26 results about "Glycosynthase" patented technology

Provided herein are host cells capable of producing a human milk oligosaccharide (HMO), such as yeast cells that include one or more heterologous nucleic acids encoding one or more enzymes of the HMO biosynthetic pathway, such as a fucosyltransferase, GDP-mannose dehydratase, lactose permease, and / or fucose synthase. Also provided are fermentation compositions including the disclosed host cells, as well as related methods of producing and recovering HMOs generated by the host cells.

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 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 present invention provides for the use of recombinant Endo-S2 mutants (named Endo-S2 glycosynthases) that exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of glycoproteins wherein a desired sugar chain is added to a fucosylated or nonfucosylated GlcNAc-IgG acceptor. As such, the present invention allows for the synthesis and remodeling of therapeutic antibodies thereby providing for certain biological activities, such as, prolonged half-life time in vivo, less immunogenicity, enhanced in vivo activity, increased targeting ability, and / or ability to deliver a therapeutic agent.

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.

A genetically modified Corynebacterium for production of fucosyllactose, wherein the Corynebacterium has been modified to express a permease for lactose import, GDP-D-mannose-4,6-dehydratase (GMD), GDP-L-fucose synthase (WcaG) and fucosyltransferase (FucT) from exogenous nucleic acid sequences. The exogenous nucleic acid sequences encode a permease for lactose import and the GMD, WcaG and FucT are chromosomally integrated. The Corynebacterium additionally may comprise chromosomally integrated exogenous nucleic acid sequences for expression of phosphomannomutase (ManB) and GTP-mannose-1-phosphate guanylyltransferase (ManC). In certain embodiments, the Corynebacterium is Corynebacterium glutamicum. The Corynebacterium of the invention may be defective for functional expression of one or more glycosyltransferases involved in corynebacterial cell wall biosynthesis.

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 relates to a novel glycosynthase, especially an endoglycoceramide synthase, and to a method for the glycosylation of sphingolipids.

The present application relates to methods of producing a protein in fucosylated and afucosylated forms at a predetermined ratio by partially or completely inactivating GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductae (GDP-L-fucose synthase, EC 1.1.1.271) and additionally by adjusting the presence or absence of fucose source during culturing.

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.

The present invention belongs to the fields of genetic engineering and enzyme engineering technology and discloses a trehalose synthase mutant. The trehalose synthase mutant is obtained by subjecting one of the amino acids 160, 192, 216, 228, or 234 of the parent trehalose synthase to a point mutation. Specifically, the mutant is obtained by designing site-directed mutagenesis primers, performing site-directed mutagenesis using a vector carrying the trehalose synthase gene as a template, constructing a plasmid vector containing the mutant, and then transforming the vector into a host cell. The trehalose synthase mutant is used in the production of trehalose. The trehalose synthase mutant of the present invention contains a trehalose synthase with improved thermal stability and has good industrial prospects for its use in trehalose production.

The application belongs to the field of enzyme-catalyzed synthesis of organic compounds, and particularly relates to a mutant of amylosucrase, a solidified enzyme, a preparation method of the solidified enzyme and a glycosidase catalytic synthesis method, wherein the mutant of amylosucrase is an amino acid sequence with at least one mutation of N210T, R131K and S450T in a wild amino acid sequence shown in SEQ ID NO. 1. The mutant has excellent activity, stability and substrate tolerance.

