57 results about "Pantoic acid" patented technology

The invention discloses a method for producing D-pantoic acid by immobilizing D-pantoic acid lactone hydrolase fusion protein, and belongs to the technical field of biology. The insoluble seed pumpkin acidic polysaccharide PPFS3 is extracted from an agricultural waste, namely seed pumpkin pulp, meanwhile, a fusion protein constructed by connecting a carbohydrate binding module and D-pantoic acid lactone hydrolase is provided, and the fusion protein and the seed pumpkin acidic polysaccharide PPFS3 have specific binding capacity; a simple, convenient and low-cost immobilized enzyme and recombinant protein purification method can be established. The formed immobilized fusion protein can be used for efficient and continuous production of D-pantoic acid.

The invention discloses a Zymomonas mobilis recombinant strain for producing D-pantothenic acid as well as a preparation method and application of the Zymomonas mobilis recombinant strain. A hydroxymethyl transferase gene panB, a ketopantoic acid reductase gene panE and a pantothenic acid synthase gene panC are integrated in a genome of the recombinant strain; an expression plasmid containing an acetolactate synthase gene Bsals, a keto acid reductoisomerase gene ilvC and a dihydroxy acid dehydratase gene ilvD is also transferred into the recombinant strain; wherein the nucleotide sequences of the gene Bsals, the gene ilvC, the gene ilvD, the gene panB, the gene panE and the gene panC are shown as SEQ ID NO. 1 to SEQ ID NO. 6 in sequence. The zymomonas mobilis ZM4 is systematically modified through a genetic engineering means, and an optimal enzyme combination adaptive to the zymomonas mobilis is defined by screening hydroxymethyltransferase, pantothenic acid synthase and ketopantoic acid reductase from different sources, so that a core foundation is laid for efficient operation of a D-pantothenic acid synthesis route, and the method has a wide application prospect. The technical bottlenecks of single metabolic pathway and low synthesis efficiency of the traditional strain are broken through.

The invention discloses a method for improving pantoic acid production capacity of an engineering strain. The invention provides a method for improving the pantoic acid yield of pantoic acid engineering bacteria. The method comprises the following step: adding amino acid into a fermentation system. Specifically, the amino acids are serine, alanine and glycine. By adding amino acid, the pantoic acid yield of the pantoic acid engineering bacteria is greatly increased, and the method has great application and popularization value for industrial production of pantoic acid and downstream products thereof.

The invention discloses a producing strain of D-pantoic acid lactone hydrolase and application of the producing strain. The producing strain is fusarium moniliforme DPLH6, and the preservation number of the producing strain is CGMCC (China General Microbiological Culture Collection Center) No. 42135. The fusarium moniliforme strain disclosed by the invention has the capability of producing the D-pantoic acid lactone hydrolase at high yield, the D-pantoic acid lactone is prepared after the D-pantoic acid lactone hydrolase fermented by the strain is immobilized by glutaraldehyde, less enzyme dosage and shorter catalysis time can be realized, and the ee value of the target product D-pantoic acid lactone is greater than 99%.

The present disclosure provides a microorganism with increased activity of lipoate protein ligase, a composition for producing pantothenic acid and / or pantoic acid comprising the microorganism, and a method for producing pantothenic acid and / or pantoic acid comprising a step of culturing the microorganism, wherein the microorganism has excellent pantothenic acid and / or pantoic acid producing ability.

The invention provides a process method for recovering DL-panthenol mother liquor and application of the process method, and belongs to the technical field of chemical engineering, the process method comprises the following steps: firstly, carrying out solvent separation on the DL-panthenol mother liquor to respectively obtain a gas phase containing ethyl acetate, methanol and water and a liquid phase containing ethyl acetate, 3-aminopropanol, DL-panthenol, pantoic acid and water; carrying out gas phase treatment on the gas phase to obtain a mixed solution containing methanol and ethyl acetate; and carrying out liquid phase treatment on the liquid phase to obtain 3-aminopropanol and pantoic acid lactone. According to the process method provided by the invention, ethyl acetate, methanol, 3-aminopropanol and pantoic acid lactone in the DL-panthenol mother liquor can be recycled in a high-purity and high-yield manner and reused in a preparation link of DL-panthenol, so that the purposes of recycling resources and reducing the cost are achieved, and the process method is suitable for industrial application.

