83 results about "Sucrose synthetase" patented technology

The invention relates to a method for preparing rebaudioside M through an enzyme method. According to the method, rebaudioside A or rebaudioside D is used as a substrate, and the substrate reacts to generate the rebaudioside M in the presence of sucrose and UDP under the catalytic action of a mixture of UDP-glucosyltransferase and sucrose synthetase or recombinant cells containing the UDP-glucosyltransferase and the sucrose synthetase, wherein the reaction is performed in a water-phase system having a pH value of 5.0-9.0 at 20-60 DEG C. The method for preparing rebaudioside M through an enzyme method has important application value; and compared with the existing technology of extracting rebaudioside M from stevia rebaudian leaves, the method provided by the invention obviously shortens the production cycle, improves the productivity and lowers the cost, and can provide products having higher purity. Thus, the method can be used in the food and beverage industry in a more economical manner.

The invention discloses a method for preparing rebaudioside-M by an enzymic method. The method comprises the steps of utilizing tomato UDP-glycosyltransferase and potato sucrose synthetase, and using rebaudioside-A and sucrose as raw materials for producing rebaudioside-M. According to the method disclosed by the invention, the high-yield recombinant strain of UDP-glycosyltransferase and sucrose synthase is obtained by virtue of a genetic engineering technology at first, then a bio-enzyme crude product is collected, a catalytic reaction is directly carried out, UDP or UDP-glucose is not added in the reaction system, the rebaudioside-A and the sucrose are used as the raw materials, and the rebaudioside-A is catalyzed to generate the rebaudioside-M through the efficient circulation of catalyzing the UDP-glucose by the sucrose synthase, and through the action of the tomato UDP-glycosyltransferase. Compared with the existing preparation method for rebaudioside-M, the process is short in steps, low in cost, important in application value, and good in economical efficiency.

The invention discloses a recombinant pichia pastoris engineering bacterium and a method for using the recombinant pichia pastoris engineering bacterium as a whole-cell catalyst for synthesizing RA (rebaudioside A). The recombinant pichia pastoris engineering bacterium is obtained through conversion of pichia pastoris after linearization of an expression vector containing exogenous DNA as follows: the DNA sequence for coding sucrose synthetase Sus1 is represented by SEQ ID NO.3, and the DNA sequence for coding UDP-glycosyl transferase UGT76G1 is represented by SEQ ID NO.4. The intracellular expression of the Sus1 and the surface expression of the UGT76G1 are realized by the aid of the recombinant pichia pastoris engineering bacterium, and the recombinant pichia pastoris engineering bacterium is used as the whole-cell catalyst and can be effectively used for synthesizing the RA, so that the synthetic efficiency of the RA is improved, the raw material utilization rate is increased, the production cost is reduced, and a new industrial way is opened for production and processing of the RA.

The invention discloses a co-catalytic reaction system for preparing rare ginsenosides. The co-catalytic reaction system comprises glycosyltransferase GT (a); sucrose synthase SUS (b); uridine diphosphate UDP (c); and saccharose (d). Experiments show that in the presence of the saccharose and little UDP, an enzyme combination composed of the glycosyltransferase and the saccharose is capable of replacing an in-vitro reaction system to synthesize UDP sugar, one of expensive materials for the rare ginsenosides, and efficiently and economically converting substrates such as protopanoxadiol or protopanaxatriol into the rare ginsenosides, wherein the UDP is about 1/4 of the UDP sugar in price and is 1% of its original dosage; thus, preparation cost of the rare ginsenosides is greatly saved, and large-scale commercial preparation of the ginsenosides is better facilitated.

