CN114045273A

CN114045273A - Glycosyl transferase OsUGT91C1 mutant and application thereof

Info

Publication number: CN114045273A
Application number: CN202111350045.8A
Authority: CN
Inventors: 朱晓峰
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2022-02-15
Anticipated expiration: 2041-11-15
Also published as: WO2023083359A1; CN114045273B

Abstract

The invention discloses a glycosyltransferase OsUGT91C1 mutant and application thereof, wherein an amino acid sequence shown in SEQ ID No.1 is obtained by mutating 208 th amino acid from Phe to Met on the basis of a glycosyltransferase OsUGT91C1 amino acid sequence; the amino acid sequence shown in SEQ ID No.2 is that the 208 th amino acid is mutated from Phe to Met and the 379 th amino acid is mutated from Phe to Ala on the basis of the amino acid sequence of glycosyltransferase OsUGT91C1, wherein the 1 st to 14 th amino acids of the two mutants have redundancy and can be completely removed or changed without influencing the normal enzyme activity. The mutant provided by the invention improves the capability of catalyzing the same normal reaction, further removes the disadvantage of side reaction of the original OsUGT91C1 enzyme, and can efficiently catalyze and generate a series of stevioside products including Reb E.

Description

Glycosyl transferase OsUGT91C1 mutant and application thereof

Technical Field

The invention belongs to the field of enzymology, and particularly relates to a glycosyltransferase OsUGT91C1 mutant and application thereof.

Background

In recent years, the harm of high calorie sugar is gradually paid attention, but because sweet taste can stimulate the generation of reward neurotransmitters such as endogenous opium, dopamine and the like, people are difficult to quit the dependence on the sweet taste. Although a plurality of artificial sweeteners in mass production can meet the requirement of low calorie, the taste, safety and psychological acceptability are still controversial, and the artificial sweeteners are difficult to effectively replace the traditional high calorie sweeteners. Stevia native to south america has been locally used as a natural source of sweetness for over a hundred years. In 2006, the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) of the United nations in combination with the Special Committee for food additives (JECFA) passed the safety evaluation of the health and harmlessness of stevioside. In 2008 the FDA approved in the united states to allow stevia sugar to be used in food products. In 2011, stevioside obtains a safety certificate of European food safety administration. With the continued increase in consumer demand for low-sugar, low-calorie foods and beverages, stevia has become one of the fastest growing natural sweeteners used globally.

Stevioside is a generic term for a number of steviol glycosides. All Steviol glycosides have a similar chemical structure pattern, i.e. there are different glucosyl modifications (a in fig. 1) at the C13-hydroxy (also denoted R1 in the figure for simplicity) and/or C19-carboxy (also denoted R2 in the figure for simplicity) core skeleton of the common Steviol (Steviol, CAS 471-80-7). According to the combination condition of glycosyl composition and glycosidic bond of C13-hydroxyl and C19-carboxyl, the Stevioside molecules can be named, but currently, Stevioside products in the market are represented by Stevioside (Stevioside, CAS 57817-89-7 accounts for 57% and is represented by ST) and Stevioside A (Rebaudioside A, CAS 58543-16-1, the content of the Stevioside can reach 32% and is represented by Reb A), and the Stevioside molecules have the disadvantage of poor mouthfeel, so that the market acceptance of the Stevioside is influenced. Research shows that stevioside D (Rebaudioside D, CAS 63279-13-0, represented by Reb D) and M (Rebaudioside M, CAS 1220616-44-3, represented by Reb M) has the sweetness which is 200-fold and 300-fold that of cane sugar, has the mouthfeel which is very close to that of cane sugar which is a sweet standard product, is considered to be the component with the best quality of stevioside, and is the key point of research and development of stevioside manufacturers. Both Peterjoy and Coca Cola were patented with the use of steviol glycosides D and M in beverages.

However, the content of steviol glycosides D and M in the leaves of natural stevia is below 1%, which is difficult to extract directly from naturally planted stevia. Due to the low content, the extraction process from stevia rebaudiana is tedious, the cost is high, and the marketable application of the stevioside D and M with the best mouthfeel is limited. Research shows that the stevia rebaudiana is lack of catalytic capability of adding No.2 glucosyl in the C19-carboxyl direction of stevioside, Reb D and Reb M cannot be effectively synthesized, and the phenomenon of low natural content of the Reb D and the Reb M is caused; meanwhile, the 2 nd glucosyl group of Reb D and Reb M in the C19-carboxyl direction obviously improves the mouthfeel compared with other stevioside. Therefore, the method for utilizing heterologous enzymatic conversion makes up the defect of insufficient capability of stevia to catalyze and add No.2 glucosyl in the C19-carboxyl direction of stevioside, and is a solution for preparing stevioside D and M in a large scale. A glycosyl transferase OsUGT91C1 (also called EUGT11) from rice can catalyze the addition of glucosyl group No.2 (the glucosyl group No.2 is the glucosyl group which forms beta (1-2) glycosidic bond with the glucosyl group No.1, and the glucosyl group No.1 is particularly the glucosyl group which directly forms beta-glycosidic bond with the C13-hydroxyl or the C19-carboxyl) in the directions of C13-hydroxyl and C19-carboxyl of stevioside (b, C and d in figure 1). The capability of adding 2 # glucosyl in both directions of C13-hydroxyl and C19-carboxyl of stevioside by utilizing glycosyltransferase OsUGT91C1 makes up the deficiency of capability of adding 2 # glucosyl in the direction of C19-carboxyl of stevioside by stevia rebaudiana, and is a determining factor for mass production of stevioside D and M. Through a series of enzymatic reactions including glycosyltransferase OsUGT91C1, the total synthetic conversion of stevioside is carried out in vivo (not necessarily stevia rebaudiana Bertoni) or in vitro by using precursor substances, or the stevioside D and M (Reb D and Reb M) is generated through the conversion of natural extracts of stevia rebaudiana Bertoni by adding glucosyl on specific positions of Stevioside (ST), stevioside A (Reb A) or other available stevioside molecules with abundant natural contents through the enzymatic reaction.

Since addition of No.2 glucosyl group in C19-carboxyl direction of steviol glycoside is key to generation of Reb D and Reb M, the inventors hoped to further improve the ability of glycosyltransferase OsUGT91C1 to catalyze addition of No.2 glucosyl group (forming β (1-2) glycosidic bond with No.1 glucosyl group) in C19-carboxyl direction of steviol glycoside for more efficient conversion to steviol glycoside D and M. Meanwhile, the inventors found that the glycosyltransferase OsUGT91C1 has poor specificity in catalyzing the enzymatic conversion process of stevioside D, M, and besides the addition of glucose group No.2 (which is a reaction required for the enzymatic conversion to generate stevioside D, M and is called a normal reaction), there are also obvious side reactions, namely, the addition of glucose group No. 6 (or glucose group No. 4) in the direction of C13-hydroxyl or C19-carboxyl, and the two forms a beta (1-6) glycosidic bond (or a beta (1-4) glycosidic bond) with glucose group No.1 (the side reactions mentioned in the patent refer to the reaction of adding glucose group No. 6 (or glucose group No. 4)) (b, C and d in FIG. 1). The side reaction wastes enzyme catalysis resources and substrates, forms obvious byproducts, influences the generation efficiency of the stevioside D and M, also brings impurities, and brings great difficulty for obtaining high-quality stevioside D, M pure products.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to improve the capability of glycosyltransferase OsUGT91C1 for adding No.2 glucosyl in the direction of catalyzing C13-hydroxyl or/and C19-carboxyl of stevioside, and on the other hand, side reaction is eliminated on the premise of improving the catalytic capability.

Based on the three-dimensional space structure of the glycosyltransferase OsUGT91C1 independently discovered by the inventor, the original amino acid sequence of the glycosyltransferase OsUGT91C1 is rationally optimized, and the inventor changes the phenylalanine at the 208 th position into methionine (Phe208Met) to obtain the novel enzyme A. The example verifies that the new enzyme A can enhance the capability of glycosyltransferase OsUGT91C1 of adding No.2 glucosyl group at C13-hydroxyl, especially at C19-carboxyl.

It was found through previous studies that side reactions could be completely eliminated by changing the 379 th phenylalanine of the glycosyltransferase OsUGT91C1 to alanine (Phe379 Ala). The inventor combines the characteristics of enhanced catalytic ability of the novel enzyme A (Phe208Met) and combines amino acid changes of two sites of Phe208Met and Phe379Ala to successfully construct a novel enzyme B (Phe208Met/Phe379Ala) which can eliminate side reactions and enhance the target catalytic ability (adding No.2 glucosyl). The new enzyme B (Phe208Met/Phe379Ala) is verified to eliminate the side reaction of adding the No. 6 glucosyl (forming beta (1-6) glycosidic bond), and has about 3-7 times of enhancement effect on the normal reaction of adding the No.2 glucosyl, and can participate in the enzymatic conversion of stevioside more efficiently and more specifically.

Further research shows that on the basis of the mutation, the deletion or the change of 1 to 14 amino acids (MDSGYSSSYAAAAG) at the front end of the glycosyltransferase OsUGT91C1 does not influence the enzyme property after mutation. The 14 amino acids at the front end are redundant and can be completely removed or changed.

