137 results about "Rebaudioside M" patented technology

The invention discloses a glycosyl transferase UGT76G1 mutant and a method for catalytically synthesizing rebaudioside M by using the glycosyl transferase UGT76G1 mutant, and relates to the technical field of biological catalytic synthesis. The glycosyl transferase UGT76G1 mutant is any one of the following components (A)-(C): (A) a protein obtained by performing any one or more of the following mutations on the basis of an amino acid sequence as shown in SEQ ID NO.1: the 89th amino acid is mutated into H from M; the 380th amino acid is mutated from L to M; the 411th amino acid is mutated from A to Y; (B) a protein which has 95% or 98% or more of identity with the amino acid sequence defined by (A) and has the same function as the amino acid sequence defined by (A); and (C) a fusion protein obtained by connecting a tag to the terminal of the protein defined in (A) or (B). The glycosyl transferase UGT76G1 mutant disclosed by the invention is obtained by mutating and screening glycosyl transferase UGT76G1, has higher catalytic activity, and can be used for efficiently synthesizing rebaudioside M by taking rebaudioside D and UDPG as substrates.

The invention discloses a glycosyl transferase mutant and an application of the glycosyl transferase mutant in synthesis of rebaudioside. According to the invention, single-point mutation and multi-point mutation are carried out on the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, and a mutant with improved catalytic activity is obtained. The catalytic activity, the substrate specificity and / or the substrate specificity of the glycosyl transferase mutant are / is changed, and the catalytic activity of enzyme to a specific substrate can be remarkably improved by mutation at a specific site. And carrying out induced expression and protein purification on the obtained mutation sequence to obtain the mutant enzyme. The mutant enzyme is used as a catalyst, and UDPG is used as a glycosyl donor, so that the catalytic reaction efficiency of substrates such as stevioside ST, rebaudioside A (RebA) and rebaudioside D (RebD) can be obviously improved. According to the glycosyl transferase UGT76G1 mutant constructed by the research, the catalytic activity of the glycosyl transferase UGT76G1 mutant is improved, and efficient production of rebaudioside M is realized by optimizing a reaction system.

Glycosyltransferase UGT76G1 and UGT91C1 mutants and a method for catalytic synthesis of rebaudioside A, D, and M by using same, pertaining to the technical field of biocatalytic synthesis. The glycosyltransferase UGT76G1 mutant can effectively synthesize rebaudioside A using stevioside STV as a substrate. The glycosyltransferase UGT91C1 mutant is obtained by mutation screening of wild-type glycosyltransferase UGT91C1, has higher enzyme activity and catalytic rate, and can effectively synthesize rebaudioside D using rebaudioside A as a substrate. The glycosyltransferase UGT76G1 mutant can also be obtained by mutation and subsequent screening of glycosyltransferase UGT76G1, has higher catalytic activity, and can effectively synthesize rebaudioside M using rebaudioside D and UDPG as substrates.

The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to a method for producing rebaudioside M by an enzyme method. The method comprises the following steps: S1, adding a mixed solution 1 of recombinant cell disruption liquid into a substrate, adding into a buffer solution to obtain a pre-reaction solution 1 (a mixture of recombinant cell disruption liquid of a recombinant strain UGT11 (S158F) and a recombinant strain AtSUS (P94N)), and collecting precipitate after reaction; and S2, after the precipitate is dissolved, adding a mixed solution 2 (a mixture of recombinant cell disruption solutions of a recombinant strain UGT76G1 (G83Q) and a recombinant strain AtSUS (P94N)) of recombinant cell disruption solutions, adding a buffer solution to obtain a pre-reaction solution 2, and collecting the reaction solution to obtain Reb M. The method provided by the invention can effectively solve the problems of low content and poor specificity of the extracted Reb M, and can realize the conversion rate of 93.33% from Reb A to Reb M.

The present disclosure provides enzymes and methods of using those enzymes to transfer sugar moieties to the substrate steviol glycosides. The designed beta-1, 3-glycosyltransferase can be used for adding a glucose monomer connected with beta-1, 3 to steviol glycosides. In particular, the designed beta-1, 3-glycosyltransferase is used in a one-pot reaction together with a beta-1, 2-glycosyltransferase and a sucrose synthase to convert stevioside and / or Reb A to Reb M.

