81 results about "Rebaudioside D" patented technology

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 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 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 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.

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 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 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.

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

Provided herein are genetically modified host cells, compositions, and methods for improved production of steviol glycosides. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleic acid expression cassette that expresses an ABC-transporter capable of transporting steviol glycosides to the extracellular space or to the luminal space of an intracellular organelle. In some embodiments, the host cell further comprises one or more heterologous nucleotide sequence encoding further enzymes of a pathway capable of producing one or more steviol glycosides in the host cell. The host cells, compositions, and methods described herein provide an efficient route for the heterologous production of steviol glycosides, including but not limited to, rebaudioside D and rebaudioside M.

The development of a novel tea beverage containing alcohol is needed. One embodiment of the present invention provides a tea beverage containing one or more steviol glycosides selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, and rebaudioside M, and tannin, wherein the content of the one or more steviol glycosides is 0.5-3,000 mg / L, the content of the tannin is 5-3,000 mg / L, and the tea beverage has an alcohol content of 1-40 v / v%.

The present invention provides: a novel beverage having foam retentivity; and a method for improving foam retentivity. The present invention provides a beverage containing rebaudioside D and a compound represented by formula (1)(in the formula, R1 represents Xyl(1-2)Glc1-, and R2 represents Glc(1-2)[Glc(1-3)]Glc1-, Glc represents glucose, and Xyl represents xylose), wherein the sum of the content of rebaudioside D and the compound represented by formula (1) is 30-600 ppm, and the content of the compound represented by formula (1) in terms of mass is 0.5-95% of the aforementioned sum.

The invention belongs to the technical field of genetic engineering and protein modification, and particularly relates to a novel combined mutant of glycosyl transferase UGT91C1-F208M. The gene sequence of the starting protein UGT91C1-F208M is Seq ID (Identity) NO. 1, and the primary sequence of the protein is Seq ID NO. 2. The mutant is obtained through a site-directed mutagenesis technology. According to the invention, combined mutation of an H93 site is further carried out on the basis of the UGT91C1-F208M, and compared with the UGT91C1-F208M, the activity of the optimal mutant UGT91C1-F208M-H93F is improved by 1.3 times, and the activity of the optimal mutant UGT91C1-F208M-H93F is improved by 1.3 times; and compared with the wild type UGT91C1, the activity is improved by 2.6 times. The activity of the mutant is remarkably improved, and the thermal stability of the mutant is kept unchanged, so that the mutant has remarkable advantages in large-scale industrial production of rebaudioside D (RD). According to the mutant disclosed by the invention, the catalytic efficiency of enzyme is remarkably improved, good thermal stability is also maintained, and a more efficient and economical solution is provided for related industrial production.

The application discloses a novel steviol glycoside composition and application thereof, and belongs to the technical field of sweeteners. The composition comprises rebaudioside M2, rebaudioside D and rebaudioside M, wherein the weight percentage of each component is as follows: rebaudioside M2 60%-85%, rebaudioside D 5%-15%, and rebaudioside M 10%-25%. The composition is highly simulated to sucrose in terms of sensory properties such as sweetness intensity, sweetness peak speed, after-sweetness residue, bitterness and astringency through ternary synergistic compounding, and is suitable for edible products such as dairy products, beverages, candies, baked products, frozen drinks, condiments and health foods, and can realize partial or complete replacement of sucrose, and has the advantages of pure naturalness, zero calorie, pure taste, wide application and the like.

A Stevia extract made from leaves of the Stevia rebaudiana plant is described. The extract has desired levels of steviol glycosides and is useful in food, beverage, and other consumable products.

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 %.