The technical scheme of the invention is as follows: the glycosyltransferase OsUGT91C1 mutant has an amino acid sequence of any one of the following a, b, C and d:

a. SEQ ID No. 1;

b. SEQ ID No. 2;

c. the 1 st to 14 th amino acids are arbitrarily deleted or changed on the basis of the amino acid sequence shown in SEQ ID No. 1;

d. the 1 st to 14 th amino acids are arbitrarily deleted or changed on the basis of the amino acid sequence shown in SEQ ID No. 2.

The amino acid sequence shown in SEQ ID No.1 is obtained by mutating 208 th amino acid from Phe to Met on the basis of a glycosyltransferase OsUGT91C1 amino acid sequence; the amino acid sequence shown in SEQ ID No.2 is obtained by mutating the 208 th amino acid from Phe to Met and the 379 th amino acid from Phe to Ala on the basis of the amino acid sequence of glycosyltransferase OsUGT91C 1.

An expression gene encoding the glycosyltransferase OsUGT91C1 mutant.

An expression vector containing the above-mentioned expression gene.

Recombinant bacteria or cells carrying the expression vector.

The glycosyltransferase OsUGT91C1 mutant is applied to the enzymatic synthesis or conversion process of stevioside D or M and other stevioside molecules.

Further, the enzymatic synthesis or conversion process of the glycosyltransferase OsUGT91C1 mutant in stevioside D or M and other stevioside molecules refers to the addition of a No.2 glucosyl group in the direction of C13-hydroxyl or/and C19-carboxyl of stevioside, wherein the No.2 glucosyl group is a glucosyl group which forms a beta (1-2) glycosidic bond with the No.1 glucosyl group, and the No.1 glucosyl group refers to the glucosyl group which directly forms a beta-glycosidic bond with the C13-hydroxyl or C19-carboxyl.

The substrates that catalyze the normal reaction of adding No.2 glucosyl group in the C13-hydroxyl or C-19 carboxyl direction are not exclusive and include, but are not limited to, Steviol glycoside substrates such as Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O-monogluside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1), and the normal reaction of adding No.2 glucosyl group in the C13-hydroxyl or C-19 carboxyl direction can occur as long as No.1 glucosyl group is present in the C13-hydroxyl or C-19 carboxyl direction, but No. 3 glucosyl group is not present.

Besides being capable of expressing, purifying and preparing the natural OsUGT91C1 or the two novel enzymes of the invention in Escherichia coli, the enzyme can also be expressed and prepared in other expression systems such as other biological systems, non-biological systems, cells, non-cells (Cell-free) and the like; the novel enzyme with the side reaction eliminated is introduced into an organism, an abiotic body, a cell and a non-cell (cell-free) through a transgenic or other gene introduction means to carry out the enzymatic conversion of stevioside, and is used for the enzymatic conversion of adding No.2 glucosyl to the stevioside in an in vitro system such as enzyme and enzyme immobilization, and the enzymatic conversion process including but not limited to the enzymatic conversion process of preparing stevioside D and M achieves the purpose of eliminating the side reaction.

The side reaction of the patent is defined as the side reaction that glucose group No. 6 (or glucose group No. 4) is added in the two directions of C13-hydroxyl and C19-carboxyl of steviol or stevioside to form beta (1-6) glycosidic bond (or beta (1-4) glycosidic bond) with glucose group No. 1; the normal reaction of the present patent is defined as a reaction in which glucose group No.2 is added in both directions of C13-hydroxyl group, C19-carboxyl group of steviol or stevioside to form a β (1-2) glycoside bond with glucose group No. 1.

The liquid chromatography-mass spectrometry combination is utilized to verify that the novel enzyme A (Phe208Met) enhances the capability of catalyzing the addition of the No.2 glucosyl, particularly the addition of the No.2 glucosyl at the C19-carboxyl position. The novel enzyme B (Phe208Met/Phe379Ala) has no side reaction while enhancing the ability to add glucose group 2. The Mie's kinetic parameters of two new enzyme catalytic reactions are measured by methods such as liquid chromatography-mass spectrometry, fluorescence conversion method and the like, and the new enzyme B (Phe208Met/Phe379Ala) is verified to eliminate the side reaction of adding No. 6 glucosyl (forming beta (1-6) glycosidic bond), have about 3-7 times of enhancement effect on the normal reaction of adding No.2 glucosyl, and can participate in the enzymatic conversion of stevioside more efficiently and more specifically. The new enzyme B catalyzes enzymatic conversion, and the obtained normal product added with No.2 glucosyl group does not contain byproducts and can be used for synthesizing stevioside D and M.

Compared with the prior art, the invention has the following beneficial effects:

the invention solves the enzymatic defect of rice-derived glycosyltransferase OsUGT91C1 in stevioside conversion, and simultaneously remarkably improves the catalytic capability of the target catalytic reaction. The new enzyme B (Phe208Met/Phe379Ala) can more efficiently and specifically complete the normal enzymatic reaction of adding No.2 glucosyl to stevioside, and is used for converting to obtain high-quality and high-purity stevioside D and M serving as health sweeteners.

Drawings

FIG. 1 shows a simplified representation of a portion of the steviol glycoside structure, associated with this patent, and a schematic representation of the normal and side reactions in the enzymatic conversion of OsUGT91C1 to steviol glycosides D and M

a part of the structure of steviol glycosides, including steviol, steviol glycosides D and M and simplified forms of structures. Steviol is represented by the complete chemical structure, and steviol glycosides D and M are represented by the complete chemical structure and the simplified diagram, both in parentheses are the sweetness multiples compared to sucrose. In the simplified scheme, the C13-hydroxyl group (simplified by R1) and the C19-carboxyl group (simplified by R2) are shown. The glucosyl group is represented by a hexagon, the 1-hydroxyl group of each glucose is marked by a black dot, the number in the middle of each glucose represents several glucosyl groups, and the type of glycosidic bond formed by the glucosyl group and the glucosyl group 1 is also represented, for example, 2 represents a beta (1-2) glycosidic bond, 3 represents a beta (1-3) glycosidic bond, 4 represents a beta (1-4) glycosidic bond, and 6 represents a beta (1-6) glycosidic bond.

b, OsUGT91C1 is a schematic diagram of a normal reaction for catalyzing addition of No.2 glucosyl at two ends of a stevioside substrate C13-hydroxyl (simplified by R1) or (and) C19-carboxyl (simplified by R2) and a side reaction for adding No. 6 glucosyl (or No. 4 glucosyl).

C, the OsUGT91C1 is a schematic diagram of a normal reaction for catalyzing addition of No.2 glucosyl group and a side reaction for adding No. 6 glucosyl group (or No. 4 glucosyl group) in a stevioside substrate C13-hydroxyl (represented by R1 in a simplified mode).

d OsUGT91C1 is a schematic diagram of a normal reaction for catalyzing addition of No.2 glucosyl group and a side reaction for adding No. 6 glucosyl group (or No. 4 glucosyl group) in a stevioside substrate C19-carboxyl group (simplified and represented by R2).

FIG. 2 SDS-PAGE analysis of neoenzyme A (Phe208Met), OsUGT91C1 in its native state, and neoenzyme B (Phe208Met/Phe379Ala)

a SDS-PAGE analysis of the novel enzyme A (Phe208 Met). M, Marker; 1, post-induction sample; 2, thallus lysate; 3, supernatant of the thallus lysate; 4, precipitating the thallus lysate; 5, the Ni column flows through the sample; 6-12, Ni column eluting protein sample.

B SDS-PAGE analysis of OsUGT91C1 in its native state and of the novel enzyme B (Phe208Met/Phe379 Ala). M, Marker; WT, native state OsUGT91C 1; 2, the Q column flows through the sample; 3-7, eluting the protein sample by the Q column. The right arrows in the two figures indicate the position of the protein band of interest.

FIG. 3 comparison of the natural state OsUGT91C1 and the novel enzyme A (Phe208Met) catalyzing the normal enzymatic reaction with addition of glucose group No.2 at both ends of the C13-hydroxy (R1) and C19-carboxy (R2) groups of the substrate Rubu to form a beta (1-2) glycosidic bond

a, b liquid phase analysis of the reaction of OsUGT91C1 in its native state, neoenzyme A (Phe208Met) with Rubu. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.25mg/mL of the enzyme was added for 0 hour as a control, 0.05mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.25mg/mL of the enzyme was reacted for 2 hours and 18 hours. The products of OsUGT91C1 showing the natural state and adding the 2-glucosyl at the C13-hydroxyl (R1) and the C19-carboxyl (R2) ends of the Rubu respectively increase along with the increase of time and enzyme amount, the novel enzyme A (Phe208Met) can also add the 2-glucosyl at the C13-hydroxyl and the C19-carboxyl of the substrate Rubu, and can catalyze to obtain more products adding the 2-glucosyl at the C19-carboxyl, the target catalytic capability at the C19-carboxyl is enhanced, and finally the product Reb E adding the 2-glucosyl at the C13-hydroxyl and the C19-carboxyl is more than the product OsUGT91C1 in the natural state; c, d natural state OsUGT91C1 and new enzyme A (Phe208Met) catalyze C13-hydroxyl (R1) and C19-carboxyl (R2) ends of substrate Rubu to add No.2 glucosyl respectively to generate a reaction scheme of Reb E, and the new enzyme A (Phe208Met) shows target catalytic capability enhanced in the direction of C19-carboxyl.

