90 results about "Sucrose synthase" patented technology

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 provides a method for producing glycosyltransferase or sucrose synthase by fermenting recombinant escherichia coli, and belongs to the technical field of biological fermentation. According to the fermentation method provided by the invention, a three-stage material supplementing control fermentation strategy is adopted, namely after fermentation is carried out for 5 hours, material supplementing operation is started, the material supplementing speed is 6.24-7.20 g / L / h, fermentation is carried out for 8-10 hours, the material supplementing speed is reduced to 4.80 g / L / h, the temperature is reduced to 25 DEG C, and induction is carried out; then maintaining the material supplementing speed until the fermentation OD600 is 65-75, and then reducing the material supplementing speed to 3.84-4.80 g / L / h until the OD600 is not obviously increased any more; the material supplementing mode has remarkable advantages in the aspects of improving fermentation OD600 and fermentation enzyme activity, shortening the fermentation period, reducing the cost and the like.

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 belongs to the technical field of bioengineering, and discloses a glycosyl transferase mutant which is obtained by manufacturing at least one point mutation with an amino acid sequence as shown in SEQ ID NO.1, mutated genes are cloned to corresponding carrier plasmids and transformed into escherichia coli for expression, and the obtained enzyme can be applied to production of stevioside D. The invention further discloses a preparation method of the glycosyl transferase mutant. The obtained glycosyl transferase mutant has the characteristics of high efficiency and stability, and can be coupled with sucrose synthase to efficiently prepare stevioside D.

Disclosed in the present invention are a sucrose synthase mutant and a method for preparing RD using same. The sucrose synthase mutant is based on the amino acid sequence shown in SEQ ID NO: 2 and contains a mutation in at least one of the following sites: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R. By means of site-directed mutagenesis of sucrose synthase, the enzyme activity of the sucrose synthase mutant is improved compared with that of the wild type. Also, some mutants can still maintain a high conversion rate in a wide pH range.

The invention discloses an engineered glycosyl transferase and application thereof in efficient preparation of triterpenoid saponin. The engineered glycosyl transferase is obtained by mutating serine at the 15th site of UGT74AC1 enzyme into alanine, mutating histidine at the 47th site into aspartic acid, mutating leucine at the 48th site into valine, mutating alanine at the 180th site into serine, mutating histidine at the 180th site into aspartic acid, mutating histidine at the 180th site into aspartic acid, mutating histidine at the 180th site into aspartic acid, mutating histidine at the 180th site into aspartic acid The mutant is obtained by mutating serine at the 332 site into proline, phenylalanine at the 367 site into tryptophan and lysine at the 420 site into arginine. According to the invention, the glycosyl transferase with high catalytic activity is obtained by modifying an enzyme engineering technology, and good application of the glycosyl transferase in synthesis of triterpenoid saponin is realized. The glycosyl transferase disclosed by the invention can also be coupled with sucrose synthase, or coupled with N-acetylhexosamine kinase and N-acetylglucosamine-1-uridine phosphate transferase, and acetylglucosamine is used as a glycosyl donor, so that cascade production of triterpenoid saponin with lower cost and higher efficiency is realized.

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 a sucrose synthase mutant and a method for preparing RD (Reduced Disorder) by using the sucrose synthase mutant. The sucrose synthase mutant is obtained by mutation of at least one of the following sites on the basis of an amino acid sequence as shown in SEQ ID NO: 2: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R; site-directed mutagenesis is carried out on sucrose synthetase, so that the enzyme activity of the sucrose synthetase mutant is improved compared with that of a wild type, and meanwhile, a part of the mutant still maintains a relatively high conversion rate in a relatively wide pH range.

The invention discloses saccharomyces cerevisiae engineering bacteria for efficiently synthesizing mogroside V on the basis of cytoplasm and lipid droplet double-cell area compartment and a construction method of the saccharomyces cerevisiae engineering bacteria. The construction method comprises the following steps: integrating truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase tHMG1, pentenyl pyrophosphate isomerase IDI1, epoxy squalene cyclization enzyme ERG1, squalene synthetase ERG9, choline kinase ScCK, isopentenyl phosphokinase AtIPK, cucurbitadienol synthetase SgCDS, cyclic epoxy hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 at the site of a saccharomyces cerevisiae genome GAL80; an ABC efflux protein PDR11, a glycosyl transferase UGTMG1, a sucrose synthase Susy and a glycosyl transferase SgUGT94-289-3 are integrated at a site Exg1 of a saccharomyces cerevisiae genome. The yield of mogroside V produced by shake flask fermentation of the saccharomyces cerevisiae engineering strain provided by the invention reaches 96.3 mg / L, so that mogroside V is efficiently synthesized from the beginning in the saccharomyces cerevisiae, and the saccharomyces cerevisiae engineering strain has important practical significance for promoting sustainable development of the mogroside industry.

