65 results about "Rebaudioside M" 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 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 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 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 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 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.

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 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 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.

A composition for effervescing and flavoring water includes 20 to 45 weight percent flavor enhancing ingredient; 10 to 35 weight percent sodium bicarbonate; 10 to 20 weight percent citric acid; 10 to 45 weight percent complex carbohydrate; one or more of: (i) a first natural sweetener comprising 0.1 to 2 weight percent Rebaudioside M; and (ii) a second natural sweetener comprising 0.1 to 2 weight percent Rebaudioside A. The composition is configured to generate bubbles in the water when added to the water and to add flavor and sweetness to the water.

The invention discloses a glycosyl transferase mutant for synthesizing rebaudioside M as well as a product and application thereof, and belongs to the technical field of gene engineering. The technical problem to be solved is that the existing glycosyl transferase and sucrose synthase are low in enzyme activity and insufficient in enzyme activity stability, so that the synthesis efficiency of rebaudioside M (RebM) is not high, and the industrial application of preparing RebM by an enzyme method is limited. According to the key points of the technical scheme, the glycosyl transferase mutant is provided, the mutant is obtained by mutating UDP-glycosyl transferase UGT76G1, and the amino acid sequence of the UDP-glycosyl transferase UGT76G is as shown in SEQ ID NO. 1; the mutation comprises mutation on the 83rd site of SEQ ID NO.1, and the amino acid sequence of the glycosyl transferase mutant is shown as SEQ ID NO.5. The invention further discloses a preparation method of the glycosyl transferase mutant.

The invention relates to the technical field of rebaudioside M crystals, in particular to a preparation process for improving fluidity of rebaudioside M crystals, which comprises the following steps: (1) taking a rebaudioside M raw material, adding a first solvent, heating and stirring to obtain a phase I solution for later use; (2) taking a second solvent of which the density is greater than that of the phase I solution as a phase II solution; taking a third solvent of which the density is smaller than that of the phase II solution as a phase III solution; and (3) adding the phase III solution in the step (2) into the phase II solution, taking the phase I solution in the step (1), controlling the temperature, adding, standing at constant temperature in a closed state until white flaky crystals appear in the phase I solution and at the interface of the phase I solution, the phase II and the phase III mixed solution, separating, leaching, and drying to obtain the rebaudioside M crystal. By adopting the preparation process, the rebaudioside M crystal with high flowability and high dissolution rate can be obtained.

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.

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.

A stevia cultivar containing a high Rebaudioside M content, designated ‘18136109’, is disclosed. The invention relates to the plant parts of stevia cultivar ‘18136109’, to the plants of stevia ‘18136109’ and to methods for producing a stevia plant produced by crossing the cultivar ‘18136109’ with itself or another stevia variety, including methods using marker assisted breeding. The invention further relates to hybrid stevia seeds and plants produced by crossing the cultivar ‘18136109’ with another stevia cultivar. Twelve highly polymorphic SNPs loci and the corresponding genomic sequences used to identify plant variety ‘18136109’-derived plant materials are also disclosed.

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 disclosure relates, at least in part, to compositions (e.g., formulations) comprising a steviol glycoside. Rebaudioside M (Reb M), combined with brazzein that have improved caloric profile as well as flavor, taste and / or mouthfeel. In some embodiments, the composition further comprises one or more additional sweeteners.

This invention relates to a novel Lactobacillus mari strain exhibiting excellent glucose transfer activity and a method for producing glucose-transferable steviol glycosides using the same. The novel Lactobacillus mari CJST01 strain exhibits superior steviol glycoside transfer activity compared to Lactobacillus mari DSM20444 strain, which is known to produce glucose-transferable steviol glycosides. This results in higher productivity and a shorter enzymatic reaction time in the manufacturing process. The steviol glycosides produced using this strain have the advantage of superior taste quality and aroma compared to alpha-1,4 linked steviol glycosides and rebaudioside M. Furthermore, glucose-transferable rebaudioside A produced using Lactobacillus mari CJST01 strain exhibits reduced off-flavors and odors and superior solubility.