The invention provides a method for producing DL-2, 4-dihydroxy-3, 3-dimethyl butyric acid (DL-pantoic acid) and / or DL-3-hydroxy-4, 4-dimethyl oxacyclopentane-2-one (DL-pantolactone), in particular to a method for producing DL-2, 4-dihydroxy-3, 3-dimethyl butyric acid and / or DL-3-hydroxy-4, 4-dimethyl oxacyclopentane-2-one (DL-pantolactone) by condensation of hydroxypivalaldehyde and formyl-CoA of C1 source, in particular to a method for producing DL-2, 4-dihydroxy-3, 3-dimethyl oxacyclopentane-2-one (DL-pantoic acid) and / or DL-3-hydroxy-4, 4-dimethyl oxacyclopentane-2-one (DL-pantoic acid). The invention relates to a method for preparing 2, 4-dimethyl oxacyclopentane-2-one. The present invention also provides a genetically engineered microorganism for use in the production of DL-2, 4-dihydroxy-3, 3-dimethylbutyric acid and / or DL-3-hydroxy-4, 4-dimethyl oxacyclopentane-2-one, and also provides a genetically engineered microorganism for use in the production of DL-3-hydroxy-4, 4-dimethyl oxacyclopentane-2-one. In addition, the key enzyme has been engineered to improve its performance in the production of DL-pantoic acid or DL-pantolactone.

Provided are a genetically engineered bacterium for high-yield production of D-pantothenic acid (D-PA), and a preparation method therefor and a use thereof. The D-PA production yield of the engineered bacterium is significantly improved by means of the following actions: overexpressing acetolactate synthases IlvB and IlvN for pyruvate diversion while deleting a transcription attenuation region and mutating a valine feedback inhibition site; overexpressing ketol-acid reductoisomerase IlvC and dihydroxy acid dehydratase IlvD, and altering the coenzyme preference of IlvC from NADPH to NADH to unlock the upstream pathway for D-PA synthesis; overexpressing PanB and overexpressing a serine-glycine transport system to promote the synthesis of 5,10-methylenetetrahydrofolate; at the same time, enhancing the expression of ketopantoic acid reductase PanE; and finally overexpressing pantothenate synthetase PanC and increasing the content of cofactor ATP required by PanC. By means of the integrated enhancement of the above multiple modules, the provided engineered bacterium achieves improved cell growth due to balanced cofactors and increased D-PA.

The invention discloses a synthesis and preparation method of magnesium tritrip acid, which comprises the following steps: S1, heating magnesium hydroxide and 4-aminobutyric acid in methanol for reaction to obtain magnesium 4-aminobutyrate; s2, reacting the magnesium 4-aminobutyrate obtained in the step S1 with pantoic acid lactone in isopropyl alcohol under heating to obtain a magnesium furonate initial product; and S3, purifying the magnesium furonate initial product to obtain a magnesium furonate finished product. According to the invention, 4-aminobutyric acid, magnesium hydroxide, pantothenic acid lactone, ethanol, methanol and the like are used as raw materials, and the magnesium furonate is prepared through the processes of acid-base neutralization, ester exchange, refined crystal transformation and recrystallization; the method has a series of advantages of good process stability, excellent reaction selectivity, high yield, cheap and easily available raw materials, simple operation, high safety, environmental friendliness and the like.

The invention discloses a pantothenic acid synthetase mutant as well as a preparation method and application thereof, and relates to the technical field of pantothenic acid synthesis. The mutant is obtained by mutating Q at the 62nd site of an amino acid sequence as shown in SEQ ID NO.2 into N and / or mutating Q at the 155th site of the amino acid sequence as shown in SEQ ID NO.2 into N. By using the pantothenic acid synthetase mutant disclosed by the invention, the enzyme activity of the pantothenic acid synthetase is remarkably improved, and the conversion rate of D-pantoic acid and the yield of D-pantoic acid are improved.

The present invention relates to a Corynebacterium mutant microorganism that produces L-glutamic acid and a method for producing L-glutamic acid using the same, and more specifically to a novel mutant, polynucleotide, and transformant of pantoate-beta-alanine ligase involved in the L-glutamic acid biosynthetic pathway, and a method for producing L-glutamic acid using the same. The pantoate-beta-alanine ligase mutant of the present invention has altered enzymatic activity due to the substitution of one or more amino acids in the amino acid sequence constituting pantoate-beta-alanine ligase, and a recombinant microorganism containing the same can efficiently produce L-glutamic acid.

