135 results about "Pyridoxal" patented technology

The invention discloses aminotransferase, a mutant and application to Sitagliptin preparation. According to the application, wet thalli obtained by performing fermentation culture on recombinant escherichia coli containing aminotransferase encoding genes are used as biocatalysts; Sitagliptin precursor ketone is used as a substrate; dimethyl sulfoxide is used as a latent solvent; phosphopyridoxal is used as a coenzyme; isopropylamine is used as an auxiliary substrate; a trolamine buffer solution with the pH being 8 to 9 is used as a reaction medium; a reaction system is formed; the biocatalytic reaction is performed under the conditions of the temperature being 30 to 45 DEG C and the stirring speed being 100 to 250 r / min; after the reaction is completed, the reaction liquid is separated and purified; the Sitagliptin is obtained. The aminotransferase and the mutant are used as biocatalysts; the latent carbonyl compound of Sitagliptin precursor ketone is directly used as the substrate; meanwhile, biocatalytic reaction is performed by using isopropylamine as the auxiliary substrate and using the pyridoxal phosphate as the coenzyme; the separation and purification is performed; Sitagliptin with high optical purity is prepared. The method has the advantages that the total yield is 76 percent; the product e.e. value reaches 99 percent.

The invention discloses a transaminase mutant as well as application thereof to preparation of sitagliptin midbody. The application adopts a wet thallus obtained by fermenting and culturing recombinant escherichia coli comprising aminotransferase coded gene as a biological catalyst, adopts sitagliptin midbody precursor ketone as a substrate, adopts dimethyl sulfoxide as a co-solvent and , adopts phosphopyridoxal as co-enzyme, adopts isopropylamine as an auxiliary substrate, adopts a pH 8-9 triethanolamine buffering solution as a reaction medium to form a reaction system to perform the biological catalytic reaction under the conditions that the temperature is 30 to 45 DEG C, and the stirring rate is 100 to 250 r / min, after the reaction is ended, the reaction solution is separated and purified to obtain sitagliptin; and the total yield of the method is about 81 percent, and a e.e. value of the product reaches 99 percent.

A pyridoxal-5′-phosphate pharmaceutical formulation suitable for oral administration is provided comprising a dissolution profile, when measured in a standard dissolution apparatus, according to the United States Pharmacopoeia dissolution test, at 37° C. in a 0.05M phosphate buffered solution having a pH of 6.8 at 75 rpm, as follows: (a) greater than about 30% at 15 minutes, (b) greater than about 85% at 30 minutes, (c), greater than about 90% at 45 minutes, or (d) greater than about 95% at 60 minutes. Additionally, in vivo oral intake of between 15 and 60 mg / kg of a pyridoxal-5′-phosphate pharmaceutical formulation can produce a maximum plasma level (Cmax) of between about 1 mg / L and 8 mg / L. A pharmaceutical formulation provided comprises (a) a core, wherein said core comprises pyridoxal-5′-phosphate or a pharmaceutically acceptable salt thereof; (b) a sub-coat surrounding the core; and (c) an enteric coat surrounding the sub-coat.

The invention provides a biological preparation method of R-3-aminobutanol. The method includes: conducting whole-gene synthesis according to an artificially designed sequence shown as SEQ ID NO:1, and performing cloning into pET24a so as to obtain a recombinant expression vector; transferring the recombinant expression vector into E.coli to obtain a recombinant D-transaminase genetic engineering bacterium; culturing the bacterium to prepare recombinant D-transaminase; adding 3-carbonyl butanol with a final concentration of 10-300mmol / L, dimethyl sulfoxide or acetonitrile with a mass percentage concentration of 2-15%, pyridoxal phosphate with a final concentration of 0.1-1mmol / L, isopropylamine or D-alanine with a final concentration of 0.02-1mol / L, and recombinant D-transaminase with a mass percentage concentration of 0.01-1% are added into the reaction solution to compose a reaction system for reaction; and at the end of the reaction, extracting R-3-aminobutanol from the reaction solution. With a low cost, the method provided by the invention can achieve a conversion rate of more than 97% and a product yield of greater than 87%, and does not generate by-product, thus being more suitable for industrial application.