The invention discloses a saccharomyces cerevisiae genetically engineered bacterium for efficiently synthesizing mogroside V through multi-dimensional cytoplasm adaptability and a construction method of the saccharomyces cerevisiae genetically engineered bacterium. The construction method of the saccharomyces cerevisiae genetically engineered bacterium comprises the following steps: integrating truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase tHMG1, pentenyl pyrophosphate isomerase IDI1, squalene synthetase ERG9, epoxy squalene cyclization enzyme ERG1, choline kinase ScCK, isopentenyl phosphokinase AtIPK, phosphoketolase BbPK, phosphotransacetylase PTA and dihydroxyacetone hydrolase EcHAD at a site of a saccharomyces cerevisiae genome GAL80, and carrying out enzymolysis, so as to obtain the saccharomyces cerevisiae genetically engineered bacterium. The invention relates to a preparation method of a cytochrome P450 enzyme, which comprises the following steps of: preparing a cycloepoxide hydrolase SgEPH3, a cytochrome P450 enzyme CYP87D18 and a cytochrome P450 enzyme reductase AtCPR1; 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 saccharomyces cerevisiae genetically engineered bacterium constructed by the invention realizes a higher level of mogroside V yield synthesized from the beginning in microorganisms, and has important industrial application potential.

The application belongs to the technical field of biology, and provides a recombinant microorganism and application of the recombinant microorganism in preparation of 2'-fucosyllactose, wherein the recombinant microorganism carries nucleic acids encoding GDP-mannose dehydratase, GDP-L-fucose synthase and alpha-(1,2) fucosyltransferase. It is found that by making the microorganism express GDP-mannose dehydratase, GDP-L-fucose synthase and alpha-(1,2) fucosyltransferase and adjusting the copy number of the above genes, efficient production of 2'-fucosyllactose can be realized, and the production cost is low, the cycle is short, and the yield is high.

The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to a sucrose synthase mutant as well as a preparation method, a product and application thereof. The mutant is characterized in that on the basis of a wild sucrose synthase amino acid sequence as shown in SEQ ID NO.1, the 94th amino acid is mutated from P to N, and the mutated amino acid sequence is as shown in SEQ ID NO.2; or the 660th amino acid is mutated into H from Y, and the mutated amino acid sequence is as shown in SEQ ID NO.3. The mutant can be used for in-vitro glycosylation reaction, plant genetic improvement or industrial biological catalysis, and solves the technical problems of poor stability and low catalytic efficiency of natural enzyme.

The invention discloses a method for preparing rare ginsenoside 20 (S)-Rg3 by directionally converting panax notoginseng stem and leaf saponin based on multienzyme synergistic catalysis, and belongs to the technical field of enzyme engineering. The method comprises the following steps: firstly, generating 20 (S)-Rg3 by using multifunctional xylosidase Tpexy3 and glucosidase Tpebgl3; further, glucosidase Dth3 and glucosidase Tpebgl1 are used for efficiently converting the saponin of the non-Rg3 into the protopanoxadiol; finally, PPD is used as a substrate, and directional synthesis of 20 (S)-Rg3 is achieved through a three-enzyme cascade reaction system composed of UGT mutants MuUGT73F13, UGT94Q2 and sucrose synthase GmSUS. According to the method, the resource advantages of the PPD type saponin in the pseudo-ginseng stems and leaves are fully utilized, the raw material utilization rate and the product added value are remarkably improved, and a feasible path is provided for green and efficient production of the rare ginsenoside.

The invention belongs to the technical field of genetically engineered bacteria, and particularly relates to a multienzyme co-expression genetically engineered bacterium as well as a preparation method and application thereof. According to the invention, genetic engineering bacteria capable of co-expressing sucrose synthase, beta-1, 2-glycosyltransferase and beta-1, 3-glycosyltransferase are constructed, so that fundamental transformation from a traditional complex process of three-bacterium three-enzyme step-by-step fermentation-in-vitro mixing to an innovative mode of one-bacterium three-enzyme one-step catalysis is successfully realized; the innovation of the technical route enables the production efficiency, the cost control and the process simplification to be remarkably improved.

The present disclosure relates to novel glycosynthase enzymes for antibody or antibody-drug conjugates (ADCs) engineering. The enzyme mutants, termed EndoSc-D233Q / E350N and EndoSc-D233Q / E350Q, contain the glycan conjugation and / or modification activity at the conserved N297 glycosylation site of Fc region of an exemplary antibody. It has been demonstrated that the glycosynthase activities of EndoSc-D233Q / E350N and EndoSc- D233Q / E350Q can be applied to various mAbs targeting different receptors. It has been found that both mAb-GlcNAc and mAb-GlucNAc(Fuc) were suitable substrates for both EndoSc mutants.

