The present invention relates to a method for enzymatically preparing a β-nicotinamide mononucleotide. The method reacts a nicotinamide ribose as a substrate to produce the β-nicotinamide mononucleotide under the catalysis of a nicotinamide ribokinase and/or a nicotinamide ribokinase-containing recombinant cell in the presence of a phosphate group donor. The method for enzymatically preparing the β-nicotinamide mononucleotide provided by the present invention has important application values. Compared with the prior art that chemically synthesizes a β-nicotinamide mononucleotide, the method of the present invention is more environmentally friendly, lower in cost, and can provide products with higher purity, and thus can be more economically used in the fields of health care products and biomedicine.
Provided in the present application is a method for generating a fatty alcohol by utilizing a fatty acid or fatty acid ester. The method applies to preparation of a saturated fatty alcohol and unsaturated fatty alcohol.
C12P 7/04 - Préparation de composés organiques contenant de l'oxygène contenant un groupe hydroxyle acycliques
C12P 7/64 - Graisses; Huiles; Cires de type ester; Acides gras supérieurs, c. à d. ayant une chaîne continue d'au moins sept atomes de carbone liée à un groupe carboxyle; Huiles ou graisses oxydées
C12N 15/63 - Introduction de matériel génétique étranger utilisant des vecteurs; Vecteurs; Utilisation d'hôtes pour ceux-ci; Régulation de l'expression
3.
METHOD FOR PREPARING REBAUDIOSIDE M BY USING ENZYME METHOD
Provided is a method for preparing rebaudioside M by using an enzyme method. In the method, rebaudioside A or rebaudioside D is used as a substrate; and in the existence of a glucosyl donor, rebaudioside M is generated by means of reaction of the substrate under the catalysis of UDP-glucosyltransferase and/or recombinant cells containing the UDP-glucosyltransferase.
C12P 19/56 - Préparation d'O-glucosides, p.ex. glucosides avec un atome d'oxygène du radical saccharide lié directement à un système cyclique condensé d'au moins trois carbocycles, p.ex. daunomycine, adriamycine
C12P 19/18 - Préparation de composés contenant des radicaux saccharide préparés par action d'une transférase glycosylique, p.ex. alpha-, bêta- ou gamma-cyclodextrines
C12N 15/00 - Techniques de mutation ou génie génétique; ADN ou ARN concernant le génie génétique, vecteurs, p.ex. plasmides, ou leur isolement, leur préparation ou leur purification; Utilisation d'hôtes pour ceux-ci
The present invention relates to a biological preparation method of (S)-3-(dimethylamino)-1-(thiophene-2-radical)-1-propyl alcohol. According to the method, (S)-3-(dimethylamino)-1-(thiophene-2-radical)-1-acetone or salt thereof is used as a substrate, and the substrate is subjected to asymmetric reduction reaction in the presence of biocatalyst, cofactor and hydrogen donor to produce (S)-3-(dimethylamino)-1-(thiophene-2-radical)-1-propyl alcohol. Particularly, the biocatalyst is a combination of ketoreductase (KRED) and glucose dehydrogenase, the hydrogen donor is glucose, and the asymmetric reduction reaction is conducted at pH 6.8-7.0 and 25ºC-35ºC. Compared with the biological method in the prior art, the present invention has higher processing stability, and is simpler, more efficient and safer. During the reaction, the generation of highly toxic organic solvents such as acetone can be avoided, the application principle of green chemistry is met, and industrialized application is facilitated.
Provided is a method for preparing a rosuvastatin intermediate which is (R)-3-hydroxy-monomethyl glutarate and hydroxyl-protected (R)-3-hydroxy- monomethyl glutarate. (R)-4-cyano-3-hydroxy-methyl butyrate serving as a substrate is subjected to reaction under the action of an enzyme to generate the (R)-3-hydroxy- monomethyl glutarate, the enzyme is a recombinant nitrilase, and the reaction is carried out in an aqueous-phase buffering solution with the pH of 7.0 to 9.0 at the temperature of 25 to 35 degrees centigrade. The method has the characteristics of mild reaction conditions and high reaction efficiency, and is easy and convenient to operate; a specific recombinant nitrilase is used, thereby solving the problems of ultralow concentration of the substrate and low conversion rate in an existing biological method.
Provided is a biological preparation method for chiral N- protection peridol, in which N-protection piperidone is used as a substrate, and the substrate is subjected to an asymmetric reduction reaction in the presence of a reconstructed ketone reduction enzyme, a cofactor, and isopropyl alcohol to generate chiral N- protection peridol. The asymmetric reduction reaction takes place in an aqueous buffer having a pH value of 7.0 to 9.0 at the temperature of 25°C to 45°C, so as to successfully prepare chiral N- protection peridol having a high optical purity. The method has temperate reaction conditions, high reaction efficiency, and simple operations; especially, the concentration of the reaction substrate can be increased to 20%, so that the method has important and practical industrial application value.
C12P 17/12 - Préparation de composés hétérocycliques comportant O, N, S, Se ou Te comme uniques hétéro-atomes du cycle l'azote comme unique hétéro-atome du cycle contenant un hétérocycle à six chaînons
7.