The invention discloses a biocatalytic method for synthesis of rebaudioside M. Firstly, rebaudioside E is synthesized by a glycosylation reaction of stevioside with UDP-glycosyltransferase from tomatos and sucrose synthetase from potatoes; then, rebaudioside M is synthesized by a glycosylation reaction of rebaudioside E with UDP-glycosyltransferase from stevia rebaudiana and sucrose synthetase from potatoes. The method uses a molecular cloning technique, escherichia coli genetically engineered bacteria for heterologous expression of UDP-glycosyltransferase and sucrose synthetase are obtained,after fermentation and enzyme production, crude extract of cells is directly used for a catalytic reaction, addditionally added saccharose is decomposed with sucrose synthetase to obtain UDP-glucose,UDP in the crude extract and the UDP-glucose serve as raw materials for the glycosylation reaction, a double-enzyme cycle reaction system is established, and the rebaudioside M is produced by effectively catalyzing stevioside. The cost of raw materials is lower, the processing steps are simple, and the method has an important application value.

The invention discloses a glycosyl transferase mutant and a method for catalytically synthesizing rebaudioside M by using the glycosyl transferase mutant. The mutant is obtained by performing mutation on the basis of a glycosyl transferase amino acid sequence shown as SEQ ID NO: 1, performing induced expression on a mutant strain to obtain a mutant enzyme, and catalyzing 20g/L RebE to synthesize 12.8 g/L RebM by using the mutant enzyme as a catalyst and the enzymic method. The kinetic parameters of the mutant S195Q on rebaudioside E and rebaudioside D and the Michaelis constant of the mutant are 56.34 +/-2.02 mu M and 214.48 +/-14.54 mu M respectively, and are 1/3 and 2/5 of those of a wild type. The glycosyl transferase mutant is coupled with sucrose synthase to realize efficient catalytic synthesis of rebaudioside M. According to the present invention, the recombinant strain of the glycosyl transferase UGT76G1 or the mutant thereof and the sucrose synthase is constructed so as to achieve the efficient catalytic synthesis of the rebaudioside M; the method has the optimal yield in the current enzymatic catalytic synthesis experiment of rebaudioside M, and is green, environment-friendly and pollution-free.

The invention belongs to the field of plant physiology, and discloses a method for extracting sucrose synthase and sucrose phosphate synthase of pear fruits and an activity determination method thereof. Small molecules such as sugar, ions and the like can be usually removed from a primary extraction enzyme solution through adding a salting-out step and a dialysis step in the extracting process of the sucrose synthase and the sucrose phosphate synthase, so that activities of the sucrose synthase and the sucrose phosphate synthase in the fruits can be accurately detected. The method provides physiological basis for disclosing accumulation differences of the sugar in the fruits, and further provides theoretical basis for improving the quality of fruit sweetness and flavor.

A recombinant DNA construct comprising a polynucleotide encoding an ODP1 polypeptide operably linked to a sucrose synthase 2 promoter where this construct can be used to increase oil content in the seeds of a cruciferous oilseed plant while maintaining normal germination is disclosed. A method for increasing oil content in the seeds of a cruciferous oilseed plant while maintaining normal germination using this construct is also disclosed.

The invention discloses a glycosyltransferase mutant and a method for catalytically synthesizing rebaudioside A by using the same. The amino acid sequence of original glycosyltransferase is SEQ ID NO:1. According to the experiment, Asn196, Asn78, Asn400, Asn69, Gln72, Gln198, Gln178, Gln160 and Thr319 are screened on the basis of prediction obtained by starting from surface residues Q, N and T ofUGT76G1 and combining data analysis results of solvent accessible surface area, B-factor and the like, and finally, a UGT-SuSy system of the better mutant strain 76G1_Q72E-N196D-T319E is screened outby virtue of single-point or multi-point iterative mutation in Thr81, so as to realize efficient catalytic synthesis of the rebaudioside A. The glycosyltransferase UGT76G1 or a mutant gene thereof isconnected with a sucrose synthase gene to obtain a recombinant plasmid, and a double-enzyme co-expressed recombinant strain is constructed. By constructing a double-enzyme system, regeneration of in-vivo UDPG is realized, the problem of sources of expensive glycosyl donors is effectively solved, the cost is reduced, and application of biotechnology industry is promoted. The mutant is simple to prepare, the rebaudioside A is synthesized through efficient catalysis in a short time, and the biological enzyme catalysis method is green, environmentally friendly and pollution-free and is more suitable for current green industrial processing and production.