The invention relates to an enzymatic reaction catalyst and application thereof in synthesis of quercetin 3, 4 '-di-O-beta-d-glucoside, and relates to the technical field of biological catalysis. The enzymatic reaction catalyst comprises glycosyl transferase and sucrose synthase, and the glycosyl transferase comprises glycosyl transferase I and glycosyl transferase II; the amino acid sequence of the glycosyl transferase I is as shown in SEQ ID NO: 4, the amino acid sequence of the glycosyl transferase II is as shown in SEQ ID NO: 5, and the amino acid sequence of the sucrose synthase is as shown in SEQ ID NO: 6. According to the invention, glycosylation reaction of hydroxyls at 3 and 4'sites of quercetin is established, and high-efficiency and low-cost enzymatic synthesis of the natural product quercetin 3, 4 '-di-O-beta-d-glucoside is realized.

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 application relates to the field of bioengineering, and particularly to a mutant of an enzyme, a mutant of a glycosyltransferase and application thereof in preparing rhodiolin. The present application provides a mutant of an enzyme, which has the following amino acid site mutations on the basis of a wild-type glycosyltransferase: the 308th site is mutated from I to Q, the 333rd site is mutated from V to R, the 363rd site is mutated from W to S and the 386th site is mutated from S to G, and the amino acid sequence of the wild-type glycosyltransferase is shown as SEQ ID NO: 1. The present application obtains a glycosyltransferase and a sucrose synthase with improved thermal stability through structural rational design and deep learning modification, and can glycosylate tyramine to generate rhodiolin in the form of regenerating ADP-glucose, and the price of ADP is only about 20% of UDP, which has a cost advantage.

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 purpose of the present invention is to provide: a plant having an increased yield of storage tissue; and a method for producing the plant. It was discovered that the yield of storage tissue in a plant can be increased by expressing, in the storage tissue of the plant, a sucrose synthase having a substrate specificity to adenosine diphosphate.

The invention belongs to the technical field of genetic engineering and functional enzyme preparations, particularly relates to sucrose synthase and a coding gene and a mutant thereof, and further discloses application of the sucrose synthase. According to the invention, sucrose synthase is obtained from candidate methylobacterium and is further mutated, so that a sucrose synthase mutant of which the enzyme activity, the conversion rate and the stability are remarkably improved is obtained, and the activity and the efficiency of a stevioside synthesis process are effectively improved.

The invention discloses a glycosyl transferase mutant with improved thermal stability and activity, and belongs to the technical field of biological catalytic synthesis. Through rational design, the 102nd site, the 110th site, the 208th site and the 251th site of glycosyl transferase are mutated to obtain a combined mutant UGT94E13M3 phase starting enzyme, the half-life period of the combined mutant UGT94E13M3 phase starting enzyme at the temperature of 45 DEG C is prolonged from 0.60 h to 8.88 h, the thermal stability is improved by 14.8 times, and the enzyme activity is improved by 1.08 times. According to the invention, the mutant and sucrose synthase are subjected to a coupling reaction, so that the Reb D conversion rate reaches 71.40%, and an efficient and green new way is provided for production of rebaudioside M8.

The present invention discloses a sucrose synthase mutant with improved thermal stability, belonging to the field of enzyme engineering. The present invention provides a sucrose synthase mutant with improved thermal stability, capable of synthesizing UDP-Glc at high temperatures, thereby improving its production efficiency and meeting the needs of industrial production.

The application discloses a sucrose synthetase mutant and a method for preparing RD by using the sucrose synthetase mutant, wherein the sucrose synthetase mutant contains at least one of the following mutations: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R on the basis of the amino acid sequence shown in SEQ ID NO:2; the sucrose synthetase mutant has higher enzyme activity than the wild type, and meanwhile, some mutants still maintain a high conversion rate in a wider pH range.

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 discloses 2-phenethyl alcohol-beta-D-glucoside catalyzed and synthesized by a cofactor self-circulating multi-enzyme cascade system and application of the 2-phenethyl alcohol-beta-D-glucoside. The system comprises phenylalanine dehydrogenase and a mutant thereof, wherein the phenylalanine dehydrogenase is used for converting L-phenylalanine (L-Phe) into phenylpyruvic acid (PPA); pPA is converted into pyruvate decarboxylase (Aro10) of phenylacetaldehyde; the alcohol dehydrogenase is used for reducing phenylacetaldehyde into 2-phenethyl alcohol (2-PE) or a mutant of the alcohol dehydrogenase; 2-PE is converted into sucrose synthase (SuSy) of 2-phenethyl alcohol-beta-D-glucoside (2PE-Glc) or a mutant of the sucrose synthase, and UDP-glucosyltransferase (UGT) or a mutant of the UDP-glucosyltransferase. A novel multi-enzyme cascade system with cofactor self-circulation capability is realized and is used for efficiently synthesizing 2PE-Glc from L-phenylalanine. The invention not only provides an effective 2PE-Glc biosynthesis strategy, but also shows the potential of a multi-enzyme cascade system in the aspect of synthesizing biologically active complex glycoconjugates.

