56 results about "Sucrose synthase" patented technology

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.

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 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 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 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 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 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.

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.

This invention belongs to the fields of genetic engineering and enzyme engineering, specifically relating to a sucrose synthase mutant, its preparation method, product, and application. The mutant is based on the amino acid sequence of the wild-type sucrose synthase shown in SEQ ID NO.1, with the 94th amino acid mutated from P to N, resulting in the amino acid sequence shown in SEQ ID NO.2; or the 660th amino acid mutated from Y to H, resulting in the amino acid sequence shown in SEQ ID NO.3. This mutant can be used for in vitro glycosylation reactions, plant genetic improvement, or industrial biocatalysis, solving the technical problems of poor stability and low catalytic efficiency of natural enzymes.

The mutant sucrose synthase satisfies the following (1) and / or (2) and has an amino acid sequence having 70% or more identity to the amino acid sequence of SEQ ID NO: 1. (1) An amino acid residue corresponding to the 348th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than said amino acid residue and having improved enzyme activity compared to before substitution. (2) An amino acid residue corresponding to the 618th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than said amino acid residue and having improved enzyme activity compared to before substitution.

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 utility model discloses a glucosyl stevioside production device, and relates to the technical field of glucosyl stevioside production, an inlet of a conversion tank is communicated with a raw material tank, a cyclodextrin tank, a beta cyclodextrin transferase tank and a first alkali liquor tank, and an outlet of the conversion tank is communicated with a reaction tank; an inlet of the reaction tank is respectively communicated with a sucrose tank, a sucrose synthase tank, a uridine diphosphate tank, a UGT glycosyltransferase tank and a second alkali liquor tank through pipelines, an outlet of the reaction tank is communicated with a cation exchange resin column, an inlet of the cation exchange resin column is communicated with an ethanol tank, an outlet of the cation exchange resin column is communicated with a concentration tank, and the concentration tank is communicated with a second alkali liquor tank. An outlet of the concentration tank is communicated with a dryer, and an outlet of the dryer is communicated with a glucosyl stevioside tank. The residue of unreacted glucoside in the glucosyl stevioside is reduced through a double-enzyme method, the conversion rate of a substrate is improved, the residue of the substrate is further reduced, and the taste of the product is integrally improved.

The invention relates to an enzymatic reaction catalyst and application thereof in synthesis of 10-hydroxycamptothecin-glucoside, and relates to the technical field of biological catalysis. The enzymatic reaction catalyst comprises glycosyl transferase with an amino acid sequence as shown in SEQ ID NO: 1 and sucrose synthase with an amino acid sequence as shown in SEQ ID NO: 2. The high-efficiency and low-cost enzymatic synthesis of 10-hydroxycamptothecin glucoside is realized through the catalytic reaction of the enzymatic reaction catalyst.

The present application relates to a method for improving corn yield and lignin content by overexpressing sucrose synthase gene. The method is to overexpress sucrose synthase gene ZmSUS1 in corn to obtain transgenic corn with high yield and high lignin content; the nucleotide sequence of the sucrose synthase gene ZmSUS1 is shown as SEQ ID NO. 1. The present application overexpresses sucrose synthase gene ZmSUS1 in corn, effectively provides precursor substances for the biosynthesis of starch and lignin, so that the corn overexpressing sucrose synthase gene ZmSUS1 has total starch content increased by 3.5-13.5%, yield increased by 19.0%-30.3%, and lignin content increased by 2.84-3.15% compared with normal corn plants, significantly improving the total starch content of corn, the yield of corn and the lignin content in straw.

