177 results about "Cofactor" patented technology

The present invention provides a microorganism useful for biologically producing 1,3-propanediol from a fermentable carbon source at higher yield than was previously known. The complexity of the cofactor requirements necessitates the use of a whole cell catalyst for an industrial process that utilizes this reaction sequence to produce 1,3-propanediol. The invention provides a microorganism with disruptions in specified genes and alterations in the expression levels of specified genes that is useful in a higher yielding process to produce 1,3-propanediol.

The present inventors produced a variety of bispecific antibodies that specifically bind to both F. IX/F. IXa and F. X, and functionally substitute for F. VIIIa, i.e., have a cofactor function to promote F. X activation via F. IXa. Among these antibodies, the antibody A44/B26 reduced coagulation time by 50 seconds or more as compared to that observed when the antibody was not added. The present inventors produced a commonly shared L chain antibody from this antibody using L chains of A44, and showed that A44L can be used as commonly shared L chains, although the activity of the resulting antibody is reduced compared to the original antibody (A44HL-B26HL). Further, with appropriate CDR shuffling, the present inventors successfully produced highly active multispecific antibodies that functionally substitute for coagulation factor VIII.

The present invention concerns an electrode carrying immobilized redox enzymes such that electric charge can flow between an electron mediator group to the enzyme cofactor by the use of boronic acid or a boronic acid derivative that acts as a linker moiety between the cofactor and the electron mediator group. The invention also concerns devices and systems that make use of the electrode of the invention, such as bio-sensors and fuel cells, the electrode being one of the components thereof.

The invention discloses an E. coli BL21(pETDuet-ydjLnox) containing a 2R,3R-butanediol dehydrogenase gene ydjL and an NADH oxidase gene nox, wherein the strain is preserved in the 'China Center for Type Culture Collection' on December 23 in 2008, and the preservation number is CCTCC NO: M 208259. The invention also discloses application of a gene recombination bacterium in producing chiral S-AC by catalyzing meso-BD, producing chiral R-AC by catalyzing 2R,3R-BD and producing chiral pure 2S,3S-BD by splitting a 2,3-BD mixture. The concentration of a chiral AC prepared from the recombinant E. coli can reach over 6 grams per liter (the ee value is more than or equal to 96 percent); and the ee value of the chiral pure 2S,3S-BD is more than or equal to 98 percent, and the regeneration of a cofactor is achieved, thus the gene recombination bacterium has great industrial application prospect.

In one embodiment of the invention, a method is provided for preparing plasmid from host cells which contain the plasmid, comprising: (a) providing a plasmid solution comprised of unligatable open circular plasmid; (b) reacting the unligatable open circular plasmid with one or more enzymes and appropriate nucleotide cofactors, such that unligatable open circular plasmid is converted to 3′-hydroxyl, 5′-phosphate nicked plasmid; (c) reacting the 3′-hydroxyl, 5′-phosphate nicked plasmid with a DNA ligase and DNA ligase nucleotide cofactor, such that 3′-hydroxyl, 5′-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) reacting the relaxed covalently closed circular plasmid with a DNA gyrase and DNA gyrase nucleotide cofactor, such that relaxed covalently closed circular plasmid is converted to negatively supercoiled plasmid. In other embodiments, DNA gyrase is replaced by reverse DNA gyrase or reaction (d) is not performed.

The invention discloses application of alcohol dehydrogenase with amino acid sequence disclosed as SEQ ID NO:2 in preparing ethyl (R)-4-chloro-3-hydroxy butyrate from ethyl 4-chloroacetoacetate by asymmetric reduction. By using alcohol dehydrogenase with amino acid sequence disclosed as SEQ ID NO:2 as a catalyst, ethyl 4-chloroacetoacetate as a substrate and NADH (nicotinamide adenine dinucleotide) as a cofactor, asymmetric reduction is carried out to prepare the ethyl (R)-4-chloro-3-hydroxy butyrate. The invention applies the alcohol dehydrogenase with amino acid sequence disclosed as SEQ ID NO:2 in preparing ethyl (R)-4-chloro-3-hydroxy butyrate from ethyl 4-chloroacetoacetate by asymmetric reduction for the first time, and has favorable effect. The enzyme activity is up to 5.6 U/mg, the yield of the substrate is up to 94%, and the enantiomeric excess value of the product is 100%. The yield is high, and the production cost is greatly lowered.

