36 results about "Glycosynthase" patented technology

The invention discloses a methanophilic genetically engineered bacterium for producing trehalose as well as a construction method and application of the methanophilic genetically engineered bacterium, and belongs to the technical field of industrial biology. The methanophilic genetically engineered bacterium capable of producing the trehalose is obtained by taking methanophilic bacteria methanophilic 5GB1, Alkalicicus glycogenes WONF2802 or methanophilic bacteria NG09 as host bacteria, introducing maltooligosyl trehalose synthase (MTS) into the host bacteria, and carrying out over-expression on maltooligosyl trehalose hydrolase (MTH), so as to obtain the methanophilic genetically engineered bacterium capable of producing the trehalose. Under the condition that methane or methanol serves as a carbon source, the yield of the produced trehalose can reach the gram level or above, a new low-carbon biosynthesis way can be provided for commercial production of the trehalose, and the methanophilic genetically engineered bacterium for producing the trehalose has a good industrial application prospect.

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 discloses recombinant yarrowia lipolytica with high yield of 3-fucosyllactose as well as a construction method and application of the recombinant yarrowia lipolytica. According to the invention, a reinforced lactose transporter lac12 gene, a reinforced GDP-mannose-4, 6-dehydratase gmd gene, a reinforced GDP-L-fucose synthase gmer gene and a 3-fucosyltransferase SUMO-fut3Bc gene with a reinforced SUMO tag are subjected to integrated expression on a recombinant yarrowia lipolytica chromosome, and a 6-phosphogluconolactonase pgl gene is subjected to overexpression, so that a recombinant yarrowia lipolytica strain is obtained. The constructed recombinant yarrowia lipolytica can maintain the genetic stability of a target gene, the yield and production efficiency of 3-fucosyllactose are remarkably improved, and the requirements of industrial production can be met.

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 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.

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 present invention belongs to the technical field of enzyme engineering, and particularly relates to a sucrose synthase mutant and its application. The sucrose synthase mutant described in the present invention is obtained by the following mutations on the amino acid sequence of sucrose synthase shown in SEQ ID NO.1: valine at position 159 is mutated to threonine. The sucrose synthase mutant has high enzyme activity and can be applied to the biocatalytic synthesis of neohesperidin. The present invention also provides a method for preparing neohesperidin using the sucrose synthase mutant. Using hesperetin-7-O-glucoside, sucrose, and uridine diphosphate as substrates, and adding the whole-cell crude enzyme solution of rhamnosyltransferase, rhamnose synthase, and sucrose synthase, neohesperidin is catalytically obtained with a relatively high conversion rate.

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 discloses a sucrose synthase for catalytic production of NDP-Glc and a recombinant gene of the sucrose synthase. The substrate Reb A / Reb D catalyzed by the sucrose synthase has poor solubility at low temperature, and the reaction efficiency can be obviously accelerated when the substrate Reb A / Reb D catalyzes and produces Reb D / Reb M in a high-temperature environment, so that the sucrose synthase also needs to be subjected to catalytic reaction at high temperature, and NDP-Glc is provided for the production of Reb D / Reb M in real time. Compared with wild type sucrose synthase, the sucrose synthase variant disclosed by the invention has the advantage that the thermal stability and the enzyme activity are obviously improved. The enzyme activity of the wild sucrose synthase is only about 29% of the highest value in a high-temperature environment of 70 DEG C, and the enzyme activity of the sucrose synthase mutant in the environment of 70 DEG C is more than 50U / mg. Therefore, the sucrose synthase variant disclosed by the invention is suitable for being used as a biocatalyst and is used for large-scale production of a sweetening agent rebaudioside D / rebaudioside M with better taste.

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 disclosure relates to a method for enhancing the quality (DAR distribution, stability and etc.) of glycosylated antibody-drug conjugates (ADCs) using active charcoal. In particular, the present disclosure adopts hydrophobic interaction chromatography (HIC) column, Protein A chromatography, active charcoal stirs and buffer exchange either alone or in tandem in order to exclude free glycosynthase enzymes. Therefore, the purity of glycosylated ADCs could be raised and ADC degradation could be prevented. Finally, the quality of OBI-902 (anti-TROP2 ADC) and OBI-904 (anti-Nectin-4 ADC) drug substance (DS) can be further determined.

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.

The invention discloses a method for multi-enzyme step-by-step preparation of glucosyl stevioside, and relates to the technical field of glucosyl stevioside preparation.The method comprises the steps that crushed rebaudioside A serves as a raw material, dextrin serves as an auxiliary material, cyclodextrin glucoside transferase is added for a glycosylation reaction, a glucosyl stevioside solution I is cooled, and a glucosyl stevioside solution II is obtained; the method comprises the following steps of: adding a saccharifying enzyme to hydrolyze, adding cane sugar, UGT glycosyl transferase, cane sugar synthase and uridine diphosphate, continuously converting unreacted rebaudioside A into stevioside RI, inactivating after the reaction, adsorbing with resin, desorbing with ethanol, dealcoholizing a desorbed solution, 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.

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.