The application discloses a sucrose synthase mutant, a preparation method and application, and the amino acid sequence of the mutant comprises the mutation of the amino acid residues of at least one site corresponding to T22A, V47L, M48I, T49A, S60K, Y68G, C73S, Q80L, L90P, V104E, T150S, S212E, D219P, A221D, Q226E, K243T, M248L, I387L, V398I, E415D, G529S, Y532F, S533P, D549E, N581R, D596A, Q623E, V658Y, I667V, T719A, T729C, Q731E, G734S, A738K, D758E in SEQ ID NO:1. The application is based on sucrose synthase SrSUS1, single-point and combined mutations are carried out on the predicted amino acid, a sucrose synthase mutant with high enzyme activity is obtained, the mutant is coupled with glycosyltransferase, high-efficiency preparation of the glycosylation product of steviol glycoside RebM is realized, and the application is suitable for industrial production.

The invention discloses a sucrose synthase gene Pnsu1 which is derived from pseudo-ginseng, and the nucleotide sequence of the sucrose synthase gene Pnsu1 is as shown in SEQ ID NO: 1; the sucrose synthase gene Pnsu1 is applied to promotion of biosynthesis of notoginsenoside, the sucrose synthase gene Pnsu1 is overexpressed in panax notoginseng cells, so that the panax notoginseng cells overexpressed with the sucrose synthase gene Pnsu1 remarkably increase the content of panax notoginseng saponins compared with normal panax notoginseng cells, a new way is provided for synthesis and accumulation of a large amount of ginsenoside, and the application of the sucrose synthase gene Pnsu1 in the field of biosynthesis of notoginsenoside is broad. And a technical method and a new strategy are provided for a ginsenoside synthesis network.

The invention belongs to the field of enzyme engineering, and particularly relates to a thermostable sucrose synthase mutant and application thereof. On the basis of a sucrose synthase amino acid sequence as shown in SEQ ID NO: 1, site-directed mutagenesis is carried out through a rationally designed molecular modification technology, and the thermal stability of the modified sucrose synthase mutant is remarkably improved. The mutant enzyme is obtained by carrying out induced expression and protein purification on the obtained mutant strain, and compared with wild type sucrose synthase, the half-life period of the mutant is prolonged from 42.88 min to 679.41 min under the condition of 50 DEG C and is 15.8 times that of the wild type sucrose synthase; at the temperature of 55 DEG C, the half-life period is prolonged from 12.94 min to 110 min and reaches 8.5 times of that of a wild type, and the strain can better adapt to industrial production.

This disclosure relates to novel glycosynthase enzymes for the engineering of antibody-drug conjugates (ADCs). The enzyme variants, designated EndoSd-D232M and EndoSz-D234M, contain glycan conjugation and / or modification activity at the conserved N297 glycosylation site in the Fc region of exemplary antibodies. The glycosynthase activity of EndoSd-D232M and EndoSz-D234M was demonstrated to be applicable to various mAbs targeting various receptors, including, but not limited to, Globo H, SSEA-4, and SSEA-3 series receptors (OBI-888; Globo H ganglioside), Herceptin (Her 2 receptor), Perjeta (Her 2 receptor), and Vectibix (EGFR receptor). Both mAb-GlcNAc and mAb-GlucNAc(F) were found to be suitable substrates for EndoSd-D232M and EndoSz-D234M.

