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CN-121427864-B - L204A mutant enzyme for preparing rebaudioside I and application thereof

CN121427864BCN 121427864 BCN121427864 BCN 121427864BCN-121427864-B

Abstract

The invention relates to the technical field of biocatalysis, and discloses an L204A mutant enzyme for preparing rebaudioside I and application thereof, wherein the enzyme is obtained by mutating UGT76G1 into L204A, namely, the leucine of the 204 th amino acid sequence of UGT76G1 is mutated into alanine, the high-efficiency in-vitro enzyme method for preparing the Rebaudioside I (RI) is realized for the first time, the catalytic efficiency is improved by more than 7 times (from about 7% to more than or equal to 50%) compared with the original enzyme, the high-efficiency performance can be stably maintained under different scales of 10mL to 5L, a solid foundation is laid for industrialized application of the RI, the preparation method is simple, the process condition is controllable, and the method is suitable for popularization and application.

Inventors

  • GOU RONG
  • YANG ZHIRONG
  • LI PENGJING
  • LIU RENBIN
  • LI SHIYOU

Assignees

  • 成都圆大生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260104

Claims (8)

  1. 1. An L204A mutant of UGT76G1, which is characterized in that, Leucine at amino acid 204 of UGT76G1 sequence was mutated to alanine.
  2. 2. A nucleic acid molecule encoding a mutant L204A of UGT76G1 as claimed in claim 1, wherein the sequence of said nucleic acid molecule is as set forth in SEQ ID No. 1.
  3. 3. An expression system comprising the nucleic acid molecule of claim 2 and a substance that is required to facilitate transcription of the nucleic acid and translation of the mRNA.
  4. 4. An expression system according to claim 3, wherein the system is a microbial cell or a plant cell.
  5. 5. Use of an L204A mutant of UGT76G1, the nucleic acid molecule of claim 2, or an expression system of claim 3 or 4 to catalyze the conversion of rebaudioside a to rebaudioside I.
  6. 6. A method of preparing rebaudioside I comprising the steps of: Adding a rebaudioside a containing feedstock followed by a catalyst comprising the UGT76G1 mutant of claim 1, controlling reaction conditions, catalyzing the conversion of rebaudioside a to rebaudioside I.
  7. 7. A method of preparing rebaudioside I according to claim 6, wherein the catalyst comprising the L204A mutant of UGT76G1 according to claim 1 is pure L204A mutant protein of UGT76G1 or a cleavage mixture or expression system suspension of an expression system according to claim 3 or 4.
  8. 8. The method of preparing rebaudioside I according to claim 6, wherein the raw material containing rebaudioside a is a plant extract or pure material.

Description

L204A mutant enzyme for preparing rebaudioside I and application thereof Technical Field The invention relates to the technical field of biocatalysis, in particular to L204A mutant enzyme for preparing rebaudioside I and application thereof. Background The statements in this section merely provide background information related to the present disclosure and may not constitute prior art. The Rebaudioside I is a natural non-caloric sweetener separated from stevia rebaudiana Bertoni (S. rebaudiana Morita), is a steviolbioside, has the English name of rebaudiosid I (RI for short), has sweetness of about 200-300 times that of sucrose, is more pure in sweetness, has a rear bitter taste and similar liquorice aftertaste which are obviously lower than those of Rebaudioside A (RA for short) and other steviolbiosides (stevioside for short), has high sweetness and low caloric characteristics, and is suitable for diabetics or weight-reducing people. Is stable to heat, acid and alkali (not decomposed below 200deg.C), and can be used in food industry such as baking and beverage. The food is not decomposed by human digestive enzymes, almost does not generate heat, is discharged through kidney shape after being eaten, has no teratogenesis or carcinogenesis risk, does not need strict limit for daily intake, and has wide food application prospect. However, its extremely low content in natural stevia leaves constitutes a primary limitation in its commercial development. Typical stevia rebaudiana dry leaves have a total stevioside content of about 10% to 20% (dry weight), while rebaudioside I is typically only 0.2% to 0.6% by weight of the dry leaf, much less than rebaudioside a (typically 3% to 5%) and stevioside (typically 5% to 10%). Even the high glycoside varieties screened by conventional breeding means have very limited elevation of rebaudioside I content. The natural low abundance results in abnormally high cost for large-scale separation and purification of rebaudioside I directly from plant raw materials, and the raw material supply is difficult to stabilize, which cannot meet the market demand. The method is an effective way for converting the higher-content rebaudioside A into the higher-utilization-value rebaudioside I, and the conventional method adopts enzymes such as beta-glucosidase and the like for glycosylation modification, but the steps of the conversion process are complicated, the conversion efficiency is low, and the utilization rate of raw materials is further reduced. The high efficiency of the biocatalyst determines the final yield of rebaudioside I and therefore the development of a high conversion efficiency biocatalyst is of great value. Disclosure of Invention Aiming at the problem that the conversion efficiency of the existing method for converting the rebaudioside A into the rebaudioside I with higher utilization value is low, the invention provides an L204A mutant enzyme for preparing the rebaudioside I and application thereof, and discovers a novel UGT76G1 mutant, wherein the efficiency of catalyzing the conversion of the rebaudioside A into the rebaudioside I is higher than 50%. The technical scheme of the invention is as follows: In one aspect, the invention provides an L204A mutant of UGT76G1, which has at least 80% similarity to UGT76G 1. Preferably, the mutant has at least 90% similarity to UGT76G 1. Preferably, the mutant is obtained from the original UGT76G1 by mutating leucine of the 204 th amino acid sequence of the UGT76G1 to alanine. Preferably, the mutation is caused by a site-directed mutagenesis primer as shown in the sequences SEQ ID NO.2 and SEQ ID NO. 3. In another aspect, the invention provides a nucleic acid molecule encoding a mutant L204A of UGT76G1 as described above. Preferably, the nucleic acid molecule has a sequence as shown in SEQ ID NO.1, or a nonsensical mutation or a neutral mutation is generated from the sequence shown in SEQ ID NO. 1. In another aspect the invention provides an expression vector comprising an isolated nucleic acid molecule as described above. In another aspect the invention provides a host cell comprising an expression vector as described above. Preferably, the host cell is E.coli, B.subtilis, E.coli, A.oryzae, B.penicillin, A.niger, streptomyces, or yeast. In another aspect, the invention provides an expression system comprising a nucleic acid molecule as described above as a template and further comprising substances required for transcription and translation of mRNA with an auxiliary nucleic acid molecule. According to a preferred embodiment, the system is a microbial cell or a plant cell. The plant is, for example, a nicotiana benthamiana cell. In another aspect, the invention provides the use of a L204A mutant of UGT76G1, a nucleic acid molecule as described above, or an expression system as described above, for catalyzing the conversion of rebaudioside A to rebaudioside I. According to a preferred embodiment, the catalysis is in vitro cata