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CN-121427867-B - M88F mutant enzyme for preparing rebaudioside I and application thereof

CN121427867BCN 121427867 BCN121427867 BCN 121427867BCN-121427867-B

Abstract

The invention relates to the technical field of biocatalysis, and discloses an M88F mutant enzyme for preparing rebaudioside I and application thereof, wherein the enzyme is obtained by mutating UGT76G1 to obtain M88F by mutating methionine of 88 th amino acid sequence of UGT76G1 into phenylalanine, and can efficiently and directionally convert substrate Rebaudioside A (RA) into higher-value Rebaudioside I (RI). In the optimized reaction system, the conversion rate is stabilized to 40%, and the catalytic efficiency is improved by more than 5-8 times compared with the common reference enzyme. The enzyme preparation has the characteristics of high catalytic activity, mild reaction condition, simple operation and the like, has good stability and reproducibility in laboratory research and large-scale production, and has wide industrialized application prospect.

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 M88F mutant enzyme for preparing rebaudioside I, wherein the mutant is UGT76G1, and wherein the mutant is produced from UGT76G1 by: M88F, mutation of methionine at position 88 of UGT76G1 to phenylalanine; the coding nucleic acid sequence of the M88F mutant enzyme is shown as SEQ ID NO. 1.
  2. 2. A nucleic acid encoding an M88F mutant enzyme for preparing rebaudioside I according to claim 1, wherein the sequence is as set forth in SEQ ID No. 1.
  3. 3. An expression vector comprising a nucleic acid according to claim 2 encoding an M88F mutant enzyme according to claim 1 for preparing rebaudioside I.
  4. 4. An expression vector according to claim 3, wherein the vector is a pET32a plasmid.
  5. 5. Use of an M88F mutant enzyme producing rebaudioside I according to claim 1 or a nucleic acid encoding an M88F mutant enzyme producing rebaudioside I according to claim 2 or an expression vector according to claim 3 or 4 for catalyzing the conversion of rebaudioside a to rebaudioside I.
  6. 6. The use of claim 5, wherein the catalysis is achieved by contacting an M88F mutant enzyme with rebaudioside a.
  7. 7. A method of preparing rebaudioside I comprising the steps of: Adding the mutant UGT76G1 of claim 1 or the prokaryotic expression system comprising the nucleic acid of claim 2 to a reaction system comprising rebaudioside a, controlling the reaction conditions such that rebaudioside a is converted to rebaudioside I.
  8. 8. The method of preparing rebaudioside I according to claim 7, wherein the host cell of the prokaryotic expression system is one of bacillus subtilis, escherichia coli, and streptomyces.

Description

M88F mutant enzyme for preparing rebaudioside I and application thereof Technical Field The invention relates to the technical field of biocatalysis, in particular to M88F 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 M88F 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 35%. The technical scheme of the invention is as follows: in one aspect, the invention provides an enzyme that produces rebaudioside I as a mutant of UGT76G1, the mutant having at least 80% similarity to UGT76G 1. Preferably, the mutant has at least 90% similarity to UGT76G 1. Preferably, the mutant is obtained by mutating the original UGT76G1 such that M88F is obtained by mutating methionine of the 88 th amino acid sequence of UGT76G1 to phenylalanine. 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 encoding an enzyme for preparing rebaudioside I as described above, wherein the nucleic acid has nonsensical mutations in its sequence. Preferably, the sequence of the nucleic acid is shown in SEQ ID NO. 1. In another aspect, the invention provides an expression vector comprising a nucleic acid encoding an enzyme for preparing rebaudioside I as described above. Preferably, the vector is a pET32a plasmid. 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 the use of an enzyme for preparing rebaudioside I as described above or a nucleic acid encoding an M88F mutant enzyme for preparing rebaudioside I as described above or an expression vector as described above for catalyzing the conversion of rebaudioside a to rebaudioside I. Preferably, the catalysis is achieved by contacting the enzyme with rebaudioside a. 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