The invention relates to the technical field of biological catalysis, in particular to an enzymatic reaction catalyst for synthesizing quercetin 4 '-O-beta-D-glucoside and application of the enzymatic reaction catalyst. The enzymatic reaction catalyst comprises glycosyl transferase and sucrose synthase, the amino acid sequence of the glycosyl transferase is as shown in SEQ ID NO: 3, and the amino acid sequence of the sucrose synthase is as shown in SEQ ID NO: 4. According to the invention, glycosyltransferase and sucrose synthase are used as enzymatic reaction catalysts, and glycosyltransferase can carry out glycosylation on 4 '-site hydroxyl of quercetin; the sucrose synthase can catalyze the decomposition of sucrose to generate single-molecule fructose and single-molecule glucose, and the glucose can be combined with UDP to generate UDPG; therefore, a quercetin 4 '-hydroxyl glycosylation reaction is established, and high-efficiency and low-cost enzymatic synthesis of the natural product quercetin 4'-O-beta-D-glucoside is realized.

This invention belongs to the field of enzyme engineering, specifically relating to a thermostable sucrose synthase mutant and its applications. Based on the amino acid sequence of sucrose synthase shown in SEQ ID NO:1, this invention utilizes rationally designed molecular modification techniques to perform site-directed mutagenesis, resulting in a significantly improved thermostability of the modified sucrose synthase mutant. The mutant enzyme was obtained by inducing expression and purifying the protein from the resulting mutant strain. Compared to the wild-type sucrose synthase, the mutant exhibits a half-life that is extended from 42.88 min to 679.41 min at 50°C, which is 15.8 times that of the wild-type; and at 55°C, the half-life is increased from 12.94 min to 110 min, reaching 8.5 times that of the wild-type, thus better suited for industrial production.

The present application relates to glycosyltransferase mutants and their use in preparing quercetin-3-O-rhamnoside, by replacing the 44 amino acids of the C-terminal PSPG domain of the amino acid sequence of the glucosyltransferase AtUGT78D2 with the 44 amino acids of the C-terminal PSPG domain of the glucosyltransferase AtUGT78D1, obtaining a domain-engineered glycosyltransferase AtUGT78D2-D1PSPG; then performing site-directed mutagenesis on the glycosyltransferase AtUGT78D2-D1PSPG, obtaining a rhamnosyltransferase mutant with good selectivity, good stability and high expression, and the present application also provides a three-enzyme coupling catalytic synthesis reaction system for quercetin-3-O-rhamnoside constructed by the UGT78D2-D1PSPG mutant, which realizes the generation of UDP-rhamnose donor from sucrose as a substrate under the action of UDP-rhamnose synthase, reduces the production cost of UDP-rhamnose donor purchase, and then catalyzes the one-step directional synthesis of quercetin-3-O-rhamnoside from quercetin under the action of glycosyltransferase. At SuSy, UDP-rhamnose synthase At RHM-NRS, glycosyltransferase At UGT78D2-D1PSPG mutant The three-enzyme coupling catalytic synthesis reaction system for quercetin-3-O-rhamnoside constructed by the UGT78D2-D1PSPG mutant realizes the generation of UDP-rhamnose donor from sucrose as a substrate under the action of UDP-rhamnose synthase, reduces the production cost of UDP-rhamnose donor purchase, and then catalyzes the one-step directional synthesis of quercetin-3-O-rhamnoside from quercetin under the action of glycosyltransferase.

The present application relates to the field of microbial fermentation, and particularly relates to enzymes, enzyme compositions, expression frames, recombinant plasmids and strains for producing DFL. The present application provides a recombinant Escherichia coli engineering strain for expressing phosphomannanase gene (manB), mannose-1-phosphoguanosine transglycosylase gene (manC), GDP mannose 4,6-dehydratase gene (gmd), GDP-L-fucose synthase gene (wcaG), alpha-1,2 fucosyltransferase and alpha-1,3-fucosyltransferase genes in series. The inventors greatly improve the yield of the DFL production engineering strain by selecting high-efficiency key enzymes alpha-1,3-fucosyltransferase from different sources and expressing them in series with different strengths of alpha-1,2-fucosyltransferase, and reduce the generation of byproduct 2'-FL, and improve the economic type of the fermentation method for synthesizing DFL.