108 results about "Rebaudioside D" 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 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 preparation method of glucosyl stevioside, and relates to the technical field of glucosyl stevioside production, rebaudioside A or stevioside is taken as a raw material, cyclodextrin is taken as an auxiliary material, cyclodextrin glucosyltransferase is added for glycosylation reaction, a glucosyl stevioside solution I is cooled, and a glucosyl stevioside solution II is obtained; the method comprises the following steps: adding sucrose, UGT glycosyl transferase, sucrose synthase and uridine diphosphate into rebaudioside A or stevioside, continuously converting unreacted rebaudioside A or stevioside into rebaudioside D or rebaudioside E, inactivating after the reaction is finished, filtering by a plate frame, removing impurities through resin, analyzing the resin by using ethanol to obtain an analysis solution, and treating the analysis solution at 78-82 DEG C for 2-4 hours to obtain the rebaudioside D or rebaudioside E; and dealcoholizing at the vacuum degree of-0.1 MPa, concentrating and drying to obtain the glucosyl stevioside. The quality of the product is improved, the cost is reduced, and the taste of the product is integrally improved.

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 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 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 belongs to the technical field of biology, and discloses a UDP-glucosyltransferase mutant and an application thereof, and a preparation method of stevioside rebaudioside D. The UDP-glucosyltransferase mutant comprises a UDP-glucosyltransferase mutant, compared with the original UDP-glucosyltransferase comprising an amino acid fragment with the sequence shown as SEQ ID NO: 1, the UDP-glucosyltransferase mutant provided by the invention comprises H18P mutation and / or R370H mutation, and the mutation endows the UDP-glucosyltransferase mutant with higher specific activity for catalyzing the conversion of rebaudioside A into rebaudioside D, so that the UDP-glucosyltransferase mutant can be used for catalyzing the conversion of rebaudioside A into rebaudioside D; the high-efficiency and sufficient conversion from the rebaudioside A to the rebaudioside D can be realized, the realization of large-scale and high-efficiency production of the rebaudioside D is facilitated, and the method has a good application prospect.

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 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.

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.

The invention provides a method for efficient biosynthesis of rebaudioside M2 using glycosyltransferase, belonging to the technical field of biocatalytic synthesis. A glycosyltransferase UGT94D1 catalyzing rebaudioside D to synthesize rebaudioside M2 is obtained by mining, providing a new, efficient and environmentally friendly way of producing rebaudioside M2. During the whole catalytic reaction process, no by-products are produced, facilitating downstream purification and further reducing production costs.

The invention discloses glycosyl transferase for catalytic production of rebaudioside D 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 basically inactivated, and the enzyme activity of the glycosyl transferase variant disclosed by the invention is more than 40U / mg. Meanwhile, in a high-temperature environment of 70 DEG C, the solubility of a reaction substrate rebaudioside A 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 D with a better taste.

Provided is a novel beverage containing a steviol glycoside and an alcohol. One embodiment of the present invention provides a beverage that contains one or more steviol glycosides selected from the group consisting of rebaudioside A, rebaudioside D and rebaudioside M and has an alcohol content of 0.05 v / v% or more and less than 0.5 v / v%.

This invention discloses a glycosyltransferase mutant and its application in the synthesis of rebaudioside. Based on the amino acid sequence of the glycosyltransferase shown in SEQ ID NO:1, this invention performs single-point and multi-point mutations to obtain mutants with enhanced catalytic activity. The catalytic activity, substrate specificity, and / or substrate specificity of the glycosyltransferase mutant are altered; mutations at specific sites can significantly improve the enzyme's catalytic activity for specific substrates. The mutant enzyme is obtained by inducing expression and purifying the protein from the obtained mutant sequence. Using the mutant enzyme as a catalyst and UDPG as a glycosyl donor, the catalytic reaction efficiency for the substrates steviol glycoside ST, rebaudioside A (RebA), and rebaudioside D (RebD) can be significantly improved. The glycosyltransferase UGT76G1 mutant constructed in this study improves its catalytic activity, and the efficient production of rebaudioside M is achieved through optimization of the reaction system.

The invention belongs to the technical field of biological enzyme engineering, and particularly relates to a glycosyl transferase mutant and application thereof in synthesis of rebaudioside M. According to the invention, site-directed mutagenesis is carried out on an amino acid sequence SEQ ID NO.1 of wild type glycosyl transferase, and mutants: I29M, L149C and F374C are obtained through screening; the invention also provides a nucleic acid sequence for coding the mutant, an expression vector and a recombinant strain which are used for catalytically converting rebaudioside D to prepare rebaudioside M, and the conversion rate is obviously improved. Experimental results show that under optimized reaction conditions, the mutant can increase the conversion rate of converting rebaudioside D into rebaudioside M to 100%, which is increased by more than 30% compared with a wild type, and the mutant has significant industrial application value.