FIG. 4 comparison of the natural state OsUGT91C1 and the neoenzyme A (Phe208Met) catalyzing the addition of a glucosyl group 2 at the C19-carboxy (R2) end of the substrate Reb A, resulting in the normal enzymatic reaction of a beta (1-2) glycosidic bond

a, b liquid phase analysis of the reaction of natural state OsUGT91C1, neoenzyme A (Phe208Met) with Reb A. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.25mg/mL of the enzyme was added for 0 hour as a control, 0.05mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.25mg/mL of the enzyme was reacted for 2 hours and 18 hours. OsUGT91C1 showing a natural state adds glucose group No.2 to the C19-carboxyl (R2) end of Reb A, the generated product Reb D increases along with the increase of time and enzyme amount, and new enzyme A (Phe208Met) can obviously completely consume the substrate Reb A and generate the target product Reb D at first, and has enhanced target catalytic capability in the direction of C19-carboxyl; c, D natural state OsUGT91C1 and new enzyme A (Phe208Met) catalyze the addition of No.2 glucosyl group at the C19-carboxyl (R2) end of the substrate Reb A to generate a reaction scheme of Reb D, and the reaction of the new enzyme A (Phe208Met) is enhanced.

FIG. 5 comparison of the side reactions of OsUGT91C1 in its native state and the addition of glucose group No. 6 (or No. 4) to the C13-hydroxy (R1) end of the substrate STB by the novel enzyme B (Phe208Met/Phe379Ala)

Liquid phase analysis of the reaction of OsUGT91C1(a), neoenzyme B (Phe208Met/Phe379Ala) (B) in the native state with the substrate STB. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.75mg/mL of the enzyme was added for 0 hour as a control, 0.15mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.75mg/mL of the enzyme was reacted for 2 hours and 18 hours. It was shown that OsUGT91C1 side reaction products in the natural state increased with time and the amount of enzyme, while the novel enzyme B (Phe208Met/Phe379Ala) produced no side reaction products. The lower part of the liquid phase diagram of a is a schematic diagram of the reaction of OsUGT91C1 catalyzing STB to generate a byproduct, and the lower part of the liquid phase diagram of B is a schematic diagram of the reaction of new enzyme B catalyzing STB, so that the byproduct is not generated, and the side reaction is not generated.

FIG. 6 comparison of the natural state of OsUGT91C1 and the novel enzyme B (Phe208Met/Phe379Ala) catalyzing the addition of glucosyl group No.2 to the C13-hydroxy (R1) terminus of the substrate S13G, the normal enzymatic reaction forming the beta (1-2) glycosidic bond, and the subsequent side reaction of addition of glucosyl group No. 6 (or 4)

Liquid phase analysis of the reaction of OsUGT91C1(a), neoenzyme B (Phe208Met/Phe379Ala) (B) in native state with substrate S13G. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.75mg/mL of the enzyme was added for 0 hour as a control, 0.15mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.75mg/mL of the enzyme was reacted for 2 hours and 18 hours. OsUGT91C1 showing a natural state firstly catalyzes a normal enzymatic reaction of adding No.2 glucosyl to the end of C13-hydroxyl (R1) of a substrate S13G to generate STB, and then side reaction is carried out on the STB to add No. 6 (or No. 4) glucosyl to generate a byproduct; in contrast, the novel enzyme B (Phe208Met/Phe379Ala) catalyzes only the normal reaction of adding a glucosyl group No.2 to the C13-hydroxy (R1) end of the substrate S13G to produce STB, does not catalyze side reactions, and does not produce byproducts; the liquid phase diagram at the lower part of the a is a schematic diagram that natural state OsUGT91C1 catalyzes S13G to generate STB firstly and then catalyzes side reaction to generate by-product, and the liquid phase diagram at the lower part of the B is a schematic diagram that new enzyme B (Phe208Met/Phe379Ala) catalyzes S13G to generate STB only and does not generate by-product.

FIG. 7 comparison of the natural state OsUGT91C1 and the novel enzyme B (Phe208Met/Phe379Ala) catalyzing the addition of No.2 glucosyl groups at the C13-hydroxy (R1) and C19-carboxy (R2) ends of the substrate Rubu to form the normal enzymatic reaction of the beta (1-2) glycosidic linkage

a, B liquid phase analysis of the reaction of OsUGT91C1 in its native state, neoenzyme B (Phe208Met/Phe379Ala) with Rubu. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.25mg/mL of the enzyme was added for 0 hour as a control, 0.05mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.25mg/mL of the enzyme was reacted for 2 hours and 18 hours. The products of OsUGT91C1 with addition of 2-glucosyl at the C13-hydroxyl (R1) and C19-carboxyl (R2) ends of Rubu in a natural state increase along with the increase of time and enzyme amount, while the new enzyme B (Phe208Met/Phe379Ala) can catalyze to obtain more products with addition of 2-glucosyl at the C19-carboxyl direction, has enhanced target catalytic capability at the C19-carboxyl direction, and finally obtains a product Reb E with addition of 2-glucosyl at both the C13-hydroxyl and the C19-carboxyl directions, which is also more than that of OsUGT91C1 in a natural state; c, d, the natural state OsUGT91C1 and the new enzyme B (Phe208Met/Phe379Ala) catalyze the C13-hydroxyl (R1) and C19-carboxyl (R2) ends of the substrate Rubu to add No.2 glucosyl respectively to generate a reaction schematic diagram of Reb E, and the new enzyme B (Phe208Met/Phe379Ala) shows higher target catalytic capacity.

FIG. 8 comparison of the Normal enzymatic reactions for the production of Reb D with OsUGT91C1 and the novel enzyme B (Phe208Met/Phe379Ala) catalyzing the addition of a glucosyl group 2 at the C19-carboxy (R2) end of the substrate Reb A, forming a beta (1-2) glycosidic linkage

a, B liquid phase analysis of the reaction of OsUGT91C1, New enzyme B (Phe208Met/Phe379Ala), and Reb A in its native state. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.25mg/mL of the enzyme was added for 0 hour as a control, 0.05mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.25mg/mL of the enzyme was reacted for 2 hours and 18 hours. OsUGT91C1 showing a natural state adds glucose group No.2 to the C19-carboxyl (R2) end of Reb A, the generated product Reb D increases along with the increase of time and enzyme amount, and a new enzyme B (Phe208Met/Phe379Ala) can obviously react substrates completely and has enhanced target catalytic capability in the direction of C19-carboxyl; c, D Natural State OsUGT91C1 and New enzyme B (Phe208Met/Phe379Ala) catalyze the addition of a glucose group at C19-carboxy (R2) end of the substrate Reb A to generate a reaction scheme for Reb D, in which this reaction is enhanced for the New enzyme B (Phe208Met/Phe379 Ala).

FIG. 9 shows that the new enzyme B (Phe208Met/Phe379Ala) is used to catalyze the obtained stevioside substrate Reb E (no by-product is generated), glucose group No. 3 is added in the C13-hydroxyl or (and) C-19 carboxyl direction through glycosyltransferase UGT76G1, and stevioside D and M are obtained

a reaction scheme of converting a glycosyltransferase UGT76G1 to form Reb D and Reb M on the basis of a stevioside substrate Reb E (without generating byproducts) obtained by catalyzing a new enzyme B (Phe208Met/Phe379 Ala). The box shows that the two new enzymes do not generate side reaction, and only normally add No.2 glucosyl group in the direction of C13-hydroxyl or (and) C-19 carboxyl to form stevioside E (Reb E); then glycosyltransferase UGT76G1 relays to continuously catalyze the process of adding glucosyl group 3 in the direction of C13-hydroxyl or (and) C-19 carboxyl to form stevioside D and M (Reb D and Reb M). B, catalyzing the obtained stevioside substrate Reb E (without generating byproducts) by using a new enzyme B (Phe208Met/Phe379Ala), and further catalyzing the conversion process from Reb E to Reb D and Reb M by using a glycosyl transferase UGT76G 1. The reaction conditions in the liquid phase diagram from top to bottom are as follows: 0.03mg/mL glycosyltransferase UGT76G1 was added for 0 hour as a control, 0.03mg/mL glycosyltransferase UGT76G1 was added for 2 hours and 18 hours, and 0.15mg/mL glycosyltransferase UGT76G1 was added for 2 hours and 18 hours.