ENZYMATIC PREPARATION METHOD FOR OXIDIZED COENZYME II
The present invention relates an enzymatic preparation method for an oxidized coenzyme II using a one pot method. Three substrates: nicotinamide nucleotide (NR), adenosine triphosphate disodium salt (ATP-Na2), and sodium hexametaphosphate are used as raw materials, to make them react in the existence of four enzymes: nicotinamide nucleoside kinase (NRK), inorganic pyrophosphatase, NAD kinase, and poly-pyrophosphokinase, in a buffer solution with pH being 4.0 to 8.5, and at a temperature of 10°C to 40°C, so as to obtain the oxidized coenzyme II. The present invention solves the technical problem that nicotinamide nucleotide has a high cost and is difficult to be obtained, the reaction time is long, a process cost is high, and a process condition is not suitable for industrial scale-up in a current enzymatic preparation method; and the oxidized coenzyme II can be obtained with high efficiency and a low cost in an industrialized scale-up production manner.
The present invention provides a method for preparing oxidized coenzyme I. The method enables nicotinamide nucleotide and adenosine triphosphate disodium salt to react in a buffer solution with pH being 5.0 to 8.0, under the catalytic action of nicotinamide nucleoside kinase and pyrophosphatase, in the existence of divalent metal ions, and at the temperature of 30°C to 40°C, so as to obtain the oxidized coenzyme I.
The present invention provides a biological preparation method of (S)-3-methylamino-1-(2-thienyl)-1-propyl alcohol, wherein 3-methylamino-1-(2-thienyl)-1-acetone is used as a substrate; under a condition that a biocatalyst ketoreductase, a cofactor and a cofactor regeneration system (comprising glucose and glucose dehydrogenase) exist, the substrate undergoes an asymmetric reduction reaction to generate the (S)-3-methylamino-1-(2-thienyl)-1-propyl alcohol; the asymmetric reduction reaction is carried out in a water-phase buffer solution with a pH of 6-8; and in a initial reaction system, concentration of the substrate is 100-150 mg/mL, and a mass ratio of the ketoreductase to the substrate is 1-2%. The present invention improves the concentration of the substrate and reduces enzyme usage.
A method for biological preparation of 6-cyano-(3R,5R)- dihydroxyhexanoic-tert-butyl-acetate. The method uses 6-cyano-(5R)-hydroxy-3-oxo-hexanoic-tert-butyl-acetate as a substrate, and the substrate undergoes a reduction reaction in the presence of a biological catalyst, a cofactor, and a hydrogen donor to produce 6-cyano-(3R,5R)- dihydroxyhexanoic-tert-butyl-acetate. The biological catalyst is a recombinant keto reductase. The cofactor is either NAD/NADH or NADP/NADPH. The hydrogen donor is isopropanol. The reduction reaction is conducted in an aqueous buffer solution of a pH between 6.5 and 7.5. The method of the present invention requires only one recombinant keto reductase to complete simultaneously substrate reduction and coenzyme regeneration cycle, greatly reduces production costs, provides mild reaction conditions, can be performed in room temperature, room pressure, and an aqueous condition, and obviates the need to use hazardous reagents. The byproduct, acetone, produced during the reaction is volatile, can be easily removed from the reaction system, thus greatly simplifying post processing works.
Provided is a method for biological preparation of (3R,5S)-6-chloro-3,5- dihydroxyhexanoic-tert-butyl-acetate. The method uses (S)-6-chloro-5-carbonyl-3-hydroxyhexanoic-tert-butyl-acetate as a substrate, and the substrate undergoes a reduction reaction in the presence of a biological catalyst, a cofactor, and a hydrogen donor to produce (3R,5S)-6-chloro-3,5- dihydroxyhexanoic-tert-butyl-acetate. The biological catalyst is a recombinant keto reductase. The cofactor is either NAD/NADH or NADP/NADPH. The hydrogen donor is isopropanol. The reduction reaction is conducted in an aqueous buffer solution of a pH between 6.0 and 9.0.
A biological preparation method of ethyl (R)-4-cyano-hydroxybutanoate. The reaction system of enzymatic synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate is properly treated, and recombinant halohydrin dehalogenase is subsequently added. Under the effect of recombinant halohydrin dehalogenase, ethyl (S)-4-chloro-3-hydroxybutanoate reacts with sodium cyanide to give a target product ethyl (R)-4-cyano-hydroxybutanoate, and the reaction takes place in an aqueous phase of pH 6-8. Ethyl (S)-4-chloro-3-hydroxybutanoate is prepared by asymmetric reduction reaction of ethyl 4-chloro-3-oxobutanoate under the presence of recombinant ketoreductase enzyme, a cofactor and a hydrogen donor, the hydrogen donor is isopropanol, and the asymmetric reduction reaction takes place in a mixed system of an aqueous phase buffer solution of pH 7.0-9.0 and toluene. In the present invention, the two reactions can share one reaction system, which can reduce the material and energy consumption, effectively reduce the cost, solve the problem of high material and energy consumption of a single-step enzyme method, and solve the problem of a coupled enzyme method that the product cannot be produced effectively due to mutual interference.
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