The invention provides a reagent for detecting divalent zinc ions. The reagent is a fluorescein derivative pyridoxal fluorescein hydrzone. The pyridoxal fluorescein hydrzone is prepared through refluxing fluorescein hydrazide and pyridoxal hydrochloride in acetic-acid-containing ethanol, and has the advantages of cheap raw materials, simple reaction condition and easy production. The invention also provides a quantitative detection method of the divalent zinc ions. The method is used for quantitatively detecting the content of the divalent zinc ions in an HEPES solution having a pH value of 7.0 based on the pyridoxal fluorescein hydrzone. The detection method is highly sensitive and selective to the divalent zinc ions, and has a simple, sensitive and rapid detection process and an accurate detection result.

A composite auxin for making the flowering phase of kaffir lily earlier contains gibberellin, auxin, cytokinin, inositol, pyridoxal and acetylcholine.

The invention discloses a biological preparation method for R-3-aminopiperidine. The biological preparation method comprises the following steps: carrying out gene synthesis according to a sequence as shown in SEQ ID NO:1, cloning the gene into an expression vector pET22a to prepare a recombinant expression vector, and transferring the vector into escherichia coli to prepare a genetically engineered bacterium of recombinant D-transaminase; culturing the bacterium to prepare recombinant D-transaminase genetically engineered bacterium; adding 3-ketopiperidine with final concentration of 10-300mmol / L, 2-15wt% dimethyl sulfoxide or acetonitrile, phosphopyridoxal with final concentration of 0.1-1mmol / L, isopropylamine or D-alanine with final concentration of 0.02-1mol / L and 0.2-0.4wt% recombinant D-transaminase to form a reaction system to carry out reaction; and extracting the R-3-aminopiperidine in the reaction liquor after the reaction is ended. According to the biological preparation method, the cost is low, the conversion ratio is over 97%, the product yield is greater than 85% and byproducts are not produced, so that the biological preparation method is more suitable for industrial application.

The invention relates to a novel biaryl-structured chiral N-methyl pyridoxal catalyst and synthesis and application thereof. The general structural formula is as shown in the specification, wherein R1is hydrogen or C1-C24 alkyl, and R2 and R3 are independently hydrogen or C1-C24 alkyl. In comparison with the prior art, the prepared pyridoxal catalyst can be used in a bionic Mannich reaction process, and fast and effective synthesis of a chiral alpha-beta-diamino acid derivative is realized.

The invention discloses an L-threonine aldolase mutant, a gene and a method for preparing L-syn-p-methylsulfonylphenylserine. The L-threonine aldolase mutant is obtained by mutation of wild type L-threonine aldolase; and with glycine and p-methylsulfonylbenzaldehyde as substrates and pyridoxal phosphate as a coenzyme, the L-syn-p-methylsulfonylphenylserine can be generated from the L-threonine aldolase mutant through a catalytic condensation reaction. The L-syn-p-methylsulfonylphenylserine produced by using the L-threonine aldolase mutant disclosed by the invention has the following advantages: 1, the production process is simple, and the reaction conditions are mild; 2, the L-threonine aldolase has high selectivity and high optical purity and does not need to be split; 3, the atom utilization rate is high and theoretically can reach 100%; 4, the production process is environment-friendly and less in pollution, and conforms to the concept of green chemistry; and 5, product separation and purification are simple.

