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CN-121427862-B - M88W mutant enzyme for preparing rebaudioside I and application thereof

CN121427862BCN 121427862 BCN121427862 BCN 121427862BCN-121427862-B

Abstract

The invention relates to the technical field of biocatalysis, and discloses an M88W mutant enzyme for preparing rebaudioside I and application thereof, wherein the enzyme is obtained by mutating an 88 th amino acid sequence methionine of UGT76G1 into tryptophan by generating the following mutation, and the enzyme can be applied to RA in-vitro conversion to prepare RI with higher utilization value, the conversion rate is higher than 90%, and the enzyme has remarkable catalytic enzyme activity. The UGT76G1 mutant disclosed by the invention has the advantages of high enzyme activity, high catalytic efficiency and simple use, and is suitable for wide 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 M88W mutant enzyme for preparing rebaudioside I, wherein the mutant is UGT76G1, and wherein the mutant is produced from UGT76G1 by: M88W, mutation of methionine at position 88 of UGT76G1 to tryptophan; The nucleic acid sequence of the M88W mutant enzyme is shown in SEQ ID NO. 1.
  2. 2. The use of an M88W mutant enzyme producing rebaudioside I according to claim 1 for catalyzing the conversion of rebaudioside a to rebaudioside I.
  3. 3. The use according to claim 2, wherein the catalysis is in vitro catalysis or in vivo catalysis.
  4. 4. A method of preparing rebaudioside I comprising the steps of: Contacting a starting composition comprising rebaudioside a with a biocatalyst capable of converting rebaudioside a into rebaudioside I to produce rebaudioside I; the biocatalyst is an M88W mutant enzyme of UGT76G1, and the mutation of M88W is specifically that methionine of the 88 th amino acid sequence of UGT76G1 is mutated into tryptophan; The nucleic acid sequence of the M88W mutant enzyme is shown in SEQ ID NO. 1.
  5. 5. The method of preparing rebaudioside I according to claim 4, wherein the biocatalyst is in the form of a high purity material, a cell lysate or a whole cell suspension.
  6. 6. The method of preparing rebaudioside I according to claim 5, wherein the cells in the cell lysate or whole cell suspension are microbial cells.
  7. 7. The method of preparing rebaudioside I according to claim 6, wherein the microbial cell is one of bacillus subtilis, escherichia coli, aspergillus oryzae, penicillium, aspergillus niger, streptomyces, yeast.
  8. 8. The method of preparing rebaudioside I according to claim 4, wherein the amount of rebaudioside I prepared is 85% to 99% of the amount of rebaudioside a in the starting composition comprising rebaudioside a.

Description

M88W mutant enzyme for preparing rebaudioside I and application thereof Technical Field The invention relates to the technical field of biocatalysis, in particular to M88W 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 steviosides (such as stevioside), 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 M88W mutant enzyme for preparing the rebaudioside I and application thereof, and discovers a novel UGT76G1 mutant, and the efficiency of catalyzing the conversion of the rebaudioside A into the rebaudioside I is higher than 90%. The technical scheme of the invention is as follows: in one aspect, the invention provides a M88W mutant enzyme for preparing rebaudioside I, which is a mutant of UGT76G1, wherein the mutant has at least 80% similarity with UGT76G 1. Preferably, the mutant has at least 90% similarity to UGT76G 1. Preferably, the mutant is obtained from the original UGT76G1 by mutating methionine of the 88 th amino acid sequence of the M88W UGT76G1 to tryptophan. 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. Preferably, the amino acid sequence of the mutant is shown as SEQ ID NO. 1. In another aspect, the invention provides the use of an enzyme as described above for preparing rebaudioside I to catalyze the conversion of rebaudioside a to rebaudioside I. According to a preferred embodiment, the catalysis is in vitro catalysis or in vivo catalysis. In another aspect, the invention provides a method for preparing rebaudioside I comprising the steps of: Contacting a starting composition comprising rebaudioside a with a biocatalyst capable of converting rebaudioside a to rebaudioside I, wherein the biocatalyst is an M88W mutant of UGT76G1, the mutation of M88W specifically being a methionine mutation of the 88 th amino acid sequence of UGT76G1 to tryptophan, to produce rebaudioside I. The mutation is caused by a site-directed mutagenesis primer shown in sequences SEQ ID NO.2 and SEQ ID NO. 3. According to a preferred embodiment, the biocatalyst is selected from the group consisting of a high purity material, a cell lysate or a whole cell suspension. According to a preferred embodiment, the cells in the cell lysate or whole cell suspension are microbial cells. For example, the microbial cell is E.coli. According to a preferred embodiment, the starting composition comprisi