FIG. 10 SDS-PAGE analysis of the truncation of neoenzyme B (Phe208Met/Phe379Ala) after removal of amino acids 1-14;

comparative SDS-PAGE analysis of the neoenzyme B (Phe208Met/Phe379Ala) and of the truncations after removal of amino acids 1-14 of neoenzyme B. M, Marker; 1, new enzyme B; 2, samples through which a truncation of new enzyme B flowed from the Q column; 3-8 samples eluted from Q column with truncation of New enzyme B

FIG. 11 shows a comparison of the normal enzymatic reaction of beta (1-2) glycosidic bond formed by OsUGT91C1, the truncation of neoenzyme B after removal of amino acids 1-14, catalyzing the addition of glucose group 2 to the C13-hydroxy (R1) and/or C19-carboxy (R2) ends of the substrate Rubu, respectively;

OsUGT91C1(a) in a natural state and a truncation (B) of the neoenzyme B after removing amino acids 1 to 14 are compared with the solution phase analysis of the Rubu reaction. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.25mg/mL of the enzyme was added for 0 hour as a control, 0.05mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.25mg/mL of the enzyme was reacted for 2 hours and 18 hours. The products of OsUGT91C1 showing the natural state, in which glucosyl group No.2 is added at the C13-hydroxyl (R1) and C19-carboxyl (R2) ends of Rubu respectively, are increased along with the increase of time and enzyme amount, while the new enzyme B truncated body has the same enzymatic catalytic reaction and has better catalytic capability than the natural OsUGT91C1 as the new enzyme B. C, the natural state OsUGT91C1 and the novel enzyme B truncated body can carry out normal target reaction for catalyzing addition of No.2 glucosyl at the ends of C13-hydroxyl (R1) and C19-carboxyl (R2) of a substrate Rubu respectively, a reaction scheme for generating Reb E is generated, and the redundancy of amino acids at 1-14 positions is further verified, so that the redundancy can be completely removed or changed.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.

Firstly, construction of natural OsUGT91C1 protein expression vector

(1) Acquiring an amino acid Sequence (NCBI Reference Sequence: XP _015629141.1) of OsUGT91C1, and optimizing and synthesizing an OsUGT91C1 coding codon according to the requirements of a protein expression system. The encoding codons optimized by the inventors based on the expression system of E.coli are as follows. Due to codon degeneracy, codon optimization options can be varied.

In order to prevent the substitution of an unimportant amino acid sequence of OsUGT91C1 and to avoid the need for the patent claims, the translated protein is considered to belong to OsUGT91C1 if the degree of identity between the amino acid sequence of the protein and the amino acid sequence of OsUGT91C1 is 50% or more.

Whether in the amino acid sequence or the spatial structure of OsUGT91C1, the aim of improving the normal reaction of adding No.2 glucosyl to OsUGT91C1 or (and) eliminating the side reaction of OsUGT91C1 is achieved by changing the equivalent site of the amino acids at positions 208 and 379 or other sites (because of the redundancy of the amino acids, the amino acids at positions 208 and 379 are not always the numbers at positions 208 and 379 by removing one or any amino acids at positions 1-14).

OsUGT91C1 (bold amino acids are the modified amino acid sites in this patent, underlined is the artificially added amino acid sequence, wherein LE is for convenient ligation into E.coli expression vector pET21b, and HHHHHHHHHH (6 histidine tag) is for later purification):

nucleotide sequence after codon optimization of OsUGT91C1 (underlined sequence is coding region of the above-mentioned artificially added amino acid sequence):

atgGATAGCGGTTATAGTAGCAGTTATGCCGCAGCCGCCGGCATGCATGTTGTGATTTGCCCGTGGCTGGCCTTTGGTCATCTGCTGCCGTGCTTAGACCTGGCCCAGCGTCTGGCCAGCCGTGGTCACCGTGTTAGCTTTGTGAGCACCCCGCGTAATATCAGCCGTCTGCCGCCGGTTCGTCCGGCATTAGCCCCGCTGGTGGCATTTGTGGCCTTACCGCTGCCGCGTGTTGAGGGTCTGCCTGATGGCGCCGAAAGTACCAACGACGTGCCGCATGACCGCCCGGATATGGTGGAGCTGCATCGTCGCGCCTTTGATGGTCTGGCAGCCCCGTTTAGCGAGTTTCTGGGCACAGCCTGCGCCGATTGGGTGATCGTTGACGTGTTTCATCACTGGGCAGCCGCAGCCGCCCTGGAACATAAAGTTCCGTGCGCAATGATGCTGCTGGGTAGCGCCCACATGATTGCCAGCATTGCCGATCGTCGCCTGGAACGCGCAGAGACCGAAAGCCCGGCAGCAGCAGGTCAAGGTCGTCCTGCCGCAGCCCCGACCTTTGAAGTGGCCCGCATGAAACTGATCCGTACCAAAGGTAGTAGCGGCATGAGCCTGGCCGAACGCTTTAGCCTGACCCTGAGCCGCAGTAGCCTGGTGGTTGGTCGCAGTTGTGTGGAATTCGAGCCGGAAACAGTGCCGCTGCTGAGCACCCTGCGCGGCAAACCGATCACCTTTCTGGGCCTGATGCCGCCGTTACATGAAGGCCGTCGTGAAGATGGTGAAGATGCCACAGTGCGTTGGCTGGATGCACAGCCGGCCAAAAGCGTTGTGTACGTTGCCCTGGGTAGCGAAGTTCCTCTGGGTGTGGAAAAGGTGCACGAACTGGCACTGGGTCTGGAACTGGCCGGTACCCGCTTCCTGTGGGCCTTACGTAAACCTACCGGTGTTAGCGATGCCGATCTGCTGCCGGCAGGTTTTGAGGAACGTACCCGTGGTCGCGGTGTTGTGGCAACACGCTGGGTTCCGCAGATGAGCATTCTGGCCCATGCCGCCGTGGGTGCCTTTCTGACCCATTGTGGCTGGAATAGCACCATCGAAGGCCTGATGTTCGGCCATCCTCTGATCATGCTGCCTATCTTCGGTGATCAGGGTCCGAACGCACGCCTGATTGAAGCAAAGAATGCCGGTCTGCAGGTGGCACGTAACGATGGCGACGGTAGCTTCGATCGTGAAGGCGTTGCCGCCGCAATTCGCGCCGTTGCAGTTGAAGAAGAGAGCAGCAAGGTGTTCCAGGCCAAAGCCAAAAAACTGCAGGAGATCGTGGCCGATATGGCATGCCATGAGCGCTACATCGATGGCTTCATCCAGCAGCTGCGCAGCTATAAAGATctcgagcaccaccaccaccaccac

(2) the synthetic OsUGT91C1 coding region was amplified by the primers listed in Table 1, and the 5 'and 3' ends of the amplified fragment were provided with Nde I and Xho I restriction enzyme sites, respectively.

TABLE 1 primer sequences of pET21b-OsUGT91C1

(3) pET21b expression vector and the OsUGT91C1 coding region amplified fragment of step 2 were digested with Nde I and Xho I, and the coding region of OsUGT91C1 was ligated between Nde I and Xho I cleavage sites of pET21b vector with T4 ligase, to constitute pET21b-OsUGT91C1 expression vector for expressing glycosyltransferase OsUGT91C1 in E.coli.

(4) And transferring all the ligation products into 100 mu LE. coli DH5 alpha competent cells, selecting positive monoclonal colonies on a plate, inoculating the positive monoclonal colonies into 10mL LB culture medium, culturing at 200rpm and 37 ℃ for 12-16 hours, extracting plasmids, sequencing to verify the correctness of the expression plasmids, and completing the construction of the natural OsUGT91C1 glycosyltransferase expression vector.

Secondly, in order to enhance the normal reaction of adding No.2 glucosyl, constructing an expression vector of a novel enzyme A (Phe208 Met); in order to improve the target catalytic activity of the enzymatic addition of glucose group 2 and eliminate side reactions, an expression vector of a novel enzyme B (Phe208Met/Phe379Ala) was constructed

1. Construction of expression vector for novel enzyme A (Phe208Met)

(1) The site-directed mutagenesis primers used for the design of the novel enzyme A (Phe208Met) and the novel enzyme B (Phe208Met/Phe379Ala) using pET21B-OsUGT91C1 in its native state as a template are shown in Table 2.

Table 2 mutant primer sequences are as follows: (underlined part indicates the mutation site)

(2) By ddH₂O dissolves the primers of Table 2, diluting the primers to a concentration of 10. mu.M. PCR amplification is carried out by using a pair of mutation primers of Phe208Met and pET21b-OsUGT91C1 as a template, wherein the system is as follows: dNTP 4. mu.L, 5 XPS buffer 10. mu.L, upstream and downstream primers 2. mu.L each, template 1. mu.L (about 10ng), PCR amplification enzyme Primer star 0.5. mu.L, and the remainder made up to 50. mu.L with ddH 2O. And (3) uniformly mixing, and performing PCR amplification by using an amplification program: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 30s, annealing at 69 deg.C for 30s, extension at 72 deg.C for 8min, amplification for 20 cycles, extension at 72 deg.C for 10min, and storage at 4 deg.C.

(3) Taking 10 mu L of the amplification product out for agarose gel to verify the amplification effect of PCR, adding 1 mu L of LDpnI enzyme into the rest 40 mu L of system, incubating for 1-2 hours at 37 ℃, taking 10 mu L of the amplification product, transferring into 100 mu L of LE. coli DH5 alpha competent cells respectively, carrying out ice bath for 30min, carrying out heat shock for 2min at 42 ℃, adding fresh LB 300 mu L after ice bath for 3min, incubating for 1 hour at 200rpm and 37 ℃ by a shaking table, taking 150 mu L of the amplification product, uniformly spreading the 150 mu L of the amplification product on Amp resistant solid LB plates, and standing and culturing overnight at 37 ℃.

(4) And selecting monoclonal colonies on two plates, inoculating the monoclonal colonies into 10mL LB culture medium, culturing at 200rpm and 37 ℃ for 12-16 hours, extracting plasmids, and verifying the correctness of the Phe208Met at a mutation site by DNA sequencing, wherein the coded amino acid sequence is shown as SEQ ID No. 1.