The invention provides a novel isolated sucrose synthase nucleic acid and its encoded protein. The present invention also provides methods and compositions relating to altering sucrose synthase levels in plants, and in particular, in plant stalks and/or plant seeds. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and transgenic seeds.

The invention relates to a composite growth regulator for increasing yield and saccharinity during the sugar accumulating period of beetroot and a usage, which are particularly suitable for the application under the arid environment, and have obvious effect on increasing yield and saccharinity through the foliage spray in late August. The regulator can not only control the injury caused by the stress of the environment, and keep the duration of the function foliage at the maximum, but also increase the activity of sucrose synthase, reduce the consumption of glycogen, promote coordination and uniform of the beetroot and the environment, individual and group, root and overground part, the external shape of the plant and internal physiological function during the sugar accumulating period, and finally reach the goal of improving the yield and saccharinity. Under the environment of the west of Jilin province, with less than 300 mm of the amount of precipitation, 0.47 percent of salt content and PH value of 8.6, the yield of beetroot reaches to 52,766.25kg/hm2, the yield is increased by 12.18 percent, and the sugar content is 17.0 percent, which is increased by 1.4 percent.

The invention discloses a method for biosynthesis of quercetin glycoside, which connects the gene of glycosyltransferase UGT73G1 or mutant thereof with sucrose synthase gene to obtain a recombinant plasmid, and constructs a recombinant bacterium containing a double enzyme system; The recombinant strain was inoculated in LB liquid medium, cultured at 25-40 DEG C until OD600 reached 2-3, adding inducer lactose to induce for 12 to 20 hour, centrifuging that fermentation broth to collect bacterial cells, crushing the bacterial cell, and centrifuging to collect supernatant to obtain crude enzyme solution; dissolving quercetin or isoquercetin in DMSO, add crude enzyme solution, sucrose, reaction 8-30 h, adding methanol to stop that reaction, and centrifuging to obtain the supernatant quercetin-3, 4'-Diglucoside; diglucoside. The amino acid sequence of the glycosyltransferase mutant is an amino acid sequence in which the amino acid sequence shown in SEQID NO: 1 is mutated, and the mutated amino acid site is selected from one or more of S151, T168, K274, T293, G361, S365, V371 and F381. The mutant is simple in preparation and high in yield, and realizes the function of quercetin The yieldof 3, 4 '-diglucoside was increased.

The invention discloses a method for synthesizing soybean oligosaccharides through biological catalysis. The method has the beneficial effects that that through establishing an in-vitro multi-enzyme cascade reaction system composed of sucrose synthetase, uridine diphosphate glucose 4-differential isomerase, inositol galactoside synthase, raffinose synthase and threonose synthase, sucrose is takenas a substrate to synthesize a single component or a mixed component in inositol galactoside, raffinose and threonose; the method realizes the recycling conversion of uridine diphosphate and inositol,and a crude enzyme solution added in the system contains a small amount of uridine diphosphate, so that the expensive uridine diphosphate can be avoided; the inositol galactoside yield reaches 50 g/L, the conversion rate is 70 percent, the raffinose yield reaches 20 g/L, the threonose yield reaches 10 g/L, and the obtained inositol galactoside, raffinose and threonose can be used in the fields offood, medicines and the like; the method disclosed by the invention is of great significance in the utilization of high attachment values of the sucrose and a biomass rich in the sucrose.

Methods and means are described to modify fiber characteristics in a fiber producing plant, such as cotton, based on the use of a novel type of sucrose synthase protein or related nucleic acids.

