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CN-119931981-B - Glycosyltransferase UGT76G1 mutant and method for catalyzing and synthesizing rebaudioside A by using same

CN119931981BCN 119931981 BCN119931981 BCN 119931981BCN-119931981-B

Abstract

The invention discloses a glycosyltransferase UGT76G1 mutant and a method for synthesizing rebaudioside A by catalyzing the same, belonging to the technical field of biocatalysis synthesis. The glycosyltransferase UGT76G1 mutant is any one of (A) to (C) obtained by mutating 109 th amino acid from L to Q, 113 th amino acid from S to C, 424 th amino acid from I to F on the basis of the amino acid sequence shown in SEQ ID NO.1, and (B) a protein which has more than 95% of identity with the amino acid sequence defined in (A) and has the same function, and (C) a fusion protein obtained by connecting the tail ends of the proteins defined in (A) or (B) with a tag. The glycosyltransferase UGT76G1 mutant has high enzyme activity, and can efficiently synthesize rebaudioside A by taking stevioside STV as a substrate.

Inventors

  • ZHU LIPING
  • ZHU LIN
  • TANG SHUANGYAN
  • SONG WEICAI
  • XU LIANGPING
  • LIANG CHAONING
  • LI SHIZHONG
  • FENG XINYI
  • WANG LUMING
  • LEI YANYAN

Assignees

  • 东台市浩瑞生物科技有限公司

Dates

Publication Date
20260505
Application Date
20240529

Claims (10)

  1. 1. A glycosyltransferase UGT76G1 mutant, wherein the UGT76G1 mutant is any one of the following (a) - (B): (A) The protein obtained by the following mutation is generated on the basis of the amino acid sequence shown in SEQ ID NO. 1: Mutating the 109 th amino acid from L to Q; the 113 th amino acid is mutated from S to C; mutation of amino acid 424 from I to F; (B) A fusion protein obtained by ligating a tag to the terminal of the protein defined in (A).
  2. 2. Glycosyltransferase UGT76G1 mutant according to claim 1, wherein the amino acid sequence of the UGT76G1 mutant is shown in SEQ ID No. 3.
  3. 3. Any of the following biomaterials: (A) An expressed gene encoding the UGT76G1 mutant of claim 1 or 2; (B) A recombinant plasmid having the expression gene of (A) linked thereto; (C) A recombinant cell comprising an expressed gene of the recombinant plasmid or UGT76G1 mutant.
  4. 4. An enzyme composition comprising a glycosyltransferase UGT76G1 mutant and a sucrose synthase AtSUS, wherein the glycosyltransferase UGT76G1 mutant is as set forth in claim 1 or 2; the amino acid sequence of the sucrose synthase AtSUS is shown in SEQ ID NO. 5.
  5. 5. A recombinant strain set for expressing the enzyme composition according to claim 4, which comprises a recombinant strain A and a recombinant strain B, wherein the recombinant strain A contains a recombinant plasmid A obtained by constructing a coding gene of the glycosyltransferase UGT76G1 mutant according to claim 1 or 2 into an expression vector, and the recombinant strain B contains a recombinant plasmid B obtained by constructing a coding gene of sucrose synthase AtSUS into a plasmid.
  6. 6. The recombinant strain set of claim 5, wherein the host bacteria include, but are not limited to, E.coli, saccharomyces cerevisiae, pichia pastoris, or Corynebacterium glutamicum.
  7. 7. A method for catalyzing and synthesizing rebaudioside a by utilizing a glycosyltransferase UGT76G1 mutant, comprising the steps of: Adding stevioside STV, UDPG, sucrose and the enzyme composition of claim 4 or the induced expression enzyme product of the complete recombinant strain of claim 5 into a catalytic reaction system, reacting, inactivating enzyme, and centrifuging to obtain a supernatant, wherein the supernatant contains rebaudioside A.
  8. 8. The method of claim 7, wherein the inducible expression of the enzyme product by the recombinant strain set of claim 5 comprises inducible expression of enzyme product a and inducible expression of enzyme product B; the method for obtaining the induced expression enzyme product A comprises the following steps: Inoculating the recombinant strain A seed solution in claim 5 into a culture medium containing kanamycin sulfate, adding L-arabinose to continuously induce and culture for 8-40 h when the OD 600 of the culture solution reaches 0.6-0.8, centrifuging, collecting thalli, breaking cells, centrifuging, and collecting supernatant to obtain an induced expression enzyme product A, wherein the seed solution is inoculated at a volume ratio of 1%, the final concentration of kanamycin sulfate is 10-100 mug/mL, and the final concentration of L-arabinose is 0.1-15 mM; The method for obtaining the induced expression enzyme product B comprises the following steps: Inoculating the recombinant strain B seed solution in claim 5 into a culture medium containing kanamycin sulfate, culturing at 30-40 ℃ for 200-300 r/min until OD 600 reaches 0.6-0.8, adding L-arabinose, continuously culturing for 8-40 h, centrifuging, collecting thalli, crushing cells, centrifuging, collecting supernatant to obtain an induced expression enzyme product B, wherein the seed solution is inoculated at a volume ratio of 1%, the final concentration of kanamycin sulfate is 10-100 mu g/mL, and the final concentration of L-arabinose is 0.1-15 mM.
  9. 9. The method according to claim 7 or 8, wherein the concentration of stevioside STV in the catalytic reaction system is 10-100 mM, the concentration of UDPG is 0.1-5 mM, the concentration of sucrose is 50-800 mM, the addition amount of the inducible expression enzyme product of the recombinant strain A is 0.1-50 mL, and the addition amount of the inducible expression enzyme product of the recombinant strain B is 0.1-50 mL.
  10. 10. The method according to claim 7, wherein the pH value in the catalytic reaction system is 5.0-8.0, the temperature is 25-60 ℃, and the reaction time is 5-30 h.

