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CN-121427865-B - M88A mutant enzyme for preparing rebaudioside I and application thereof

CN121427865BCN 121427865 BCN121427865 BCN 121427865BCN-121427865-B

Abstract

The invention relates to the technical field of biocatalysis, and discloses an M88A mutant enzyme for preparing rebaudioside I and application thereof, wherein the enzyme is obtained by mutating methionine of an 88 th amino acid sequence of UGT76G1 into alanine, the key conversion rate of RI is greatly improved from 7% to 85% (the improvement is over 12 times), the utilization rate and economy of raw materials are obviously improved, perfect reproduction can be realized under any test scale (10 mL,100mL and 5L), the conversion rate is always anchored to more than 85%, and the unmatched stability and amplifying potential are shown.

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 (7)

  1. 1. An M88A mutant enzyme for preparing rebaudioside I, wherein the mutant is UGT76G1, and wherein the mutant is produced from UGT76G1 by: M88A methionine of 88 th amino acid of UGT76G1 sequence is mutated into alanine; The nucleic acid sequence of the M88A mutant enzyme is shown in SEQ ID NO. 1.
  2. 2. A genetically engineered strain comprising a genetically engineered expression plasmid capable of expressing an M88A mutant enzyme for preparing rebaudioside I according to claim 1, said genetically engineered expression plasmid having a promoter operably linked thereto a nucleic acid sequence encoding an M88A mutant enzyme for preparing rebaudioside I according to claim 1.
  3. 3. Use of an M88A mutant enzyme producing rebaudioside I according to claim 1 or a genetically engineered strain according to claim 2 for catalyzing the conversion of rebaudioside a to rebaudioside I.
  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 M88A mutant of UGT76G1, and the mutation of the M88A mutant of UGT76G1 is specifically that methionine of 88 th amino acid of a UGT76G1 sequence is mutated into alanine; The nucleic acid sequence of the M88A mutant enzyme is shown in SEQ ID NO. 1.
  5. 5. The method of preparing rebaudioside I according to claim 4, wherein the high purity material, cell lysate or whole cell suspension comprises the biocatalyst.
  6. 6. The method of preparing rebaudioside I according to claim 5, wherein the cell is one of bacillus subtilis, escherichia coli, aspergillus oryzae, penicillium, aspergillus niger, streptomyces, yeast.
  7. 7. The method of preparing rebaudioside I according to claim 6, wherein the starting composition comprising rebaudioside a is a plant extract or pure material.

Description

M88A mutant enzyme for preparing rebaudioside I and application thereof Technical Field The invention relates to the technical field of biocatalysis, in particular to M88A 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 M88A 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 80%. 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 derived from the original UGT76G1 by mutating: M88A methionine of the 88 th amino acid sequence of UGT76G1 is mutated into 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 an isolated nucleic acid molecule encoding an enzyme that produces rebaudioside I as described above, the sequence encoding which has nonsensical mutations. 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 genetically engineered strain comprising a genetically engineered expression plasmid capable of expressing an enzyme that produces rebaudioside I as described above, said genetically engineered expression plasmid having a promoter operably linked thereto a nucleic acid sequence encoding an enzyme that produces rebaudioside I as described above. Preferably, the nucleic acid sequence is shown as SEQ ID NO.1, and can also be a sequence obtained by nonsensical mutation or mesogenic mutation of the sequence shown as SEQ ID NO. 1. Preferably, the genetically engineered strain is escherichia coli, bacillus subtilis, escherichia coli, aspergillus oryzae, penicillium, aspergillus niger, streptomycete or yeast. 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 catal