The invention discloses a production method for biosynthesis of rebaudioside M. Reb A / Reb D has poor solubility at a low temperature, and the reaction efficiency can be obviously accelerated when the Reb A / Reb D is catalytically produced in a high-temperature environment, so that the whole reaction system is carried out in the high-temperature environment, and the production efficiency can be improved. Under the reaction temperature environment of 70 DEG C, the wild type first glycosyltransferase or / and the wild type second glycosyltransferase are / is basically inactivated, and the catalytic enzyme combination cannot meet the requirement of industrial biosynthesis of rebaudioside M. The wild-type catalytic enzyme is mutated, so that the wild-type catalytic enzyme can keep high catalytic activity at 70 DEG C. Therefore, the production cost of the rebaudioside M produced by the production method disclosed by the invention is obviously lower than that of the prior art, and the method is suitable for industrial popularization.

The invention relates to the technical field of gene engineering, in particular to a glycosyl transferase mutant, a sucrose synthase mutant and application of the glycosyl transferase mutant and the sucrose synthase mutant in synthesis of rebaudioside M. Wild glycosyl transferase and wild sucrose synthase are modified according to a directed evolution theory to obtain a glycosyl transferase mutant and a sucrose synthase mutant. Rebaudioside M is obtained by catalyzing rebaudioside D through a glycosyl transferase mutant and sucrose synthase mutant co-expression strain, and the catalytic efficiency is superior to that of a wild enzyme. The rebaudioside M is obtained by completely catalyzing 110 g / L of rebaudioside D through the co-expression strain of the glycosyl transferase mutant and the sucrose synthase mutant, only 20 h is needed, the conversion rate reaches 97%, the yield of the rebaudioside M reaches 122 g / L, the raw material conversion rate and the yield of the rebaudioside M are effectively increased, and meanwhile the synthesis time is shortened.

The invention discloses a recombinant plasmid, a recombinant strain and a method for synthesizing rebaudioside M by a one-step method, and belongs to the technical field of biological catalytic synthesis. The recombinant plasmid contains a coding gene of a glycosyl transferase UGT91C1 mutant, a coding gene of a glycosyl transferase UGT76G1 mutant and a coding gene of sucrose synthase. The mutant of the glycosyl transferase UGT91C1 is 2-12E, and the coding gene sequence of the glycosyl transferase UGT91C1 is as shown in SEQ ID NO. 1; the glycosyl transferase UGT76G1 mutant is 101H10, and the coding gene sequence of the glycosyl transferase UGT76G1 mutant is as shown in SEQ ID NO. 2; the sequence of the coding gene of the sucrose synthase is as shown in SEQ ID NO. 3. The rebaudioside M is synthesized by using a three-enzyme co-expression recombinant strain containing the plasmid through a one-step method, so that the loss of fermentation materials can be reduced, the utilization efficiency of equipment is improved, the energy consumption is reduced, and the large-scale production of the rebaudioside M is facilitated.

The invention discloses a preparation method of rebaudioside D and rebaudioside M and an application of beta-1, 2-glucosyltransferase in the rebaudioside D and rebaudioside M. The beta-1, 2-glucosyltransferase has an amino acid sequence as shown in SEQ ID NO: 4, or has a mutant amino acid sequence which has at least 99% sequence identity compared with the amino acid sequence as shown in SEQ ID NO: 4. According to the invention, the yield of an intermediate product rebaudioside D when the beta-1, 2-glucosyltransferase catalyzes rebaudioside A to generate rebaudioside M is improved, and the yield of a final product rebaudioside M is correspondingly improved.

The invention relates to the technical field of industrial microorganisms and food industry, and discloses an immobilized enzyme composition and application thereof in efficient preparation of rebaudioside M. The immobilized enzyme composition provided by the invention comprises immobilized UDP-glycosyl transferase UGT and immobilized UDP-glycosyl transferase UGT76G1 according to a mass ratio of (1 to 2): (1 to 3). By adopting the immobilized enzyme composition disclosed by the invention, the rebaudioside A can be synergistically and efficiently promoted to be converted into the rebaudioside M, and the conversion rate is up to 85% or above and can be up to 97% at most; the working efficiency of the enzyme is remarkably improved, and the rebaudioside M is efficiently prepared; meanwhile, the enzyme is good in stability and can be repeatedly used for at least five times, and the catalytic efficiency is still higher than 95%. The method has great popularization and application values.