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express recombinant genes encoding UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol glycosides, e.g., Rebaudioside A and / or Rebaudioside D, which can be used as natural sweeteners in food products and dietary supplements.

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.

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.

The present invention relates to novel rebaudioside D and rebaudioside M-like steviol glycosides and a method for producing same. The novel rebaudioside D and rebaudioside M-like steviol glycosides obtained by the production method according to the present invention exhibit improved water solubility, and thus can be easily applied to beverages and foods with a high water content, and can significantly improve the industrial utilization of steviol glycosides.

The present disclosure provides enzymes and methods of using those enzymes to transfer sugar moieties to substrate steviol glycosides. Specifically, designed beta-1,2-glycosyltransferases and sucrose synthases are used in one-pot reactions to convert stevioside and Reb A to Reb E and Reb D. Additionally, designed beta-1,2-glycosyltransferases can be used in one-pot reactions with sucrose synthases and beta-1,3-glycosyltransferases to produce Reb M from stevioside and / or Reb A.

The present invention discloses a glycosyltransferase mutant with improved thermal stability and activity, belonging to the technical field of biocatalytic synthesis. The mutant is obtained by simultaneously mutating the 158th, 218th, and 369th positions on the basis of the glycosyltransferase YojK-I241T / G327N with the amino acid sequence shown in SEQ ID NO. 3. The present invention determines the mutation sites capable of improving the thermal stability and activity of YojK through rational design, and efficiently obtains the combined mutant YojK M1 . Compared with YojK M0 , the half-life of this variant at 50 °C is increased from 2.16 h to 58.64 h, and the enzyme activity is increased by 1.39 times; finally, through fed-batch reaction, it is shown that the conversion rate of this mutant is as high as 87.7% under the condition of 25 mmol / L Reb A, providing an efficient and green new route for the production of rebaudioside D.

The application belongs to the technical field of enzyme engineering, and particularly relates to a glycosyltransferase mutant and application thereof in synthesis of steviol glycoside. The glycosyltransferase mutant is reformed on the basis of an amino acid sequence SEQ ID NO. 1, and key mutation sites include: the 77th methionine is mutated into tyrosine M77Y and the 142th methionine is mutated into tryptophan M142W; the application also discloses a nucleic acid sequence coding the mutant, an expression vector, a recombinant strain, and application thereof in industrialized production of steviol glycoside, and realizes efficient synthesis of rebaudioside D; experimental results show that the efficiency of catalyzing Reb A to synthesize Reb D is compared with that of the wild-type glycosyltransferase, the catalytic efficiency of M77Y is the highest, the conversion rate is more than 70%, which is more than 5 times of the wild type; the conversion rates of M142W and M142W / M77Y are more than 40% and more than 29% respectively, which are 3 times and more than 2 times of the wild type respectively.

The present invention discloses a glycosyltransferase and its application in the production of glycosides. The present invention clones a glycosyltransferase from a low-temperature resistant Bacillus subtilis using molecular cloning technology, constructs an expression vector with the cloned glycosyltransferase molecule and successfully expresses it in vitro. The enzyme activity test shows that the glycosyltransferase expressed in vitro can catalyze rebaudioside A to rebaudioside D, and the glycosyltransferase has the characteristic of low-temperature resistance, can still catalyze the reaction under the condition of 25°C, has a wide pH value reaction range, and can achieve the catalytic reaction within the range of pH6-8. The new glycosyltransferase disclosed in the present invention has the advantages of low-temperature resistance and wide pH value reaction, and is suitable for the production of various glycoside glycosylation products.

An aqueous steviol glycoside solution includes 5 wt% or more of one or more steviol glycosides and choline chloride in an amount such that a weight ratio of choline chloride to water is between 4 and 7. The one or more steviol glycosides and the choline chloride may be present at a weight ratio of 0.65 or lower. The one or more steviol glycosides and water may be present at a weight ratio of 4 or lower. The one or more steviol glycosides may include rebaudioside D and rebaudioside M. A method for increasing the solubility of a steviol glycoside composition includes adding one or more steviol glycosides and choline chloride to an aqueous solution to form a steviol glycoside composition with a weight ratio of choline chloride to water is in a range of 4 to 7.