2. Construction of expression vector for novel enzyme B (Phe208Met/Phe379Ala)

(1) Obtaining an expression vector of the new enzyme A (Phe208Met) which is subjected to sequencing verification in the step 4 of the 2.2.2.1 as a template, and constructing an expression vector of the new enzyme B (Phe208Met/Phe379 Ala);

(2) by ddH₂O dissolves the primers of Table 2, diluting the primers to a concentration of 10. mu.M. PCR amplification was performed using a pair of mutant primers for Phe379Ala and the expression vector for the novel enzyme A (Phe208Met) as template, as described above: dNTP 4. mu.L, 5 XPS buffer 10. mu.L, upstream and downstream primers 2. mu.L each, template 1. mu.L (about 10ng), PCR amplification enzyme Primer 0.5. mu.L, and ddH for the remainder₂The content of O is filled to 50 mu L. And (3) uniformly mixing, and performing PCR amplification by using an amplification program: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 30s, annealing at 68 deg.C for 30s, extension at 72 deg.C for 8min, amplification for 20 cycles, extension at 72 deg.C for 10min, and storage at 4 deg.C.

(4) Single colonies on two plates are picked and inoculated in 10mL LB culture medium, after 12-16 hours of culture at the temperature of 37 ℃ at 200rpm, plasmids are extracted, DNA sequencing verifies the correctness of two mutation sites Phe208Met and Phe379Ala, and the coded amino acid sequence is shown in SEQ ID No. 2.

Thirdly, the induced expression and purification of natural OsUGT91C1 and new enzyme A and B

(1) The expression plasmids corresponding to the three enzymes are transformed into an E.coli BL21(DE3) expression strain, a single clone is picked the next day and inoculated into 10mL of fresh LB culture medium, after the culture is carried out overnight at the temperature of 200rpm and 37 ℃, glycerol with the final concentration of 8 percent is added for seed preservation, and the strain can be preserved at the temperature of minus 80 ℃ for a long time.

(2) The glycerol E.coli BL21(DE3) strain preserved in the previous step was inoculated into 100mL of fresh LB medium containing Amp 50. mu.g/mL and cultured overnight at 200rpm and 37 ℃. The next day, the cells were inoculated into 1L of fresh LB medium containing Amp at an inoculation ratio of 1% and 50. mu.g/mL, cultured at 180rpm and 37 ℃ until OD600 became 1.0, the cells were cooled to 16 ℃, IPTG was added to a final concentration of 0.5mM, and expression was induced at 160rpm and 16 ℃ to 20 ℃ for 18 hours.

(3) After the induction expression is finished, centrifuging at 4000rpm for 15min, discarding the supernatant, and collecting the thalli. After the cells were resuspended in resuspension buffer (20mM Tris-HCl buffer pH 7.8,0.5M NaCl,30mM imidazole), they were repeatedly disrupted at 1000bar pressure for 3 times using a high pressure disrupter. 13500rpm, at 4 ℃ for 60min, the centrifuged supernatant was applied to an NTA-Ni column, and the non-specifically bound hetero-proteins were washed away with the above-mentioned resuspension buffer, and the target protein with the histidine tag was eluted with an elution buffer (20mM Tris-HCl buffer pH 7.8,0.5M NaCl,250mM imidazole). The eluted protein is further replaced into 20mM HEPES buffer pH 7.2 and 50mM NaCl, and after quick freezing by liquid nitrogen, the protein can be stored at-80 ℃ for a long time. The above procedure allowed for purification of OsUGT91C1, neoenzyme A, neoenzyme B in their native state, and purity was checked by SDS-PAGE (FIG. 2).

Fourthly, determination of the enzymatic Activity of the novel enzyme A (Phe208Met) and the novel enzyme B (Phe208Met/Phe379Ala)

1. Enzymatic product analysis showed that the novel enzyme A (Phe208Met) improved the catalytic ability to add a glucosyl group No.2 (forming a β (1-2) glycosidic bond with glucosyl group No. 1) in the direction of the C13-hydroxyl group, particularly the C19-carboxyl group

(1) Taking the substrate Rubu (CAS 64849-39-4) of OsUGT91C1 as an example, the OsUGT91C1 in a natural state can add glucose group No.2 (forming beta (1-2) glycosidic bond with glucose group No. 1) in both directions of C13-hydroxyl and C19-carboxyl of the substrate Rubu (normal reaction) (a in figure 3), the new enzyme A (Phe208Met) can also add No.2 glucosyl to both the C13-hydroxyl and the C19-carboxyl of the substrate Rubu, moreover, more products with the addition of the No.2 glucosyl group in the direction of C19-carboxyl can be obtained through catalysis, the target catalytic capability in the direction of C19-carboxyl is enhanced, and finally, the product Reb E (Rebaudioside E, CAS 63279-14-1) with the addition of the No.2 glucosyl group in both the directions of C13-hydroxyl and C19-carboxyl is more than the OsUGT91C1 (b in figure 3) in a natural state.

The reaction system of the example is as follows: at 20-40 ℃, 200 μ L of reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme A (Phe208Met)), at concentrations of 0.05mg/mL (1X) and 0.25mg/mL (5X), respectively, 0.3mM substrate Rubu. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butanol with the same volume, carrying out vortex oscillation, stopping the reaction and extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of the extract phase of the n-butanol at the upper layer, carrying out vacuum drying, carrying out heavy suspension with acetonitrile with 25 percent of the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive, and the same results can be obtained as long as the OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

The substrates for the natural OsUGT91C1 and the novel enzyme A (Phe208Met) to catalyze the normal reaction of adding glucose group No.2 to C13-hydroxyl or C-19 carboxyl are not exclusive, and include, but are not limited to, stevia substrates such as Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O-monogluside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1), and the normal reaction of adding glucose group No.2 to C13-hydroxyl or C-19 carboxyl can occur as long as glucose group No.1 is present in C13-hydroxyl or C-19 carboxyl direction but No. 3 is absent.

(2) Taking the substrate Reb A (Rebaudioside A, CAS 58543-16-1) of OsUGT91C1 as an example, the OsUGT91C1 in a natural state can add glucose group No.2 (forming beta (1-2) glycosidic bond with glucose group No. 1) in the C19-carboxyl direction of the substrate Reb A (normal reaction) (a in FIG. 4), while the new enzyme A (Phe208Met) can obviously take the lead of completely consuming the substrate Reb A and generating the target product Reb D (Rebaudioside D, CAS 63279-13-0), has enhanced target catalytic ability in the C19-carboxyl direction and enhanced ability of adding glucose group No.2 in the C19-carboxyl direction to form the corresponding product (b in FIG. 4).

The reaction system of the example is as follows: at 20-40 ℃, 200 μ L of reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme A (Phe208Met)), 0.3mM substrate Reb A at concentrations of 0.05mg/mL (1X) and 0.25mg/mL (5X), respectively. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butanol with the same volume, carrying out vortex oscillation, stopping the reaction and extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of the extract phase of the n-butanol at the upper layer, carrying out vacuum drying, carrying out heavy suspension with acetonitrile with 25 percent of the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive, and the same results can be obtained as long as the OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

The substrates of OsUGT91C1 and the novel enzyme A in a natural state catalyzing the normal reaction of adding the 2 nd glucosyl group in the C13-hydroxyl or C19 carboxyl direction are not exclusive, and include, but are not limited to, stevia substrates such as Rubu (Rubusoside, CAS 64849-39-4), S13G (CAS 60129-60-4) and Reb A (CAS 58543-16-1), and the normal reaction of adding the 2 nd glucosyl group in the C13-hydroxyl or C19 carboxyl direction can occur as long as the 1 st glucosyl group exists in the C13-hydroxyl or C19 carboxyl direction but the 3 rd glucosyl group does not exist.

2. Enzymatic product analysis showed that the novel enzyme B (Phe208Met/Phe379Ala) eliminates the side reaction of addition of glucose group No. 6 (or glucose group No. 4), but does not affect the catalytic ability to add glucose group No.2 (forming a beta (1-2) glycosidic bond with glucose group No. 1) in the C13-hydroxy and C19-carboxy directions

(1) Taking the substrate STB (Steviolbioside, CAS 41093-60-1) of OsUGT91C1 as an example, OsUGT91C1 in a natural state can catalyze the side reaction of adding glucose group 6 (or glucose group 4) on the substrate STB respectively (a in FIG. 5), the side reaction product of OsUGT91C1 in a natural state increases with time and the amount of enzyme, while the new enzyme B (Phe208Met/Phe379Ala) does not generate the side reaction product, and the side reaction of adding glucose group 6 (or glucose group 4) is eliminated (B in FIG. 5).