The invention discloses an aminopherase mutant and an application thereof in the production of L-glufosinate-ammonium. The application of the aminopherase mutant in the production of L-glufosinate-ammonium is as follows: a reaction system is formed by using a wet thallus obtained through the fermentation culture of recombinant escherichia coli containing a aminopherase mutant coding gene as a biocatalyst, 2-carbonyl-4 (methyl hydroxyl phosphoryl)-butyrate as a substrate, pyridoxal phosphate as a coenzyme, an amino donor as a cosubstrate, and a buffer solution with the pH value of 6-9 as a reaction medium, and is subjected to biocatalytic reaction at the temperature of 40 to 50 DEG C and the stirring speed of 150 to 250r / min to obtain the L-glufosinate-ammonium. According to the method, thetotal yield is 98%, and the e. e. value of the product reaches 99%.

A genetically engineered bacterium for preparing L-Phe is prepared from transaminase gene and constitutive promoter through linking, and inserting them into carrier to configure constitutive expression plasmid. The L-Phe can be prepared through converting the phenylphruvic acid in proper host bacteria by relative said genetically engineered bacterium. It has high mole conversion rate.

The invention relates to a nutrient composition for eliminating exercise-induced fatigue, and discloses a mixture. The mixture comprises R-lipoic acid, acetylcarnitine, biotin, niacinamide, lactoflavin, pyridoxal, kreatine, coenzyme Q10, resveratrol and taurine. The mixture has a good effect on fatigue resistance, and has safe use, small toxic and side effects, stability and controllability.

The invention discloses a method for preparing a serum-free soybean protein peptide animal cell medium, and relates to a serum-free or low-serum collective cell medium. The serum-free soybean protein peptide animal cell medium is prepared from calcium chloride, potassium chloride, anhydrous magnesium sulfate, sodium chloride, anhydrous sodium dihydrogen phosphate, L-glutamine, D-glucose, phenol red, D-calcium pantothenate, choline chloride, folic acid, i-inositol, niacinamide, pyridoxal hydrochloride, lactoflavin, thiamine hydrochloride, soybean protein peptide and distilled water. The method of the invention solves the problem that the conventional serum-free medium cannot be directly used for cultivating cells in the poor growth condition, and a large number of the cells can adapt to the conventional serum-free medium only when the serum concentrations thereof are gradually reduced, and the cells are easily affected by external factors, so fatal damages are easily caused. The method of the invention has the advantages that the cells can directly be cultivated in the medium of the invention without gradual domestication and cultivation, so the culture period of the cell is shortened and the interference of other factors is avoided; meanwhile, the virus pollution brought by serums is reduced, and the survival rate of the cells is over 94 percent.

The invention discloses a culture medium and utility and manufacturing method to produce lactic acid, which comprises the following parts: fitful thiamine, lactochrome, niacin, alcaine pyridoxal and biotin.

A serum-free vegetable protein peptide general cell culture medium relates to a serum-free or low-serum general cell culture medium. The serum-free vegetable protein peptide general cell culture medium solves the problems that the existing serum-free culture medium can not directly culture the cells with adverse growth status, most of cells can be adaptable to the existing serum-free culture medium after the gradual reduction of the serum concentration, and the existing serum-free culture medium is vulnerable to the external factors and easy to cause fatal damage. The serum-free vegetable protein peptide general cell culture medium is prepared by calcium chloride, potassium chloride, magnesium sulfate anhydrous, sodium chloride, anhydrous sodium dihydrogen phosphate, L-glutamine, D-glucose, phenol red, D-calcium pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, soybean protein peptide and distilled water. Cells can be directly cultured on the culture medium of the invention without gradual habituation and culture, thus shortening culture period and avoiding the interference by other factors, reducing the virus pollution which is caused by serum; the cell survival rate reaches more than 94 percent.

The invention discloses a mutant of threonine aldolase, a coding gene of the mutant, a recombinant vector constructed by the coding gene, a recombinant genetic engineering bacterium obtained by converting the recombinant vector, and application of the mutant of the threonine aldolase in preparation of 2- / 3- / 4-substituted phenylserine derivatives. According to the invention, the mutant of the threonine aldolase is taken as a biocatalyst, 2- / 3- / 4-substituted benzaldehyde is taken as a substrate, 5-phosphopyridoxal is taken as a coenzyme, glycine / glycine ester is taken as a cosubstrate, the enzyme catalysis reaction is performed under proper conditions and in a proper medium, a series of phenylserine derivatives with different substituent groups are prepared through separation, and the method has the total yield range of 76-99%, the product ee value of over 99%, and the de value range of 71-92%.