The present disclosure relates to a composition for producing steviol glycosides containing Rebaudioside M at high purity, using a biocatalyst containing UDP-glycosyltransferase and UGT76G1, and a method for producing steviol glycosides using the same.

The invention provides a method for preparing rebaudioside M by constructing a UGT91C1-UGT76G1-SuSy fusion enzyme on the basis of an artificially constructed substrate microchannel principle and using relatively cheap raw materials to efficiently catalyze a cascade reaction. EAAAK 3 and (GGGGS) 4 are used as connecting peptides, UGT76G1 is fused at the C end of UGT91C1, SuSy is fused at the C end of UGT76G1, mass transfer of an intermediate product is enhanced, and the intermediate product is prevented from being diffused to a reaction main body. The invention also discloses a construction method of the ugt91C1-C1-ugt76G1-suusy fusion gene, a construction method of a recombinant plasmid, a construction method of a recombinant strain, a protein expression and purification method and a method for catalytically preparing a product. The ugt91C1-C1-ugt76G1-suusy fusion gene has a very good application prospect in biological industrial catalytic production.

The invention relates to the technical field of gene engineering, in particular to a glycosyl transferase mutant and application thereof in synthesis of rebaudioside M. Wild type glycosyl transferase is modified through a directed evolution theory to obtain the glycosyl transferase mutant, so that the efficiency of synthesizing rebaudioside D through reaction of rebaudioside A is effectively improved. Besides, recombinant bacteria capable of simultaneously expressing the glycosyl transferase mutant, a second glycosyl transferase gene and a sucrose synthase gene are constructed, a wet cell obtained by induced culture of the recombinant bacteria or a crude enzyme liquid extracted by crushing the wet cell is used as a catalyst, rebaudioside A is used as a substrate, sucrose is used as an auxiliary substrate, and the rebaudioside M is catalytically synthesized by a one-pot method. According to the present invention, the complete catalysis of 100 g / L of rebaudioside A to produce rebaudioside M only needs 24 h, the conversion rate reaches 97.5%, the yield of rebaudioside M reaches 130 g / L, the raw material conversion rate and the yield of rebaudioside M are effectively improved, and the synthesis time is shortened.

The invention discloses an escherichia coli recombinant bacterium for whole-cell catalytic synthesis of rebaudioside M as well as a construction method and application of the escherichia coli recombinant bacterium. The Escherichia coli recombinant bacterium for the whole-cell catalytic synthesis of the rebaudioside M, which is constructed by the invention, contains a glycosyl transferase UGT76G1 coding gene mutant, a glycosyl transferase UGT91C1 coding gene mutant, a sucrose synthase atSuS1 gene and a polyphosphoglucokinase cgPPGK gene; a glucose-1-phosphate uridine transferase galU gene, a nucleoside diphosphate kinase ndk gene and a nucleoside diphosphate kinase ndk gene are over-expressed; meanwhile, an encoding gene araA gene of L-arabinose isomerase and an encoding gene ushA gene of 5 '-nucleotidase / UDG hydrolase are knocked out. According to the recombinant escherichia coli constructed by the invention, Reb D is prevented from being directly used as a synthetic substrate, so that the raw materials are easier to obtain, and the conversion rate of the substrate and the synthetic rate and yield of rebaudioside M are remarkably improved.

The invention relates to an engineered glycosyltransferase and a steviol glycoside glucosylation method. The present invention provides engineered glycosyltransferases (GTs), polypeptides having GT activity and polynucleotides encoding the enzymes, as well as vectors and host cells comprising the polynucleotides and polypeptides. The present invention provides engineered sucrose synthases (SuS), polypeptides having SuS activity and polynucleotides encoding the enzymes, as well as vectors and host cells comprising the polynucleotides and polypeptides. The invention also provides compositions comprising the GT enzymes and methods of using the engineered GT enzymes to prepare products having beta-glucose ligation. The present invention also provides compositions and methods for producing rebaudioside (e.g., rebaudioside M, rebaudioside A, rebaudioside I, and rebaudioside D). The invention also provides compositions comprising the SuS enzymes and methods of using the same. Methods for producing a GT enzyme and a SuS enzyme are also provided.