Examples of side reactions are as follows: at 20-40 ℃, 200 μ L of reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme B (Phe208Met/Phe379Ala)), at concentrations of 0.15mg/mL (1X) and 0.75mg/mL (5X), respectively, 0.3mM STB. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butanol with the same volume, carrying out vortex oscillation, stopping the reaction and extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of the extract phase of the n-butanol at the upper layer, carrying out vacuum drying, carrying out heavy suspension with acetonitrile with 25 percent of the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive, and the same results can be obtained as long as the OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

The substrate for catalyzing side reactions by OsUGT91C1 in a natural state is not exclusive, and includes but is not limited to side reactions on the following stevioside substrates, STB (Steviolbioside, CAS 41093-60-1), Reb E (Rebaudioside E, CAS 63279-14-1) and the like, and the side reaction of adding glucose group No. 6 (or glucose group No. 4) in the corresponding C13-hydroxyl or C-19 carboxyl direction can occur as long as glucose groups No.1 and No.2 exist in the C13-hydroxyl or C-19 carboxyl direction, but No. 3 (glucose group No. 3 refers to a glucose group which forms a beta (1-3) glycosidic bond with glucose group No. 1).

(2) Taking a substrate S13G (Steviol-13-O-monogluside, CAS 60129-60-4) of OsUGT91C1 as an example, OsUGT91C1 in a natural state firstly catalyzes a normal enzymatic reaction of adding No.2 glucosyl to a C13-hydroxyl (R1) end of a substrate S13G to generate STB, and then performs a side reaction on the STB, and adds No. 6 (or No. 4) glucosyl to generate a byproduct (a in FIG. 6); in contrast, the novel enzyme B (Phe208Met/Phe379Ala) catalyzes only the normal reaction of addition of a glucosyl group # 2 to the C13-hydroxy (R1) end of the substrate S13G to STB, does not catalyze side reactions, and does not produce by-products (B in FIG. 6).

The reaction conditions of the examples are as follows: at 20-40 ℃, 200 μ L of reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme B (Phe208Met/Phe379Ala)), at concentrations of 0.15mg/mL (1X) and 0.75mg/mL (5X), respectively, 0.3mM substrate S13G. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butanol with the same volume, carrying out vortex oscillation, stopping the reaction and extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of the extract phase of the n-butanol at the upper layer, carrying out vacuum drying, carrying out heavy suspension with acetonitrile with 25 percent of the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive, and the same results can be obtained as long as the OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

The substrates of OsUGT91C1 and the novel enzyme B (Phe208Met/Phe379Ala) in the natural state catalyzing the normal reaction of adding glucosyl group No.2 in the C13-hydroxy or C-19 carboxy direction are not exclusive, and include, but are not limited to, stevioside substrates such as 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 the normal reaction of adding glucosyl group No.2 in the C13-hydroxy or C-19 carboxy direction can occur as long as glucosyl group No.1 exists in the C13-hydroxy or C-19 carboxy direction but glucosyl group No. 3 does not exist; the substrate for catalyzing side reactions by OsUGT91C1 in a natural state is not exclusive, and includes but is not limited to side reactions on the following stevioside substrates, STB (Steviolbioside, CAS 41093-60-1), Reb E (Rebaudioside E, CAS 63279-14-1) and the like, and the side reaction of adding glucose group No. 6 (or glucose group No. 4) in the corresponding C13-hydroxyl or C-19 carboxyl direction can occur as long as glucose groups No.1 and No.2 exist in the C13-hydroxyl or C-19 carboxyl direction, but No. 3 (glucose group No. 3 refers to a glucose group which forms a beta (1-3) glycosidic bond with glucose group No. 1).

3. Enzymatic product analysis showed that the novel enzyme B (Phe208Met/Phe379Ala) enhances the catalytic ability to add glucose residue No.2 (forming a beta (1-2) glycosidic bond with glucose residue No. 1) in the direction of the C13-hydroxy group, particularly the C19-carboxy group

(1) Taking the substrate Rubu (Rubusoside, CAS 64849-39-4) of OsUGT91C1 as an example, the OsUGT91C1 in a natural state can add glucose group No.2 (forming beta (1-2) glycosidic bond with glucose group No. 1) in both directions of C13-hydroxyl and C19-carboxyl of the substrate Rubu (normal reaction) (a in figure 7), the novel enzyme B (Phe208Met/Phe379Ala) can also add glucose group No.2 to both the C13-hydroxyl and C19-carboxyl groups of the substrate Rubu, moreover, more products with the addition of the 2 nd glucosyl group in the C19-carboxyl direction can be obtained through catalysis, the target catalytic capability in the C19-carboxyl direction is enhanced, and finally, the product Reb E (Rebaudioside E, CAS 63279-14-1) with the addition of the 2 nd glucosyl group in both the C13-hydroxyl direction and the C19-carboxyl direction is also obviously more than the OsUGT91C1 (b in a figure 7) in a natural state.

The reaction system of the example is as follows: at 20-40 ℃, 200 μ L of reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme B (Phe208Met/Phe379Ala)), 0.3mM substrate Rubu, at concentrations of 0.05mg/mL (1X) and 0.25mg/mL (5X), respectively. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butanol with the same volume, carrying out vortex oscillation, stopping the reaction and extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of the extract phase of the n-butanol at the upper layer, carrying out vacuum drying, carrying out heavy suspension with acetonitrile with 25 percent of the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive, and the same results can be obtained as long as the OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

The substrates of OsUGT91C1 and the novel enzyme B (Phe208Met/Phe379Ala) in the native state catalyzing the normal reaction of adding No.2 glucosyl group in the C13-hydroxy or C-19 carboxy direction are not exclusive, and include, but are not limited to, stevia substrates such as Rubu (CAS 64849-39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1), and the normal reaction of adding No.2 glucosyl group in the C13-hydroxy or C-19 carboxy direction can occur as long as No.1 glucosyl group exists in the C13-hydroxy or C-19 carboxy direction, but No. 3 glucosyl group does not exist.

(2) Taking the substrate Reb A (Rebaudioside A, CAS 58543-16-1) of OsUGT91C1 as an example, the OsUGT91C1 in a natural state can add glucose group No.2 (forming beta (1-2) glycosidic bond with glucose group No. 1) in the C19-carboxyl direction of the substrate Reb A (normal reaction) (a in FIG. 8), while the neoenzyme B (Phe208Met/Phe379Ala) can obviously take the substrate Reb A completely consumed and generate the target product Reb D (Rebaudioside D, CAS 63279-13-0) at first, has enhanced target catalytic ability in the C19-carboxyl direction, and improves the ability of adding glucose group No.2 in the C19-carboxyl direction to form the corresponding product (B in FIG. 8).

The reaction system of the example is as follows: at 20-40 ℃, 200 μ L of reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme B (Phe208Met/Phe379Ala)), 0.3mM substrate Reb A at concentrations of 0.05mg/mL (1X) and 0.25mg/mL (5X), respectively. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butanol with the same volume, carrying out vortex oscillation, stopping the reaction and extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of the extract phase of the n-butanol at the upper layer, carrying out vacuum drying, carrying out heavy suspension with acetonitrile with 25 percent of the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive, and the same results can be obtained as long as the OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

4. The rate of normal reactions in which neoenzyme A (Phe208Met) and neoenzyme B (Phe208Met/Phe379Ala) catalyze the addition of a glucose group number 2 (forming a β (1-2) glycosidic bond with a glucose group number 1) to a steviol glycoside substrate was determined by the method of fluorescence conversion.

The new enzyme A (Phe208Met) enhances the reaction speed of adding No.2 glucosyl (forming beta (1-2) glycosidic bond with No.1 glucosyl) to stevioside substrates required for catalysis, and is more than 2 times of the activity of the natural OsUGT91C 1; the side reaction of adding No. 6 (or No. 4) glucosyl is eliminated by the novel enzyme B (Phe208Met/Phe379Ala), and simultaneously, the reaction speed of catalyzing the normally required addition of No.2 glucosyl (forming beta (1-2) glycosidic bond with No.1 glucosyl) to the stevioside substrate is enhanced, and is 3-7 times of the activity of the natural OsUGT91C1 (Table 3).

TABLE 3 kinetic parameters of OsUGT91C1, neoenzyme A (Phe208Met), neoenzyme B (Phe208Met/Phe379Ala) in its native state catalyzing the addition of glucosyl group 2 (normally required catalytic reaction) at the C13-hydroxy (R1) and C19-carboxy (R2) ends, respectively, of the substrates C13-hydroxy (R1)

(1) The assay was performed directly using a commercial glycosyltransferase activity kit, example UDP-Glo from Promega^TMThe same results can be obtained by using the Glycosyltransferase Assay kit and other methods for detecting the reaction speed of Glycosyltransferase.

UDP-Glo by Promega^TMThe Glycosyltransferase Assay kit is used for detecting the reaction speed of glucosyltransferase taking UDP-Glucose as a glycosyl donor, and the OsUGT91C1 and the two mutant neoenzymes related to the invention are both suitable.

Under the action of glucosyltransferase, the glycosyl donor UDP-Glucose transfers Glucose to a substrate to generate UDP. In this detection method, the amount of UDP or the like produced by the enzymatic glycosyltransfer reaction can be detected by converting the amount (1:1) of UDP or the like produced into ATP, which can cause luciferase to emit a quantitative fluorescence. When UDP was produced in the concentration range of 0 to 25. mu.M, the fluorescence intensity was linearly related to the molar concentration of UDP. According to the standard curve of the corresponding relation between the UDP concentration and the fluorescence intensity, the generation amount of the UDP is calculated, and the conversion into the catalytic reaction speed of the glycosyltransferase is realized.