A genetically engineered bacterium producing 2-phenylethanol and an applying method thereof are disclosed. The recombinant bacterium is constructed by coexpressing aromatic amino acid aminotransferase and exogenous phenylpyruvate decarboxylase (Aro10) (or indole-3-pyruvic acid decarboxylase), phenylethanol dehydrogenase or phenylacetaldehyde reductase in escherichia coli to obtain the recombinant bacterium producing the 2-phenylethanol. Glutamate dehydrogenase is coexpressed on this basis to achieve self-balancing cofactor circulation, thus increasing the yield of the 2-phenylethanol. The conversion ratio of the 2-phenylethanol prepared through the recombinant bacterium is high. The bacterium and the method have important application value in production of the 2-phenylethanol in the future.

The invention discloses an application of carbonyl reductase with the amino acid sequence as shown in SEQ ID NO:2 to prepare (S)-4-chloro-3-hydroxybutanoic ethyl ester by asymmetric reduction of 4-chloracetyl ethyl acetate. The carbonyl reductase with the amino acid sequence as shown in SEQ ID NO:2 is taken as a catalyst, the ethyl 4-chloracetyl ethyl acetate is taken as a substrate, and NADPH is taken as a cofactor, and the (S)-4-chloro-3-hydroxybutanoic ethyl ester is prepared by the asymmetric reduction. The carbonyl reductase with the amino acid sequence as shown in SEQ ID NO:2 is applied to preparing the (S)-4-chloro-3-hydroxybutanoic ethyl ester by the asymmertric reduction of the 4-chloroacetyl ethyl acetate for the first time, which produces good effect; the yield of the substrate is up to 95%, and the enantiomer excess value of the product is 100%, the yield is high, and the production cost is greatly reduced.

A method for producing lacto-N-biose I and galacto-N-biose inexpensively and conveniently is provided. The method for producing lacto-N-biose I or galacto-N-biose, characterized in that the method comprises causing: (i) a combination of a carbohydrate raw material with an enzyme that catalyzes phosphorolysis of the carbohydrate raw material to give a-glucose-1-phosphate; and (ii) a combination of an enzyme that converts α-glucose-1-phosphate to UDP-glucose and an enzyme that converts UDP-galactose to galactose-1-phosphate with their cofactors, and/or a combination of an enzyme (UDP-Gly synthase) that converts α-glucose-1-phosphate and UDP-galactose to UDP-glucose and α-galactose-1-phosphate, respectively, with its cofactor(s) to act in the presence of N-acetylglucosamine or N-acetylgalactosamine, phosphoric acid, lacto-N-biose phosphorylase (EC 2.4.1.211), and UDP-glucose-4-epimerase (EC 5.1.3.2).

The invention discloses an application of aldo-keto reductase in catalytic generation of (R)-4-chlorine-3-hydroxy ethyl butyrate. With aldo-keto reductase of which an amino acid sequence is as shown in SEQ ID NO:2 as a catalyst, with 4-ethyl acetoacetate as a substrate and NADPH as a cofactor, the (R)-4-chlorine-3-hydroxy ethyl butyrate is prepared by asymmetric reduction. The aldo-keto reductase of which the amino acid sequence is as shown in SEQ ID NO:2 is applied to the 4-ethyl acetoacetate to prepare the (R)-4-chlorine-3-hydroxy ethyl butyrate in an asymmetric reduction manner for the first time, so that a good effect is obtained; the enzyme activity can be up to 8.1U/mg; the conversion ratio of the aldo-keto reductase on the substrate can be up to 99.8%; the enantiomer excess value of the product is greater than 995; the yield is high; and the production cost is greatly reduced.