The present disclosure provides a combination of steviol glycosides having various flavor characteristics. In one aspect, the present disclosure provides a rebaudioside composition comprising at least one of rebaudioside A, rebaudioside D, and rebaudioside M. The present disclosure provides a rebaudioside composition comprising at least one of rebaudioside A, rebaudioside D, and rebaudioside M. In one embodiment, the present disclosure provides a rebaudioside composition comprising rebaudioside A, rebaudioside D, and rebaudioside M. In one embodiment, the present disclosure provides a rebaudioside composition comprising rebaudioside A, rebaudioside D, and rebaudioside M. The content of rebaudioside A, rebaudioside D, and / or rebaudioside M in the rebaudioside composition of the present disclosure may be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, etc.

The invention discloses glycosyl transferase for catalytic production of rebaudioside M and a recombinant gene of the glycosyl transferase. Compared with wild type glycosyl transferase, the glycosyl transferase variant disclosed by the invention has the advantages that the thermal stability and the enzyme activity are obviously improved. In a high-temperature environment of 70 DEG C, the wild type glycosyl transferase is completely inactivated, and the enzyme activity of the glycosyl transferase variant disclosed by the invention is more than 50U / mg. Meanwhile, in a high-temperature environment of 70 DEG C, the solubility of a reaction substrate rebaudioside D is improved, the thermal motion of enzyme molecules and substrate molecules is intensified, and the collision frequency and energy between the enzyme molecules and the substrate molecules are increased, so that the catalytic reaction speed can be further increased. Therefore, the glycosyl transferase variant disclosed by the invention is suitable for being used as a biocatalyst and is used for large-scale production of a sweetening agent rebaudioside M with a better taste.

The invention discloses an engineering strain for producing uridine diphosphate, a construction method and a method for synthesizing rebaudioside M, and belongs to the technical field of gene engineering. According to the engineering strain, genes such as a pyrimidine nucleoside operon gene pyrBCAKDFE, a carbonic anhydrase gene cynT and a carbamyl phosphate synthetase gene pyrAA / ABE951 * are integrated on a genome, and genes such as an aspartate carbamyl transaminase gene pyrB, an orotic acid phosphoribose transferase gene pyrE and a phosphoribose pyrophosphate synthase gene prsD128A are deleted. The engineering strain can efficiently synthesize uridine diphosphate, and can act together with a functional protein expression recombinant strain to catalytically synthesize rebaudioside M by taking sucrose and rebaudioside A as substrates.

A preparation method for rebaudioside M and the use of β-1,2-glucosyltransferase in same. The β-1,2-glucosyltransferase has an amino acid sequence shown as SEQ ID NO: 4, or a mutant amino acid sequence having at least 99% sequence identity to the amino acid sequence shown as SEQ ID NO: 4. The present invention improves the yield of an intermediate product rebaudioside D when β-1,2-glucosyltransferase is used to catalyze rebaudioside A to generate rebaudioside M, thus correspondingly improving the yield of the final product rebaudioside M.

The present application relates to methods for preparing rebaudioside D and rebaudioside M by reaction of uridine diphosphate (UDP)-glycosyltransferases; and compositions for preparing rebaudioside D and rebaudioside M, comprising uridine diphosphate (UDP)-glycosyltransferases.

To provide: reduced-calory or non-caloric sweeteners and sweetened compositions that have temporal and flavor profiles similar to those of sucrose and are suitable for use in consumer products; beverages or beverage products containing the compositions; and methods of improving at least one organoleptic property of beverages or beverage products.SOLUTION: A sweetener composition or a flavor modifying composition comprises (i) brazzein and (ii) at least one additional sweetener selected from a steviol glycoside sweetener and a mogroside sweetener. The steviol glycoside sweetener is selected from Rebaudioside M ("Reb M"), RebM80, Rebaudioside D ("Reb D"), A95 and Rebaudioside A ("Reb A"). The mogroside sweetener is siamenoside I.SELECTED DRAWING: None

The invention discloses a method for preparing amorphous rebaudioside M. The preparation method of the amorphous rebaudioside M, disclosed by the invention, comprises the following steps: carrying out instantaneous cooling on a high-temperature rebaudioside M aqueous solution to obtain the amorphous rebaudioside M, the instantaneous cooling comprises a step of adding a high-temperature rebaudioside M aqueous solution into a low-temperature polar protic solvent; wherein the temperature of the high-temperature rebaudioside M water solution is 90-100 DEG C, and the temperature of the low-temperature polar protic solvent is 1-8 DEG C. The preparation method of the amorphous rebaudioside M, disclosed by the invention, has one or more of the following advantages: high yield, low energy consumption, mild reaction conditions, simplicity in operation, low cost and suitability for industrial production.