(2) A series of solutions of different concentrations of UDP were prepared and the corresponding fluorescence intensities were determined according to the above-mentioned conversion reaction. The natural state OsUGT91C1 and the two novel enzymes related to the invention detect information such as speed, enzyme kinetic constants and the like of various catalytic reactions by using different substrates, and are used for evaluating the effect of the novel enzyme A (Phe208Met) and the novel enzyme B (Phe208Met/Phe379Ala) on promoting normal reaction of adding No.2 glucosyl. The catalytic ability of the neoenzyme A (Phe208Met) and the neoenzyme B (Phe208Met/Phe379Ala) to add the No.2 glucosyl group to the C13-hydroxyl group and the C19-carboxyl group is respectively detected by using Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1) as substrates, and compared with the catalytic ability of natural OsUGT91C 1. The substrates of OsUGT91C1 and the novel enzymes A (Phe208Met), B (Phe208Met/Phe379Ala) in their native state catalyzing the normal reaction of adding glucose group 2 in the C13-hydroxy or C-19 carboxy direction are not exclusive, and include, but are not limited to, stevioside substrates such as Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O-monogluside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1), and the normal reaction of adding glucose group 2 in the C13-hydroxy or C-19 carboxy direction can occur as long as glucose group 1 is present in the C13-hydroxy or C-19 carboxy direction, but no glucose group 3 is present.

(3) The enzyme kinetic constants of the natural state OsUGT91C1 and the new enzyme A (Phe208Met) and the new enzyme B (Phe208Met/Phe379Ala) for different substrates are shown in Table 3, and the new enzyme A (Phe208Met) enhances the reaction speed of the addition of the No.2 glucosyl group (forming a beta (1-2) glycosidic bond with the No.1 glucosyl group) for catalyzing the normal reaction, which is more than 2 times of the activity of the natural state OsUGT91C 1; the side reaction of adding No. 6 (or No. 4) glucosyl is eliminated by the novel enzyme B (Phe208Met/Phe379Ala), and simultaneously, the reaction speed of catalyzing the normally required addition of No.2 glucosyl (forming beta (1-2) glycosidic bond with No.1 glucosyl) to the stevioside substrate is enhanced, and is 3-7 times of the activity of the natural OsUGT91C1 (Table 3).

And fifthly, the novel enzyme B (Phe208Met/Phe379Ala) has no side reaction, improves the target catalytic capacity, and can catalyze and complete the normal reaction of adding No.2 glucosyl in the direction of C13-hydroxyl or (and) C-19 carboxyl of a stevioside substrate to generate a series of stevioside products including Rebaudioside E (CAS 63279-14-1). Under the condition, the glycosyltransferase UGT76G1 is utilized to add glucose group No. 3 in the direction of C13-hydroxyl or (and) C-19 carboxyl, thus obtaining the stevioside D and M (Rebaudioside D, CAS 63279-13-0 and Rebaudioside M, CAS 1220616-44-3) (figure 9).

Sixthly, the amino acid sequences of the novel enzyme A (Phe208Met) and the novel enzyme B (Phe208Met/Phe379Ala) have redundancy, and any one of the amino acids at positions 1 to 14 may be removed or changed.

Taking the example of the novel enzyme B (Phe208Met/Phe379Ala), the novel enzyme B is subjected to removal of all amino acids from positions 1 to 14 (MDSGYSSSYAAAAG) to obtain a novel enzyme B truncated body which can still be normally expressed and purified and shows the activity of the normal reaction of adding glucosyl group No.2 to the direction of C13-hydroxyl or C-19 carboxyl of a steviol glycoside substrate.

1. To verify the redundancy of amino acids 1 to 14 (MDSGYSSSYAAAAG), an expression vector was constructed in which all amino acids 1 to 14 (MDSGYSSSYAAAAG) (truncation of neozyme B) were deleted on an expression vector for neozyme B (Phe208Met/Phe379Ala)

(1) Using the expression plasmid for the novel enzyme B (Phe208Met/Phe379Ala) as a template, primers in which all amino acid truncations at positions 1 to 14 were removed were designed as shown in Table 4.

Table 4 mutant primer sequences are as follows:

(2) by ddH₂O dissolves the primers of Table 4, diluting the primers to a concentration of 10. mu.M. The new enzyme B (Phe 208M) was added to the primer pair for 1-14-F removal and 1-14-R removal, respectivelyet/Phe379Ala) as a template for PCR amplification, the same system is: dNTP 4. mu.L, 5 XPS buffer 10. mu.L, upstream and downstream primers 2. mu.L each, template 1. mu.L (about 10ng), PCR amplification enzyme Primer 0.5. mu.L, and ddH for the remainder₂The content of O is filled to 50 mu L. And (3) uniformly mixing, and performing PCR amplification by using an amplification program: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 30s, annealing at 69 deg.C for 30s, extension at 72 deg.C for 8min, amplification for 20 cycles, extension at 72 deg.C for 10min, and storage at 4 deg.C.

(3) And taking 10 mu L of each amplification product out for agarose gel to verify the amplification effect of PCR, adding 1 mu L of DpnI enzyme into the remaining 40 mu L of system, incubating at 37 ℃ for 1-2 hours, taking 10 mu L of the mixture, transferring the 10 mu L of the mixture into 100 mu L of E.coli DH5 alpha competent cells, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 2min, carrying out ice bath for 3min, adding 300 mu L of fresh LB, carrying out shake culture at 200rpm and 37 ℃ for 1 hour, taking 150 mu L of the mixture, uniformly spreading the mixture on Amp-resistant solid LB plates, and standing and culturing at 37 ℃ overnight.

(4) And selecting monoclonal colonies on two plates, inoculating the colonies in 10mL LB culture medium, culturing at 200rpm and 37 ℃ for 12-16 hours, extracting plasmids, and verifying the expression plasmid of the new enzyme B truncated body by DNA sequencing to successfully remove all amino acids at 1-14 th positions except the amino acids at 1-14 th positions and maintain the amino acid sequences at the rest positions consistent with SEQ ID No. 2.

2. Inducible expression and purification of the truncation after removal of all amino acids 1 to 14 by the novel enzyme B (Phe208Met/Phe379Ala)

(1) The corresponding expression plasmid is transformed into an E.coli BL21(DE3) expression strain, a single clone is selected and inoculated in 10mL of fresh LB culture medium the next day, after culturing overnight at 200rpm and 37 ℃, glycerol with the final concentration of 8% is added for seed preservation, and the strain can be preserved at-80 ℃ for a long time.

(3) After the induction expression is finished, centrifuging at 4000rpm for 15min, discarding the supernatant, and collecting the thalli. After the cells were resuspended in resuspension buffer (20mM Tris-HCl buffer pH 7.8,0.5M NaCl,30mM imidazole), they were repeatedly disrupted at 1000bar pressure for 3 times using a high pressure disrupter. 13500rpm, at 4 ℃ for 60min, the centrifuged supernatant was applied to an NTA-Ni column, and the non-specifically bound hetero-proteins were washed away with the above-mentioned resuspension buffer, and the target protein with the histidine tag was eluted with an elution buffer (20mM Tris-HCl buffer pH 7.8,0.5M NaCl,250mM imidazole). The eluted protein is further replaced into 20mM HEPES buffer pH 7.2 and 50mM NaCl, and after quick freezing by liquid nitrogen, the protein can be stored at-80 ℃ for a long time. The above procedure resulted in the purification of a truncated form of the novel enzyme B and the purity was checked by SDS-PAGE (FIG. 10). As can be seen from FIG. 10, when the novel enzyme B is removed from the 1 st to 14 th amino acid truncates, normal expression and purification can be achieved. Since the novel enzyme A and the novel enzyme B have the same amino acids at positions 1 to 14, the example in which the amino acids at positions 1 to 14 are completely removed for the novel enzyme B shows that the amino acids at positions 1 to 14 have redundancy. Similarly, amino acids 1 to 14 of the novel enzyme A are also redundant.

3. The truncation of the novel enzyme B does not affect the normal reaction of adding glucosyl group No.2 in both directions of C13-hydroxyl and C-19 carboxyl of a stevioside substrate, has higher catalytic activity than that of natural trans-glycosyltransferase OsUGT91C1 as the novel enzyme B, and further shows the redundancy of amino acids 1-14 in the novel enzyme A and the novel enzyme B.

(1) Taking a stevioside substrate Rubu (Rubusoside, CAS 64849-39-4) as an example, the OsUGT91C1 in a natural state can add glucose group No.2 (forming a beta (1-2) glycosidic bond with glucose group No. 1) in both directions of C13-hydroxyl and C19-carboxyl of the substrate Rubu (normal reaction) (a in FIG. 11), and the truncated body of the neoenzyme B can also add glucose group No.2 in both directions of C13-hydroxyl and C19-carboxyl of the substrate Rubu (B in FIG. 11).