The invention relates to a method for producing R-3-aminobutanol. The method comprises the following steps: artificially designing a sequence shown in SEQID NO: 1, carrying out full gene synthesis, cloning the synthesized gene fragments into an expression vector pET22b to prepare a recombinant expression vector, transferring the recombinant expression vector into Escherichia coli to prepare a genetically engineered bacterium of a recombinant D-amino acid dehydrogenase, culturing the genetically engineered bacterium to prepare the recombinant D-amino acid dehydrogenase, sequentially adding a substrate of which the final concentration is 10-300mmol/L, a 2-15wt% cosolvent, a cofactor of which the final concentration is 0.1-1mmol/L, an amino donor of which the final concentration is 0.02-1mmol/L and the 0.01-1wt% D-amino acid dehydrogenase into a reaction solution to constitute a reaction system, reacting, after the reaction is completed, and extracting R-3-aminobutanol in the reaction solution. The method disclosed by the invention has the advantages that the cost is low, the conversion rate is greater than 95%, the product yield is greater than 85%, no by-product is generated and method is more suitable for industrial applications.

The invention relates to a method for preparing alpha-aminobutyric acid by utilizing series connection amino acid dehydrogenase and codon optimization formate dehydrogenase recombinant escherichia coli. First, codon optimization is performed on a Candida boidinii formate dehydrogenase gene sequence according to the codon preference of the escherichia coli. After codon optimization on formate dehydrogenase, the expression quantity of formate dehydrogenase in the escherichia coli can be improved remarkably, the yield of alpha-aminobutyric acid is increased, and in the escherichia coli, co-expression formate dehydrogenase and L-amino acid dehydrogenase can promote circulation of cofactors in mycetome without needing the adding of any exogenous cofactors. In addition, the process of utilizing whole-cell transformation bulk chemical L-threonine to produce alpha-aminobutyric acid is simple and quick, and the cost is low. In a 5 L fermentation tank, the yield of alpha-aminobutyric acid obtained through the method can be 81.5 g/L, and an actually effective strategy is provided for industrial production of alpha-aminobutyric acid.

Bispecific antibodies whose FIX activation-inhibiting activity is not elevated and whose FVIII cofactor function-substituting activity is elevated have been successfully discovered.

In one embodiment of the invention, a method is provided for preparing plasmid from host cells which contain the plasmid, comprising: (a) providing a plasmid solution comprised of unligatable open circular plasmid; (b) reacting the unligatable open circular plasmid with one or more enzymes and appropriate nucleotide cofactors, such that unligatable open circular plasmid is converted to 3′-hydroxyl, 5′-phosphate nicked plasmid; (c) reacting the 3′-hydroxyl, 5′-phosphate nicked plasmid with a DNA ligase and DNA ligase nucleotide cofactor, such that 3′-hydroxyl, 5′-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) reacting the relaxed covalently closed circular plasmid with a DNA gyrase and DNA gyrase nucleotide cofactor, such that relaxed covalently closed circular plasmid is converted to negatively supercoiled plasmid. In other embodiments, DNA gyrase is replaced by reverse DNA gyrase or reaction (d) is not performed.

The invention discloses a method for synthesizing a chiral bisaryl alcohol compound. The method provided by the present invention specifically uses a mutant of a carbonyl reductase SSCR derived from Sporobolomyces Salmonicolor and a mutant of alcohol dehydrogenase TbSADH derived from Thermoanaerobacter brockii to catalyze asymmetric reduction of (4-chlorophenyl)pyridine-2-ketone to form (S)-(4-chlorophenyl) pyridine-2-methanol. The experiment proves that the method provided by the invention has high yield and high ee value, can reduce the addition amount of coenzyme, and does not need to add enzyme such as glucose alcohol dehydrogenase for cofactor circulation, the reaction condition is mild, and the operation is simple.