Provided is a stevia plant having at least one of genetic characteristics (1) to (5).

A glucosyltransferase catalyzing RA to produce RM and a use thereof are provided. The glucosyltransferases derived from Cornus florida and Juglans regia L. are screened and obtained. It was found that the glucosyltransferase derived from the Cornus florida exhibited a relatively high enzymatic activity in catalyzing the RA to produce the RD, while the glucosyltransferase derived from the Juglans regia L. exhibited a relatively high enzymatic activity in catalyzing the RD to produce the RM. When these two enzymes were used together in catalyzing the RA to produce the RM, a relatively high conversion rate was achieved. The use of the glucosyltransferases derived from the Cornus florida and the Juglans regia L. for preparing the RM of SGs, has not been reported yet. The glucosyltransferase provides an excellent candidate protein resource and its corresponding gene sequence for the enzymatic conversion process of the SGs.

The present invention provides engineered glycosyltransferase (GT) enzymes, polypeptides having GT activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention provides engineered sucrose synthase (SuS) enzymes, polypeptides having SuS activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention also provides compositions comprising the GT enzymes and methods of using the engineered GT enzymes to make products with β-glucose linkages. The present invention further provides compositions and methods for the production of rebaudiosides (e.g., rebaudioside M, rebaudioside A, rebaudioside I, and rebaudioside D). The present invention also provides compositions comprising the SuS enzymes and methods of using them. Methods for producing GT and SuS enzymes are also provided.

Disclosed is a method for efficiently preparing rebaudioside M by using a bio-enzymatic method. The preparation method comprises: adding rebaudioside A, uridine diphosphate glucose, glycosyltransferase, and a salt into a buffer solution, mixing to obtain a mixed solution, performing an enzyme-catalyzed reaction while stirring, and obtaining said rebaudioside M. The amino acid sequence of the glycosyltransferase is one or more of those shown in SEQ ID NO. 1-12. The glycosyltransferase can also be coupled with sucrose synthase for catalysis, so as to use sucrose as a glycosyl donor to, in one step, catalyze rebaudioside A to generate rebaudioside M.

The present invention discloses a method for producing a target stevioside. According to the method for producing the target stevioside, the distribution change amount of the target stevioside reaches 90% or above within 2 hours, so that a precipitate containing the target stevioside is formed. The target stevioside is rebaudioside D and / or rebaudioside M. The target stevioside is rebaudioside M.

Disclosed herein are edible compositions comprising a steviol glycoside composition comprising from 5% to 30% by weight of rebaudioside M (reb M), from 15% to 35% by weight of rebaudioside AM (reb AM), and from 10% to 25% by weight of rebaudioside N2 (reb N2), based on the total amount of steviol glycosides in the edible composition, wherein the edible composition has improved solubility and / or foaming profile.

The invention belongs to the technical field of biological catalysis, and particularly relates to a glycosyl transferase mutant and application thereof in catalytic synthesis of rebaudioside M. Engineering bacterium whole cells or broken enzyme liquid for co-expressing a glycosyl transferase G1 mutant, a glycosyl transferase C1 mutant and sucrose synthase SUS is used as a catalyst, Reb A is used as a substrate, sucrose is used as a glycoside donor, Reb M is synthesized under mild conditions, the space-time yield of the generated Reb M is up to 3.33 g / L / h, and the method has the advantages of being high in space-time yield, convenient to purify, green, environmentally friendly and the like, and is suitable for industrial production. The industrial application value is extremely high.

The present invention relates to a novel Lactobacillus marzei CJST01 strain having excellent steviol glycoside transglycosylation activity, and a method for producing a steviol glycoside using the same. Compared with the known Lactobacillus margaritae DSM20444 strain capable of generating the steviol glycoside transglycosylation effect, the novel Lactobacillus margaritae CJST01 strain has excellent steviol glycoside transglycosylation activity, so that the enzyme reaction time in the production process is shortened. The steviol glycosides produced by using the strain have the advantage of a better taste quality or flavor than alpha-1, 4-linked steviol glycosides and rebaudioside M. In addition, the transglycosylated rebaudioside A produced by using the Lactobacillus margaritae CJST01 strain has a reduced foreign taste and flavor, and excellent solubility.