(2) The reaction system of the example is as follows: at 20-40 ℃, 200 μ L of reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme B truncates) at concentrations of 0.05mg/mL (1X) and 0.25mg/mL (5X), 0.3mM substrate Rubu, respectively. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butanol with the same volume, carrying out vortex oscillation, stopping the reaction and extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of the extract phase of the n-butanol at the upper layer, carrying out vacuum drying, carrying out heavy suspension with acetonitrile with 25 percent of the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive, and the same results can be obtained as long as the OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

(3) The substrates of the OsUGT91C1 and the neozyme B truncated body in the natural state catalyzing the normal reaction of adding the 2 nd glucosyl group in the C13-hydroxyl or C-19 carboxyl direction are not exclusive, and include, but are not limited to, stevioside substrates such as 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 the normal reaction of adding the 2 nd glucosyl group in the C13-hydroxyl or C-19 carboxyl direction can occur as long as the 1 st glucosyl group exists in the C13-hydroxyl or C-19 carboxyl direction but the 3 rd glucosyl group does not exist.

Sequence listing

<110> Sichuan university

<120> glycosyltransferase OsUGT91C1 mutant and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 462

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Asp Ser Gly Tyr Ser Ser Ser Tyr Ala Ala Ala Ala Gly Met His

1 5 10 15

Val Val Ile Cys Pro Trp Leu Ala Phe Gly His Leu Leu Pro Cys Leu

20 25 30

Asp Leu Ala Gln Arg Leu Ala Ser Arg Gly His Arg Val Ser Phe Val

35 40 45

Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro Pro Val Arg Pro Ala Leu

50 55 60

Ala Pro Leu Val Ala Phe Val Ala Leu Pro Leu Pro Arg Val Glu Gly

65 70 75 80

Leu Pro Asp Gly Ala Glu Ser Thr Asn Asp Val Pro His Asp Arg Pro

85 90 95

Asp Met Val Glu Leu His Arg Arg Ala Phe Asp Gly Leu Ala Ala Pro

100 105 110

Phe Ser Glu Phe Leu Gly Thr Ala Cys Ala Asp Trp Val Ile Val Asp

115 120 125

Val Phe His His Trp Ala Ala Ala Ala Ala Leu Glu His Lys Val Pro

130 135 140

Cys Ala Met Met Leu Leu Gly Ser Ala His Met Ile Ala Ser Ile Ala

145 150 155 160

Asp Arg Arg Leu Glu Arg Ala Glu Thr Glu Ser Pro Ala Ala Ala Gly

165 170 175

Gln Gly Arg Pro Ala Ala Ala Pro Thr Phe Glu Val Ala Arg Met Lys

180 185 190

Leu Ile Arg Thr Lys Gly Ser Ser Gly Met Ser Leu Ala Glu Arg Met

195 200 205

Ser Leu Thr Leu Ser Arg Ser Ser Leu Val Val Gly Arg Ser Cys Val

210 215 220

Glu Phe Glu Pro Glu Thr Val Pro Leu Leu Ser Thr Leu Arg Gly Lys

225 230 235 240

Pro Ile Thr Phe Leu Gly Leu Met Pro Pro Leu His Glu Gly Arg Arg

245 250 255

Glu Asp Gly Glu Asp Ala Thr Val Arg Trp Leu Asp Ala Gln Pro Ala

260 265 270

Lys Ser Val Val Tyr Val Ala Leu Gly Ser Glu Val Pro Leu Gly Val

275 280 285

Glu Lys Val His Glu Leu Ala Leu Gly Leu Glu Leu Ala Gly Thr Arg

290 295 300

Phe Leu Trp Ala Leu Arg Lys Pro Thr Gly Val Ser Asp Ala Asp Leu

305 310 315 320

Leu Pro Ala Gly Phe Glu Glu Arg Thr Arg Gly Arg Gly Val Val Ala

325 330 335

Thr Arg Trp Val Pro Gln Met Ser Ile Leu Ala His Ala Ala Val Gly

340 345 350

Ala Phe Leu Thr His Cys Gly Trp Asn Ser Thr Ile Glu Gly Leu Met

355 360 365

Phe Gly His Pro Leu Ile Met Leu Pro Ile Phe Gly Asp Gln Gly Pro

370 375 380

Asn Ala Arg Leu Ile Glu Ala Lys Asn Ala Gly Leu Gln Val Ala Arg

385 390 395 400

Asn Asp Gly Asp Gly Ser Phe Asp Arg Glu Gly Val Ala Ala Ala Ile

405 410 415

Arg Ala Val Ala Val Glu Glu Glu Ser Ser Lys Val Phe Gln Ala Lys

420 425 430

Ala Lys Lys Leu Gln Glu Ile Val Ala Asp Met Ala Cys His Glu Arg

435 440 445

Tyr Ile Asp Gly Phe Ile Gln Gln Leu Arg Ser Tyr Lys Asp

450 455 460

<210> 2

<211> 462

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Asp Ser Gly Tyr Ser Ser Ser Tyr Ala Ala Ala Ala Gly Met His

1 5 10 15

Val Val Ile Cys Pro Trp Leu Ala Phe Gly His Leu Leu Pro Cys Leu

20 25 30

Asp Leu Ala Gln Arg Leu Ala Ser Arg Gly His Arg Val Ser Phe Val

35 40 45

Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro Pro Val Arg Pro Ala Leu

50 55 60

Ala Pro Leu Val Ala Phe Val Ala Leu Pro Leu Pro Arg Val Glu Gly

65 70 75 80

Leu Pro Asp Gly Ala Glu Ser Thr Asn Asp Val Pro His Asp Arg Pro

85 90 95

Asp Met Val Glu Leu His Arg Arg Ala Phe Asp Gly Leu Ala Ala Pro

100 105 110

Phe Ser Glu Phe Leu Gly Thr Ala Cys Ala Asp Trp Val Ile Val Asp

115 120 125

Val Phe His His Trp Ala Ala Ala Ala Ala Leu Glu His Lys Val Pro

130 135 140

Cys Ala Met Met Leu Leu Gly Ser Ala His Met Ile Ala Ser Ile Ala

145 150 155 160

Asp Arg Arg Leu Glu Arg Ala Glu Thr Glu Ser Pro Ala Ala Ala Gly

165 170 175

Gln Gly Arg Pro Ala Ala Ala Pro Thr Phe Glu Val Ala Arg Met Lys

180 185 190

Leu Ile Arg Thr Lys Gly Ser Ser Gly Met Ser Leu Ala Glu Arg Met

195 200 205

Ser Leu Thr Leu Ser Arg Ser Ser Leu Val Val Gly Arg Ser Cys Val

210 215 220

Glu Phe Glu Pro Glu Thr Val Pro Leu Leu Ser Thr Leu Arg Gly Lys

225 230 235 240

Pro Ile Thr Phe Leu Gly Leu Met Pro Pro Leu His Glu Gly Arg Arg

245 250 255

Glu Asp Gly Glu Asp Ala Thr Val Arg Trp Leu Asp Ala Gln Pro Ala

260 265 270

Lys Ser Val Val Tyr Val Ala Leu Gly Ser Glu Val Pro Leu Gly Val

275 280 285

Glu Lys Val His Glu Leu Ala Leu Gly Leu Glu Leu Ala Gly Thr Arg

290 295 300

Phe Leu Trp Ala Leu Arg Lys Pro Thr Gly Val Ser Asp Ala Asp Leu

305 310 315 320

Leu Pro Ala Gly Phe Glu Glu Arg Thr Arg Gly Arg Gly Val Val Ala

325 330 335

Thr Arg Trp Val Pro Gln Met Ser Ile Leu Ala His Ala Ala Val Gly

340 345 350

Ala Phe Leu Thr His Cys Gly Trp Asn Ser Thr Ile Glu Gly Leu Met

355 360 365

Phe Gly His Pro Leu Ile Met Leu Pro Ile Ala Gly Asp Gln Gly Pro

370 375 380

Asn Ala Arg Leu Ile Glu Ala Lys Asn Ala Gly Leu Gln Val Ala Arg

385 390 395 400

Asn Asp Gly Asp Gly Ser Phe Asp Arg Glu Gly Val Ala Ala Ala Ile

405 410 415

Arg Ala Val Ala Val Glu Glu Glu Ser Ser Lys Val Phe Gln Ala Lys

420 425 430

Ala Lys Lys Leu Gln Glu Ile Val Ala Asp Met Ala Cys His Glu Arg

435 440 445

Tyr Ile Asp Gly Phe Ile Gln Gln Leu Arg Ser Tyr Lys Asp

450 455 460

Claims

1. The glycosyltransferase OsUGT91C1 mutant has an amino acid sequence of any one of the following a, b, C and d:

a. SEQ ID No. 1;

b. SEQ ID No. 2;

2. An expressed gene encoding the glycosyltransferase OsUGT91C1 mutant of claim 1.

3. An expression vector comprising the expressed gene of claim 2.

4. A recombinant bacterium or cell carrying the expression vector of claim 3.

5. Use of the glycosyltransferase OsUGT91C1 mutant of claim 1 in the enzymatic synthesis or conversion of steviol glycoside D or M and other steviol glycoside molecules.

6. The use according to claim 5, wherein the glycosyltransferase OsUGT91C1 mutant is used for enzymatic synthesis or conversion of stevioside D or M and other stevioside molecules by adding a 2-glucosyl group in the direction of C13-hydroxyl or/and C19-carboxyl of stevioside, wherein the 2-glucosyl group is a glucosyl group which forms a beta (1-2) glycosidic bond with the 1-glucosyl group, and the 1-glucosyl group is a glucosyl group which directly forms a beta-glycosidic bond with C13-hydroxyl or C19-carboxyl.