The invention relates to the technical field of enzyme catalysis, and specifically relates to a transaminase catalyst; and the invention further relates to a method for synthesizing (R)-1-tert-butoxycarbonyl-3-aminopiperidine by adopting an enzymatic way, as well as a production method of the (R)-1-tert-butoxycarbonyl-3-aminopiperidine. The method for synthesizing the (R)-1-tert-butoxycarbonyl-3-aminopiperidine by adopting the enzymatic way comprises a step of, in the presence of pyridoxal phosphate and the transaminase catalyst, allowing reaction of N-tert-butoxycarbonyl-3-piperidinone as a reaction substrate with an amino donor so as to produce the (R)-1-tert-butoxycarbonyl-3-aminopiperidine. The method for synthesizing the (R)-1-tert-butoxycarbonyl-3-aminopiperidine by adopting the enzymatic way utilizes relatively few reagents, and is mild in reaction conditions, so that tedious steps needed for chemical process synthesis are greatly simplified; and moreover, a target product withan ee value up to 99.77% or above can be obtained without performance of separation. Therefore, the transaminase catalyst and the process method utilizing the transaminase catalyst to synthesize the (R)-1-tert-butoxycarbonyl-3-aminopiperidine provided by the invention have broad application prospects as well as great market values.

The invention discloses a glutamic acid decarboxylase recombinant bacterium, which is an Escherichia coli introduced with L-glutamic acid decarboxylase GAD; and the L-glutamic acid decarboxylase GAD gene is from yeast Kodamaea ohmeri NH-9 and has a nucleotide sequence shown as a SEQ ID No:1. The invention also discloses the construction method and application of the above glutamic acid decarboxylase recombinant bacterium. The invention has the following advantages: (1) hydrochloric acid pyridoxal has a price half of that of 5 ' -phosphoric acid pyridoxal, so as to reduce technology cost; (2) the method has high enzyme activity, short reaction time, mild conditions, substrate concentration up to 510 g / L and concentration of a product gamma-aminobutyric acid up to 352 g / L; (3) the bacteria and the buffer after rotary evaporation can be reused, and the conversion rate is 100%, and the yield is no less than 99%; and (4) the technology is simple, green and can realize a product purity higher than 99%.

The invention belongs to the technical field of biological catalysis, and relates to a biological preparation method of agmatine sulfate, which comprises the following steps: by using an L-arginine sulfate solution as a substrate raw material, carrying out decarboxylation reaction in the presence of a biocatalyst and a coenzyme to obtain agmatine sulfate, and discharging gas CO2, wherein the biocatalyst adopts L-arginine decarboxylase, and the coenzyme adopts phosphopyridoxal. The L-arginine sulfate solution used as the substrate is stirred and mixed with the L-arginine decarboxylase and the coenzyme (phosphopyridoxal) to perform decarboxylation reaction under certain conditions, thereby obtaining the agmatine sulfate. The conversion rate is greater than or equal to 98%, and the continuous production yield can reach 90%. The technical route has the advantages of simple preparation technique, high production efficiency, low cost and the like. The fermentation and conversion techniques of the recombinant L-arginine decarboxylase can achieve the industrial preparation level, thereby basically solving the problems of high chemical synthesis cost, low yield, and great environmental hidden danger and the like.

