38 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.

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 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.

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.

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 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.

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 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 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.

A glucosyltransferase mutant catalyzing RD to produce RM and a use thereof are provided. The glucosyltransferase mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO: 1 to at least one of the following mutations: mutating proline at position 84 to tryptophan; and / or mutating methionine at position 88 to valine; and / or mutating leucine at position 126 to phenylalanine; and / or mutating asparagine at position 196 to histidine; and / or mutating leucine at position 379 to isoleucine. The use of the mutant in preparation of the RM is also provided. The enzyme activity of the mutant is significantly higher than that of the wild type, and the conversion rate for the preparation of the RM is high.

The invention belongs to the technical field of enzyme engineering, and particularly relates to a beta-1, 3-glycosyl transferase mutant and application thereof in catalytic synthesis of rebaudioside M. The beta-1, 3-glycosyl transferase mutant disclosed by the invention is obtained by carrying out at least one mutation on beta-1, 3-glycosyl transferase with an amino acid sequence shown as SEQ ID NO.1: mutating isoleucine at the 14th site into alanine, and mutating isoleucine at the 14th site into alanine; and / or asparagine at the 50th site is mutated into alanine; and / or the isoleucine at the 92nd site is mutated into leucine; and / or leucine at the 141 site is mutated into cysteine or isoleucine. The mutant is applied to catalytic synthesis of rebaudioside M, and the catalytic efficiency and the thermal stability of the mutant are obviously superior to those of a wild type enzyme.

This invention discloses a glycosyltransferase UGT76G1 mutant, which is (a) a protein with the amino acid sequence shown in SEQ ID NO.3; or (b) a protein derived from (a) having one or more amino acid sequences substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO.3, and possessing enzymatic activity catalyzing the conversion of rebaudioside D to rebaudioside M. This invention also discloses the gene sequence encoding the glycosyltransferase UGT76G1 mutant of claim 1. This invention significantly improves the enzyme activity of glycosyltransferase UGT76G1 through site-directed mutagenesis. Compared with wild-type glycosyltransferase UGT76G1, the optimal mutant enzyme activity is increased by 4.72 times, thus significantly reducing the amount of enzyme used. This invention heterologously expresses the glycosyltransferase UGT76G1 mutant and sucrose synthase SUS in Pichia pastoris, without purification, and constructs a "UDP-UDPG" cyclic regeneration system using crude enzyme solution, avoiding the need to add additional UDPG as a glycosyl donor, thus significantly reducing costs.

To provide a method for producing rebaudioside D and rebaudioside M by reaction of glycosyltransferase, and to provide a composition for producing rebaudioside D and rebaudioside M containing glycosyltransferase.SOLUTION: Provided is a method for preparing rebaudioside D, the method including preparing rebaudioside D by reacting glucose-linked nucleotide diphosphate with rebaudioside A in the presence of glycosyltransferase B (UGT-B), wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins consisting of an amino acid sequence represented by a specific sequence.SELECTED DRAWING: Figure 1

This invention belongs to the field of biosynthesis technology, specifically relating to a method for the production of rebaudioside M and the transformation of complex substrates. The method includes: S1, adding a reaction additive to the substrate, adding UGT11mut enzyme and AtSUS enzyme, reacting to obtain an enzyme reaction solution; S2, adding UGT76G1mut enzyme to the enzyme reaction solution, continuing the reaction, collecting the reaction solution to obtain rebaudioside M; the weight ratio of UGT11mut enzyme, AtSUS enzyme, and UGT76G1mut enzyme is 1:1:2. This method achieves a rebaudioside A pure substrate conversion rate exceeding 95%, a rebaudioside M yield of 95.88 g / L, and a RA60 conversion rate exceeding 90%. The process is mild, controllable, green, and low-cost, suitable for large-scale industrial production.

The invention discloses a purification method of rebaudioside M. The method comprises the following steps: performing acid heat treatment on enzyme catalytic reaction liquid containing rebaudioside M to obtain filtrate; and performing ultrafiltration concentration on the filtrate to obtain a concentrated solution, collecting an extraction solvent phase and a water phase by using a three-phase extraction method in which a composite solvent consisting of DMSO and tert-butyl alcohol is added, performing salting-out, stirring, standing and layering, collecting an upper-layer solvent phase, concentrating, and drying to obtain a rebaudioside M crystal product. By adopting a three-phase extraction method, the rebaudioside M with high content, high purity and high yield is obtained, the purification condition is simple, the consumed time is short, the energy consumption is low, the cost is low, and the method has a good market prospect.

A food or beverage product is provided comprising a steviol glycoside composition, said composition comprising 5-30 wt % rebaudioside M (reb M), 15-35 wt % rebaudioside AM (reb AM), and 10-25 wt % rebaudioside N2 (reb N2) based on the total amount of steviol glycosides in the product, wherein the steviol glycoside composition is present in an amount below its sweetness threshold. It is found that the food or beverage product has a modified or enhanced flavor compared to a food or beverage product not comprising the steviol glycoside composition.

The application discloses a method for synthesizing rebaudioside M. The method comprises the following steps: in a reaction system containing rebaudioside, beta-1,2-glucosyltransferase, beta-1,3-glucosyltransferase and a glycosyl donor, rebaudioside M is obtained by catalyzing the reaction of rebaudioside and the glycosyl donor by means of beta-1,2-glucosyltransferase and beta-1,3-glucosyltransferase. The method adopts the combined catalysis of beta-1,2-glucosyltransferase and beta-1,3-glucosyltransferase, effectively converts rebaudioside into rebaudioside M, and has low raw material cost and high conversion rate.

The invention discloses a composition containing rebaudioside A, rebaudioside D and rebaudioside M. The composition comprises rebaudioside A, rebaudioside D and rebaudioside M. The content of the rebaudioside A accounts for less than 1% of the total content of the rebaudioside A, the rebaudioside D and the rebaudioside M, the content of the rebaudioside D accounts for less than 9.5% of the total content of the rebaudioside A, the rebaudioside D and the rebaudioside M, and the content of the rebaudioside M accounts for more than 84% of the total content of the rebaudioside A, the rebaudioside D and the rebaudioside M.

The application belongs to the technical field of enzyme engineering, and particularly relates to a beta-1,3-glycosyltransferase mutant and application thereof in catalyzing synthesis of rebaudioside M. The beta-1,3-glycosyltransferase mutant is obtained by mutating the beta-1,3-glycosyltransferase with an amino acid sequence shown in SEQ ID NO. 1 by at least one mutation, i.e. mutating isoleucine at the 14th position into alanine; and / or mutating asparagine at the 50th position into alanine; and / or mutating isoleucine at the 92nd position into leucine; and / or mutating leucine at the 141st position into cysteine or isoleucine. The mutant is applied to catalyzing synthesis of rebaudioside M, and is significantly superior to the wild-type enzyme in catalytic efficiency and thermal stability.