The invention is applicable to the technical field of functional tea drink processing, and provides a preparation method of monascus summer tea, which comprises the following steps: carrying out synergistic pretreatment of pulsed electric field and compound enzymolysis on summer tea; inoculating a compound microbial agent composed of monascus, aspergillus niger and lactobacillus plantarum for fermentation; the synthesis of different metabolites is accurately regulated and controlled by adopting a four-stage dynamic temperature change process; a targeted activator system composed of L-arabinose, vitamin C and betaine is added in a key metabolism window stage; an in-situ enzymatic glycosylation system composed of cyclodextrin glycosyl transferase and sucrose synthase is introduced, and monacolin K and tea polyphenol are modified. According to the invention, pH / temperature dual-response targeted activation, fructo-oligosaccharide synergistic in-situ glycosylation and stable network construction are fused with multi-strain synergistic fermentation and dynamic metabolism regulation for the first time, so that the yield, bioavailability, flavor quality and storage stability of monacolin K in a final product are remarkably 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 invention belongs to the field of gene and enzyme engineering, and particularly relates to an AtSuSy mutant as well as a preparation method, a product and application thereof. The novel sucrose synthase mutant is obtained by mutation of a 782 site of wild sucrose synthase from Arabidopsis thaliana, the amino acid sequence of the wild sucrose synthase is as shown in SEQ ID NO. 1, and the amino acid sequence of the mutant is as shown in SEQ ID NO. 3. Enzymatic property characterization is carried out through plasmid construction, protein expression and purification, the residual activity of the mutant E782P after incubation for 10 min at 65 DEG C is 66.91% which is far higher than 26.51% of wild type sucrose synthase, meanwhile, the relative activity of the mutant E782P is 114.09% of that of wild type sucrose synthase, the activity is improved while the thermal stability is improved, and the mutant E782P has the potential of industrial application.

This invention proposes a UDP-glucosyltransferase mutant and a method for preparing rebaudioside D. Site-directed mutagenesis of UDP-glucosyltransferase is performed, and the mutant plasmid is transferred to *E. coli* to obtain recombinant engineered bacteria. The recombinant engineered bacteria express the protein through fermentation to obtain a crude enzyme solution of the UDP-glucosyltransferase mutant. ADP-dependent enzyme activity and temperature-dependent enzyme activity of the UDP-glucosyltransferase mutant were measured. The mutant ADP-dependent enzyme activity was significantly increased, and it exhibited thermostability. Rebaudioside D was prepared using rebaudioside A, sucrose, ADP, the UDP-glucosyltransferase mutant, and AtSUS sucrose synthase hypercatalytic synthase.

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 application belongs to the technical field of bioengineering, and discloses a glycosyltransferase mutant and application thereof in synthesis of steviol glycoside. The glycosyltransferase mutant is obtained by making at least one point mutation in an amino acid sequence such as SEQ ID NO. 1. The mutated genes are respectively cloned into pETDuet-1 and pCDFDuet-1 carrier plasmids containing sucrose synthase to obtain carrier plasmid A and carrier plasmid B, and then transformed into E. coli for expression. The obtained enzyme can be applied to the production of steviol glycoside D. Meanwhile, carrier plasmid C containing glycosyltransferase UGT76G4 and sucrose synthase and carrier plasmid D containing glycosyltransferase NtUGT-M8 and sucrose synthase are prepared. Carrier plasmid A and carrier plasmid C are simultaneously transformed into E. coli for the production of steviol glycoside M. Carrier plasmid B and carrier plasmid D are simultaneously transformed into E. coli for the production of steviol glycoside M2. The recombinant bacteria obtained by the application can efficiently prepare steviol glycoside D, steviol glycoside M and steviol glycoside M2, respectively.

The invention relates to the technical field of extract refining, and particularly discloses a method for preparing stevioside RM. The method comprises the following steps: (1) crushing dry stevia rebaudiana leaves, mixing with water, extracting, stirring, extracting, centrifugally filtering, performing column chromatography purification, washing with water, eluting with ethanol to obtain an eluent, and concentrating the eluent to obtain a stevioside solution; (2) adding an auxiliary stable mixed aid, cane sugar, cane sugar synthase, beta-1, 2-UDP-glucosyltransferase from dendrobenthamia grandiflora and beta-1, 3-UDP-glucosyltransferase from walnut into the stevioside solution obtained in the step (1), carrying out decoloration and desalination treatment after catalytic reaction, eluting with an ethanol-water solution, and carrying out vacuum drying to obtain the stevioside. And finally, carrying out vacuum concentration, recrystallization and drying treatment to obtain the product. According to the method, the stability of an enzyme action system in the stevioside RM preparation process can be remarkably improved.