Description

Glycosyltransferase UGT76G1 mutant and method for catalyzing and synthesizing rebaudioside A by using same Technical Field The invention relates to the technical field of biocatalysis synthesis, in particular to a glycosyltransferase UGT76G1 mutant and a method for synthesizing rebaudioside A by catalysis. Background Rebaudioside a is a natural sweetener extracted from stevia plant leaves, is calorie-free, does not participate in human blood glucose metabolism, is friendly to diabetics, and is approved as a food additive by several food safety authorities internationally, and is considered to be safe within a prescribed range of use. Rebaudioside a is known for its taste more closely resembling sucrose than other steviol glycosides in stevia rebaudiana extracts. It has no obvious after-bitter taste or metallic taste, purer and sweet taste, good stability at high temperature, and no obvious sweet taste change or degradation caused by heating, and is suitable for the formula of hot processed food and beverage. Rebaudioside a is very high in sweetness, generally considered to be 200 to 450 times the sweetness of sucrose, and because of its extremely high sweetness, a sweetness effect comparable to sucrose can be achieved with very little amount, thereby significantly reducing caloric intake of a food or beverage. Because of the low calorie nature of rebaudioside a, rebaudioside a is widely used in the development of various low-sugar, sugarless, reduced-sugar products, such as beverages, candies, baked goods, condiments, table sugar, and the like, meeting the needs of consumers for healthy diets. Typically, rebaudioside a is extracted from stevia leaves by separation and purification techniques, but the yield of rebaudioside a obtained by this method is relatively low and cannot meet the high demands of the market. Disclosure of Invention Accordingly, the present invention is directed to a glycosyltransferase UGT76G1 mutant and a method for synthesizing rebaudioside a by catalyzing the same, which are used for overcoming the problem of low rebaudioside a yield in the prior art. In a first aspect, the present invention provides a glycosyltransferase UGT76G1 mutant, the UGT76G1 mutant being any one of the following (a) - (C): (A) A protein obtained by mutating any one or more of the following on the basis of the amino acid sequence shown in SEQ ID NO. 1: Mutating the 109 th amino acid from L to Q; the 113 th amino acid is mutated from S to C; mutation of amino acid 424 from I to F; (B) A protein having 95% or more identity with the amino acid sequence defined in (A) and having the same function; (C) A fusion protein obtained by ligating a tag to the terminal of the protein defined in (A) or (B). Compared with the prior art, the glycosyltransferase UGT76G1 mutant provided by the invention has higher enzyme activity compared with the wild glycosyltransferase UGT76G1, and can efficiently synthesize rebaudioside A by taking stevioside STV as a substrate. Wherein the optimal mutant 68S can be in 18, and 60mM stevioside STV is converted into 60mM rebaudioside A, namely, all stevioside STV is converted into rebaudioside A, so that the method has excellent application prospect in industrial production. Further, the amino acid sequence of the glycosyltransferase UGT76G1 mutant is shown as SEQ ID NO. 3. In a second aspect, the present invention provides a biomaterial as described in any one of the following: (A) An expressed gene encoding the above glycosyltransferase UGT76G1 mutant; (B) A recombinant plasmid having the expression gene of (A) linked thereto; (C) A recombinant cell comprising an expressed gene of the recombinant expression plasmid or UGT76G1 mutant. The expression gene is obtained by mutating SEQ ID NO.2 by one or more of the following: the 325 th to 327 th bit of SEQ ID NO.2 is replaced by CAG; the 337-339 bits of SEQ ID NO.2 are replaced by TGC by AGC; Positions 1270-1272 of SEQ ID NO.2 are replaced by TTG by ATC; preferably, the nucleotide sequence of the expressed gene is shown as SEQ ID NO. 4. In a third aspect, the invention provides an enzyme composition comprising a glycosyltransferase UGT76G1 mutant and a sucrose synthase AtSUS; Sucrose synthase AtSUS is (B1) or (B2), wherein the amino acid sequence of (B1) is shown as SEQ ID NO. 5; (B2) A protein having 95% or more identity with the amino acid sequence defined in (B1) and having the same function. In a fourth aspect, the invention provides a complete set of recombinant strains for expressing the enzyme composition, which comprises a recombinant strain A and a recombinant strain B, wherein the recombinant strain A contains a recombinant plasmid A, the recombinant plasmid A is obtained by constructing a mutant coding gene of glycosyltransferase UGT76G1 into an expression vector, the recombinant strain B contains a recombinant plasmid B, the recombinant plasmid B is obtained by constructing a coding gene of sucrose synthase AtSUS into a plasmid, and