The invention relates to the technical field of beverage preparation, and particularly discloses a preparation technology of a flavor and vision synergistic sugar-reducing tea beverage. The preparation technology of the flavor and vision synergistic sugar-reducing tea beverage comprises the following steps: step 1), performing supercritical extraction on tea leaves to obtain a supercritical extracting solution; (2) adding a compound enzyme into the supercritical extracting solution for enzymolysis and inactivation to obtain a tea-flavor zymolyte; (3) soaking tea leaves in water, filtering out the tea leaves to prepare tea soup, and adding the tea flavor zymolyte into the tea soup to obtain a flavor-enhanced tea soup base; and step 4) adding a compound flavor agent and carmine pigment into the flavor-enhanced tea soup base, and uniformly mixing to obtain the flavor and vision synergistic sugar-reduced tea beverage, the compound flavor agent being a compound of erythritol, mogroside, beta-ionone, stevioside and rebaudioside M. The invention also discloses a preparation method of the flavor and vision synergistic sugar-reduced tea beverage. The method has the advantages that the addition amount of erythritol is reduced, and meanwhile the high sweetness and flavor of the tea beverage are kept.

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 invention relates to the technical field of dairy product processing, in particular to rebaudioside M yoghourt and a preparation method thereof.The rebaudioside M yoghourt is prepared from, by mass, 14%-18% of skimmed milk powder, 0.02%-0.04% of rebaudioside M, 81%-85% of water and 0.1%-0.3% of leavening agent; rebaudioside M and skimmed milk powder are used as raw materials, pasteurization is carried out, then fermentation is carried out on a mixed material liquid through leavening agent thalli, and then post-curing and cold storage are carried out to obtain the rebaudioside M fermented milk. The problems that in existing yogurt preparation, potential health risks such as cancer risk increasing and obesity causing exist due to the fact that a large number of artificial sweeteners are used, most of the artificial sweeteners are directly discharged without being absorbed by the human body, and the substances directly enter the environment and are difficult to degrade in the environment and possibly threaten the ecological environment are solved.

The invention discloses a genetic engineering strain for synthesizing rebaudioside M as well as a preparation method and application of the genetic engineering strain, and relates to the technical field of bioengineering. The genetic engineering strain disclosed by the invention contains a coding gene of glycosyl transferase YojK, a coding gene of a glycosyl transferase UGT76G1 mutant P84A / P91A with an amino acid sequence as shown in SEQ ID NO.2, a coding gene of sucrose synthase Sus3, a coding gene of sucrose permease CscB and a coding gene of fructokinase Csck. The genetically engineered bacterium disclosed by the invention can be used for synthesizing rebaudioside M by utilizing a cheap raw material RebA, and the yield of rebaudioside M reaches 30.13 g / L. In addition, the RebM synthesis method directly adopts a fermentation method, enzyme purification is not needed, the technological process is simplified, and the production cost is reduced. The established escherichia coli genetic engineering strain capable of utilizing sucrose shows a good industrial application prospect in the synthesis of RebM.

The invention discloses a glycosyl transferase SrUGT76G1 mutant and application of the glycosyl transferase SrUGT76G1 mutant in synthesis of rebaudioside M, and relates to the technical field of biological enzyme engineering. The amino acid sequence of the glycosyl transferase SrUGT76G1 mutant is as shown in SEQ IE NO. 2. According to the mutant disclosed by the invention, the enzyme activity of the glycosyl transferase SrUGT76G1 is remarkably improved, and compared with wild type glycosyl transferase SrUGT76G1, the enzyme activity of the optimal mutant is improved by 6.6 times, so that the use amount of enzyme can be remarkably reduced. And moreover, the catalytic process is simple, no by-product is generated in the reaction, and the subsequent separation is simple, so that the method has the advantages of high economical efficiency, mild reaction conditions, environment friendliness, simple product post-treatment and the like, and shows a good industrial application prospect in the synthesis of rebaudioside M.

Provided herein are genetically modified host cells, compositions, and methods for improved production of steviol glycosides. The host cells are genetically modified to contain a heterologous nucleic acid that expresses novel and optimized variants of UGT76G1. The host cell further contains 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 rebaudioside M.