The invention discloses a method for improving the resistance of rice blast by using sucrose synthase (OsSUS3), and belongs to the technical field of molecular biology of sucrose synthase rice. The method comprises the following steps: firstly, cloning OsSUS3 gene cDNA from rice; secondly, constructing an OsSUS3 gene over-expression vector, converting rice middle flowers 11 to obtain transgenetic seedlings, wherein the OsSUS3 gene converted rice seeds Sus3(Oryza sativa Sus3) are preserved in China Center for Type Culture Collection on Jan. 3rd, 2017, and the preservation number is CCTCC NO:P201702; and thirdly, carrying out rice blast resistance measurement on the rice material of the obtained over-expression OsSUS3 gene. According to the method, the expression quantity of the OsSUS3 gene is increased, so that the rice blast scab length of transgenic rice is decreased by 17%, and the number of scabs is decreased by 37%.

The invention relates to a method for producing rebaudioside A through double-bacterium fermentation catalysis. The method is characterized by comprising the following steps: (1) connecting a glycosyltransferase UGT76G1 gene and a sucrose synthase AtSUSY gene to a pUC18 plasmid vector, transferring the pUC18 plasmid vector to DH5alpha escherichia coli competent cells, inoculating an LB culture medium, and culturing at 25-37 DEG C and 200-250 rpm for 10-18 h; (2) inoculating to a seed tank according to the inoculum size of 0.5%-15%, and culturing for 5-16 h at the speed of 150-400rpm and the ventilation ratio of 0.1-1.5 V/V.min at the temperature of 25-37 DEG C; (3) inoculating into a fermentation tank according to the inoculum size of 1%-10%, and culturing for 20-40 h at the speed of 100-1,000 rpm, the ventilation ratio of 0.2-2 V/V.min and the pH of 6.6-8.5 at the temperature of 25-37 DEG C; (4) adding an inducer when the OD600 value reaches 20-100, wherein the concentration of the inducer is 0.1-1.5 mmol/L; (5) carrying out filter pressing, resuspending, crushing and filter pressing on the fermentation liquid to obtain a crude enzyme liquid; and (6) mixing stevioside, uridine diphosphate, a phosphate buffer solution and the crude enzyme solution according to a mass ratio of (40-100):(1-4):(400-600):(50-100), and performing reaction at 25-40 DEG C for 24-48 h. The method has the advantages that two crude enzyme solutions are obtained through one-time fermentation, and operation steps are few; the enzyme activity is high and the production cost is low.

The invention provides a growth regulator for the fruiting period of peanuts. The growth regulator comprises the following components in percentage: 30-35% of growth promoter and 65-70% of growth nutrient solution. The regulator can favorably remove oxygen free radicals and delay senility; and the regulator obviously improves the activity of leaf nitrate reductase and the activities of glutamine synthetase and glutamate synthetase, and improves the activities of leaf sucrose synthetase and sucrose phosphate synthetase, thereby being beneficial to the accumulation of dry matters in pods and the synthesis of protein and fat in seed kernels. The growth regulator obviously increases the yield of peanut pods; and the yield is increased because the weight of a single pod is obviously increased, the full-pod rate is improved, and the dual-kernel pod rate and the kernel rate are greatly improved.

The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant genetically engineered bacterium for producing gastrodin, a construction method and application. According to the recombinant genetically engineered bacterium, glycosyl transferase is introduced into a host, glycosyl transferase is expressed through a host strain to catalyze a substrate to synthesize gastrodin, sucrose synthase and polyphosphate kinase are introduced into the host, synthesis and cyclic regeneration of UDPG are achieved, the amount of glycosyl donor of a glycosylation reaction is increased, a whole-cell catalytic conversion method is constructed, on the basis that no extra UPDG is added, the conversion rate of gastrodin reaches up to 95% or above, the gastrodin yield is increased, and the gastrodin production cost is reduced.

The invention relates to a method for improving production and transformation efficiency of erythrite and an application of the method. The method comprises the steps that glucose is used as a raw material, yeast is used as a fermentation and transformation bacterial strain, and in the fermentation and conversion process, sucrose synthase and malt cyclodextrin-glucan transferase are added, so thatthe conversion rate of glucose into erythrite can be increased. The method is simple to operate and low in cost, and the conversion rate is notably increased.