The invention relates to an immobilized metal ion affinity chromatography (IMAC) functionalized hybrid monolithic material with a high specific surface area, prepared through a sol-gel technology, andan application thereof in phosphorylated peptide enrichment. A hybrid monolithic material containing phosphate functional groups on the surface is prepared through a sol-gel reaction by taking tetraethoxysilane (TEOS) and diethylphosphatoethyltriethoxysilane (DPTS) as precursors, the hybrid monolithic material is hydrolyzed in concentrated hydrochloric acid (HCl) to generate phosphoric acid functional groups on the surface, and the hydrolyzed hybrid monolithic material is chelated with Ti<4+> to obtain the Ti<4+>-IMAC hybrid monolithic material. The Ti<4+>-IMAC hybrid monolithic material canadsorb micromolecular pyridoxal 5-phosphate under the action of metal chelation, and can also be used for carrying out high-sensitivity and high-selectivity enrichment on phosphorylated peptide fragments in beta-casein and human cervical carcinoma cell (HeLa) enzymatic hydrolysate.

The invention provides an animal-source-free cell culture medium, which is prepared by proportionally mixing the components including calcium chloride, potassium chloride, anhydrous magnesium sulfate, sodium chloride, anhydrous sodium dihydrogen phosphate, L-arginine hydrochloride, L-cystine hydrochloride, L-glutamine, L-Leucine, L-isoleucine, L-histidine hydrochloride, L-tyrosine, D glucose, choline chloride, folic acid, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride and the like. Cells can be directly cultivated on the cell culture medium provided by the invention, rather than gradual domestication and cultivation, so that the density and the harvesting frequency of the cultured cells are increased and the expression quantity is increased so as to increase the vaccine yield in the field of medicine, thereby effectively avoiding the interference of animal-originated characteristics to the cultivated animal or human cells. The animal-source-free cell culture medium solves the problems that the cells cultivated by the prior serum-free medium have premature deaths and unsaturated forms.

The invention discloses corneal metaphase preserving liquid containing recombinant human serum albumin and a preparation method thereof. The corneal metaphase preserving liquid containing recombinanthuman serum albumin contains glutamine, calcium pantothenate, choline chloride, folic acid, inositol, nicotinamide, pyridoxal.HCl, riboflavin, thiamine.HCl, KCl, NaCl, NaH2PO4.2H2O, glucose, dextran 40, sodium pyruvate, vitamin C, chondroitin sulphate sodium, sodium hyaluronate, non-animal recombinant human serum albumin, gentamicin sulfate, sodium bicarbonate, piperazine-1-erhanesulfonic acid, phenol red indicator, and water for injection. The non-animal recombinant human serum albumin is added to replace serum to improve corneal endothelial cell survival rate; and then the non-animal recombinant human serum albumin cooperates with a compound system of the chondroitin sulphate sodium, the sodium hyaluronate and the dextran 40, the survival rate of the corneal endothelial cell is greatly ensured.

The invention relates to a method for preparing chiral beta-trifluoromethyl-beta-hydroxy-alpha-amino acid and derivatives thereof, which specifically comprises the following steps: weighing tert-butylglycinate and derivatives thereof, trifluoromethyl ketone, acid and chiral N-methyl pyridoxal catalyst, adding a solvent, and carrying out a reaction to generate the target product chiral beta-trifluoromethyl-beta-hydroxy-alpha-amino acid and derivatives thereof. Compared with the prior art, the pyridoxal catalyst does not need to protect and deprotect amino, and simple, so that convenient and efficient synthesis of the chiral beta-trifluoromethyl-beta-hydroxyl-alpha-amino acid derivative is achieved.

The invention discloses a glutamic acid decarboxylase recombinant bacterium, which is an Escherichia coli introduced with L-glutamic acid decarboxylase GAD; and the L-glutamic acid decarboxylase GAD gene is from yeast Kodamaea ohmeri NH-9 and has a nucleotide sequence shown as a SEQ ID No:1. The invention also discloses the construction method and application of the above glutamic acid decarboxylase recombinant bacterium. The invention has the following advantages: (1) hydrochloric acid pyridoxal has a price half of that of 5 ' -phosphoric acid pyridoxal, so as to reduce technology cost; (2) the method has high enzyme activity, short reaction time, mild conditions, substrate concentration up to 510 g / L and concentration of a product gamma-aminobutyric acid up to 352 g / L; (3) the bacteria and the buffer after rotary evaporation can be reused, and the conversion rate is 100%, and the yield is no less than 99%; and (4) the technology is simple, green and can realize a product purity higher than 99%.