The invention belongs to the technical field of bioengineering, and relates to construction of genetic engineering strains, in particular to recombinant escherichia coli and a construction method andapplication thereof. According to the recombinant escherichia coli, an undecylenyl phosphate glucose phosphotransferase gene wcaJ is knocked out, manB, manC, gmd, wcaG and srlD genes are recombined and expressed, a pntAB gene, an ndK gene and a gsK gene are further recombined and expressed, and a pfkA gene is knocked out, so that a synthesis path of forming fructose 6-phosphate is enhanced and theglycolysis process of 6-fructose phosphate is weakened, the accumulation of fructose 6-phosphate is facilitated, and thus more precursor substances are provided for the synthesis of GDP-L-fucose; inaddition, the regeneration of the cofactors NADPH and GTP is enhanced, so that the capacity of producing GDP-L-fucose by the recombinant escherichia coli strain is further improved.

The invention obtains a Gaussia Luciferase (GLuc) active fragment by deleting 37 amino acids at the N-terminal of the GLuc, which is named as GLuc 148. Being the same as the wild type Gluc, the GLuc 148 has high luminescence activity and can achieve stable biochemical properties without the presence of complementary factors, but the GLuc 148 has a smaller molecular weight and performs non-secretory type expression in eukaryocytes. Thus, the GLuc 148 is more suitable for being used as a report factor and a fluorescent energy donor, and can be used in the technical fields of cell and in-vivo imaging, high-flux screening, microorganism detecting and the like.

The present invention relates to a coupled biotransformation process of converting chenodeoxycholic acid (CDCA) and related compounds to ursodeoxycholic acid (UDCA) and related compounds. It also relates to the cloning, expression, and biochemical characterization of a novel NADP⁺-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) from Clostridium difficile, cofactor switch mutants thereof, and their application for the oxidation of bile acids. A further aspect of the invention relates to novel NADP-dependent cofactor switch mutants of the NADP⁺-dependent 7α-HSDH of E. coli and their application for the oxidation of bile acids.

The invention discloses two pseudomonas rhodesiae engineering bacteria and application thereof in preparation of 2, 5-furandicarboxylic acid. The 2, 5-furandicarboxylic acid is prepared by constructing a mixed bacteria multi-cell catalytic system by constructing genetic engineering bacteria of pseudomonas rhodesiae and oxidizing 5-hydroxymethylfurfural as a biocatalyst. The pseudomonas rhodesiae engineering bacteria and the application thereof in preparation of the 2, 5-furandicarboxylic acid have the advantages that (1) the mixed bacteria multi-cell catalytic system is adopted for the first time, and FDCA is synthesized by a one-pot method; and (2) the FDCA synthesis process is simple and controllable, three-step continuous oxidation reaction is carried out in the same system, the reaction system is simple, the reaction conditions are mild, expensive cofactors are not needed, intermediate oxidation products are not accumulated, the yield and the productivity of the FDCA are high, and the industrial application potential is achieved.

The invention discloses escherichia coli recombinant bacteria capable of high-producing 2, 5-dimethylpyrazine and a construction method of the escherichia coli recombinant bacteria, and belongs to thetechnical field of gene engineering. According to the escherichia coli recombinant bacteria and the construction method thereof, a genetic engineering method is applied, L-threonine dehydrogenase TDHis overexpressed in escherichia coli K-12 capable of high-producing L-threonine, and NADH oxidase NoxE derived from lactococcus microorganisms and aminoacetone oxidase AAOSO derived from streptococcus microorganisms are expressed in a heterologous manner, and meanwhile 2-amino-3-ketobutyric acid CoA ligase KBL and primary amine oxidase TynA are knocked out, so that a novel and efficient 2, 5-dimethylpyrazine synthetic route is constructed, and the problem of unbalance of cofactors in the recombinant bacteria is solved. By taking escherichia coli E. coli THR as an example, the accumulation amount of 2, 5-dimethylpyrazine reaches 1.2 +/-0.2 g/L through a 36h shaking flask fermentation experiment of the recombinant bacteria. According to the method, L-threonine high-producing strains are used as starting strains, the synthetic route of 2, 5-dimethylpyrazine in escherichia coli is successfully reconstructed, the defect of unbalanced intracellular cofactors is improved, and a new idea is provided for breeding 2, 5-dimethylpyrazine.