The utility model discloses a preparation device of glucose stevioside, and relates to the technical field of glucose stevioside production, an inlet of a conversion tank is respectively communicated with a raw material tank, an auxiliary material tank and a cyclodextrin glucoside transferase tank, an outlet of the conversion tank is communicated with a hydrolysis tank, an outlet of the hydrolysis tank is communicated with a reaction tank, and the reaction tank is communicated with a reaction tank. The inlet of the reaction tank is respectively communicated with a sucrose tank, a sucrose synthase tank, a uridine diphosphate tank, a first UGT glycosyltransferase tank, a second UGT glycosyltransferase tank and an alkali liquor tank; an outlet of the reaction tank is communicated with a first macroporous adsorption resin column, an inlet of the first macroporous adsorption resin column is communicated with a first ethanol tank, an outlet of the first macroporous adsorption resin column is communicated with a first concentration tank, and a lower outlet of the first concentration tank is communicated with a first dryer; and an outlet of the first dryer is communicated with a first glucosyl stevioside tank. And the residual substrate is converted into stevioside with relatively better taste, so that the taste and quality of the product are improved.

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 utility model discloses a kind of multi-enzyme step-by-step preparation glucosyl steviol glycoside's device, it is related to glucose steviol glycoside production technical field, the inlet of first reaction tank is respectively communicated with raw material tank, dextrin tank, cyclodextrin glucoside transferase tank and first sodium hydroxide solution tank, the outlet of first reaction tank is communicated with hydrolysis tank, the outlet of hydrolysis tank is communicated with second reaction tank, the inlet of second reaction tank is respectively communicated with sucrose tank, sucrose synthase tank, uridine diphosphate tank, UGT glycosyltransferase tank and lye tank, the outlet of second reaction tank is communicated with concentration tank by pipeline, the lower outlet of concentration tank is communicated with drier, the outlet of drier is communicated with glucosyl steviol glycoside tank. Make residual substrate conversion is relatively more optimal steviol glycoside in taste, improve the taste, quality and yield of product.

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 invention discloses a method for biosynthesizing rebaudioside based on an in-vivo constructed UDP (User Datagram Protocol) recovery system, and belongs to the technical field of biology. A UDP recovery system is constructed in saccharomyces cerevisiae, specifically, the saccharomyces cerevisiae M24 is taken as a host, synthetic pathway coding genes including a sucrose synthase gene SUS3 and a polyphosphate dependent glucokinase gene PPGK are integrally expressed through a CRISPR Cas9 editing technology, and the genetically engineered bacterium is used for fermentation production of rebaudioside. The gene for endogenous regulation and control of UDP glucose synthesis is enhanced, a UDP recovery system is constructed at the same time, supply of UDP glucose is improved so as to improve the catalytic efficiency of enzyme, and therefore the yield of rebaudioside is increased. The breakthrough provides key technical support for industrial fermentation synthesis of rebaudioside, especially rebaudioside M, and has significant economic benefits and environmental protection values.

The present invention relates to a sucrose synthase variant, a UDP-glucosyltransferase variant, and uses thereof for producing steviol glycosides. The UDP-glucosyltransferase variant, the sucrose synthase variant, and a fusion enzyme using same according to the present invention have improved thermal stability and enzyme activity, and thus can be widely applied to the field of steviol glycoside synthesis, and have excellent industrial value and significance in being capable of increasing production efficiency and lowering production costs.

According to the invention, the glycosyl transferase srUGT76G1 is subjected to structural modification, so that a mutant with high enzyme activity is successfully obtained. Besides, the mutant and sucrose synthase SUS are combined for use, a UDPG cyclic regeneration system is established, extra addition of expensive UDPG is avoided, and therefore a new method is provided for Reb M production.

The invention discloses a bacterial-derived glycosyl transferase mutant which is obtained by mutating an amino acid residue at a specified position of an amino acid sequence as shown in SEQ ID No. 1, and discloses a method for catalytically synthesizing salidroside based on the mutant. According to the invention, sucrose synthase GmSUS from soybean and a mutant of glycosyl transferase BparUGT are selected for co-expression to form a UDPG cyclic regeneration system, so that the system can realize cyclic regeneration of UDPG only by using a trace amount of UDP and cheap sucrose, the production cost is greatly reduced, and the scheme better meets industrial application requirements. The optimal mutant and a UDPG circulating system are applied to enzymatic synthesis of the salidroside, the space time yield reaches 2.43 g / L / h and is the highest level reported at present, and the method has industrial application value.

Disclosed in the present invention is a method for efficient biosynthesis of a novel steviol glycoside derivative rebaudioside M9, belonging to the technical field of biocatalytic synthesis. The present invention provides a glycosyltransferase UGT94E13 and a mutant thereof capable of catalyzing the monoglycosylation activity of rebaudioside M2, and the mutant is used for preparing a brand-new steviol glycoside-rebaudioside M2 monoglycosylated derivative, named rebaudioside M9. The present invention constructs an enzyme cascade reaction between a glycosyltransferase mutant and a sucrose synthase, and realizes efficient catalytic synthesis of rebaudioside M9 with rebaudioside M2 as a substrate. 43.56 g / L (30 mmol / L) rebaudioside M2 is used as a substrate and is subjected to a reaction for 8 h, and 42.81 g / L (29.14 mmol / L) rebaudioside M9 can be obtained with a yield of 97.14%, which provides a new route and a new method for the production of new rebaudioside M9 and other rebaudioside derivatives.