A pyridoxal-5'-phosphate pharmaceutical formulation suitable for oral administration is provided comprising a dissolution profile, when measured in a standard dissolution apparatus, according to the United States Pharmacopoeia dissolution test, at 37 DEG C. in a 0.05M phosphate buffered solution having a pH of 6.8 at 75 rpm, as follows: (a) greater than about 30% at 15 minutes, (b) greater than about 85% at 30 minutes, (c), greater than about 90% at 45 minutes, or (d) greater than about 95% at 60 minutes. Additionally, in vivo oral intake of between 15 and 60 mg / kg of a pyridoxal-5'-phosphate pharmaceutical formulation can produce a maximum plasma level (Cmax) of between about 1 mg / L and 8 mg / L. A pharmaceutical formulation provided comprises (a) a core, wherein said core comprises pyridoxal-5'-phosphate or a pharmaceutically acceptable salt thereof; (b) a sub-coat surrounding the core; and (c) an enteric coat surrounding the sub-coat.

The present invention provides pharmaceutical compositions comprising a nicotinic acid derivative or a fibric acid derivative and a pyridoxal-5′-phosphate or a pyridoxal-5′-phosphate related compound and methods for using pharmaceutical compositions for reducing the risk of cardiovascular and other diseases.

The invention discloses a universal basal culture medium for zooblasts. The universal basal culture medium for zooblasts comprises the following components: alanine, glutamic acid, arginine, aspartic acid, cystine, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, disodium tyrosine, valine, calcium chloride, magnesium sulfate, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, biotin, choline chloride, inositol, niacinamide, D-semi-calcium pantothenate, pyridoxal, thiamine, riboflavin, ascorbic acid, folic acid, D-glucose, thymidine and reduced glutathione. The universal basal culture medium for zooblasts is good in universality.

The invention discloses a method for synthesizing L-tryptophan by immobilized enzyme. In the method, immobilized tryptophanase is taken as an enzyme source, L-cysteine and indole are taken as substrates, the L-tryptophan is produced through enzymatic conversion, the immobilized enzyme is removed through filtration, and the L-cysteine is obtained through further separation. In the method, the tryptophanase is immobilized through GE resin, and the optimized catalysis reaction conditions are that: the pH is 8.5, the reaction time is 3 hours, the concentration of the substrates is 10g / L of L-cysteine and 10g / L of indole, the cell concentration of the enzyme source is 20mL / L, and the concentration of phosphopyridoxal coenzyme is 0.15g / L. Under the optimum reaction conditions, the conversion rate of the cysteine substrate is 81.5 percent. Because the immobilized enzyme is adopted for direct enzymatic synthesis, the method is easy and convenient to implement, high in catalysis efficiency and low in fermentation cost, and the products are easy to separate; therefore, the method has good industrial prospect in the field of industrial production of L-tryptophan.

The invention overcomes the problems of long reaction time and low yield of the existing transamination method involving pyridoxal, and provides a designed and synthesized pyridoxal derivative for improving the transamination efficiency of N-terminal amino acid of protein, particularly the high-steric hindrance amino acids such as leucine, isoleucine, and valine. The pyridoxal derivative is characterized by having a structure shown by the formula VIII. The pyridoxal derivative disclosed by the invention improves the transamination efficiency of the N-terminal amino acid of the protein, particularly the transamination reaction of the polypeptide containing high-steric hindrance amino acids (Leu, Ile and Val) at N terminal, thereby being favorable for further pegylation modification of protein.