149A (zh) * 2019-10-30 2020-01-07 山东三元生物科技股份有限公司 一种莱鲍迪苷d的制备方法及其产品和应用
CN111518782B (zh) * 2020-03-24 2022-09-02 广东广业清怡食品科技股份有限公司 一种糖基转移酶ugtzj1突变体及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150315623A1 (en) * 2014-05-05 2015-11-05 Conagen Inc. Non-caloric sweetener
CN113584016A (zh) * 2021-08-17 2021-11-02 安徽金禾实业股份有限公司 一种通过易错pcr技术及高通量筛选提高葡萄糖基转移酶eugt11酶活方法
CN114045273A (zh) * 2021-11-15 2022-02-15 四川大学 糖基转移酶OsUGT91C1突变体及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein NCBI; 7 August 2018 (2018-08-07), ANONYMOUS : "UDP-glycosyltransferase 91C1 [Oryza sativa Japonica Group]", XP093065284, Database accession no. XP_015629141.1 *
HUANG XU-XU; ZHAO SHU-MAN; ZHANG YU-YING; LI YAN-JIE; SHEN HAN-NUO; LI XUGANG; HOU BING-KAI: "A novel UDP-glycosyltransferase 91C1 confers specific herbicide resistance through detoxification reaction in Arabidopsis", PLANT PHYSIOLOGY AND BIOCHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 159, 27 December 2020 (2020-12-27), AMSTERDAM, NL , pages 226 - 233, XP086468514, ISSN: 0981-9428, DOI: 10.1016/j.plaphy.2020.12.026 *
WANG, ZHENYANG ET AL.: "Heterologous expression of EUGT11 from Oryza sativa in Pichia pastoris for highly efficient one-pot production of rebaudioside D from rebaudioside A", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 163, 22 September 2020 (2020-09-22), XP086300466, DOI: 10.1016/j.ijbiomac.2020.09.132 *

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