The invention discloses a pantothenic acid synthetase mutant as well as a preparation method and application thereof, and relates to the technical field of pantothenic acid synthesis. The mutant is obtained by mutating V at the 62nd site of an amino acid sequence as shown in SEQ ID NO.2 into A. By using the pantothenic acid synthetase mutant disclosed by the invention, the enzyme activity of the pantothenic acid synthetase is remarkably improved, and the conversion rate of D-pantoic acid and the yield of D-pantoic acid are improved.

The present application relates to the technical field of biological materials, in particular to a cyclic peptide derivative, a preparation method and application thereof.The cyclic peptide derivative provided by the present application simultaneously comprises a hydrophobic cyclic peptide structure and a hydrophilic pantothenic acid structure, which are combined by a covalent ester bond; this structure endows the cyclic peptide derivative with excellent stability, effectively improving the problems of instability and irritation of pantothenic acid; after entering the skin, the derivative can be hydrolyzed into pantothenic acid under the action of esterase, and can play a role without undergoing pantoic acid oxidation conversion.Secondly, the cyclic peptide derivative in the present application has good biological safety; in an inflammatory cell model, the derivative can more effectively reduce the expression of inflammatory factors, and antioxidant activity research shows that it has strong free radical inhibition effect, and can play superior anti-inflammatory and antioxidant effects.

The present invention belongs to the field of chemical engineering technology, and particularly relates to a method for synthesizing high-purity DL-panthenol. The method comprises the following steps: S1. Dewatering pretreatment of DL-pantolactone to make the water content in the DL-pantolactone raw material < 0.1%; S2. Using the dewatered DL-pantolactone and 3-aminopropanol as raw materials, with the molar ratio of the two being 1.02 to 1.05:1, and carrying out an acylation reaction in a temperature range of 60 to 90 °C after mixing, controlling the reaction time to be 2 to 3 h to obtain a DL-panthenol reaction solution; S3. After the acylation reaction is completed, adding a mixed solvent composed of ethyl acetate and water to the reaction system, heating to dissolve until clear, and then cooling to 10 to 20 °C for crystallization; filtering the crystallization solution to obtain wet DL-panthenol, and drying the wet product to obtain DL-panthenol. The DL-panthenol obtained by this method has a chromatographic purity > 99.5%, a yield as high as 96 to 98%, and there is no need to cool to a lower temperature during the synthesis.

The invention discloses an L-pantoic acid lactone dehydrogenase mutant and an application of the L-pantoic acid lactone dehydrogenase mutant. The L-pantoic acid lactone dehydrogenase mutant is subjected to any one or a combination of at least two of the following mutations on the basis of an amino acid sequence SEQ ID NO.1: T271D, A27S, T163K, V240I or L253I. The L-pantoic acid lactone dehydrogenase mutant is constructed on the basis of the L-pantoic acid lactone dehydrogenase derived from amycolatopsis methyloralis, the mutant shows excellent catalytic activity, the cell apparent activity of the mutant is improved by more than 10 times compared with that of a wild type, and the mutant provides a new biocatalyst for efficient preparation of D-pantoic acid lactone and derivatives thereof.

The invention discloses a ketopantothenic acid reductase mutant and application thereof, the mutant is NADH dependent ketopantothenic acid reductase obtained by mutation on the basis of a wild ketopantothenic acid reductase amino acid sequence, and the mutation site comprises the 71st site and / or the 75th site. Compared with NADPH dependent wild type ketopantothenic acid reductase, the ketopantothenic acid reductase mutant can be used for reducing ketopantoic acid to generate D-pantoic acid in vitro by using NADH, so that the production cost of the D-pantoic acid is reduced.

This invention provides a method for separating optical isomers from racemic mixtures via electrodialysis. Specifically, this invention applies electrodialysis technology to enzyme resolution processes, primarily for the separation of products after enzyme resolution. Taking the preparation process of D-pantolactone as an example, the key point is to separate D-pantolactone and L-pantolactone from the enzyme resolution solution using electrodialysis, replacing existing organic solvent extraction methods. The process is simple and easy to implement, yields high-purity D-pantolactone, significantly reduces the amount of organic solvent used, lowers production costs, is environmentally friendly, and can greatly improve the working environment and operational safety. The method provided by this invention has great application potential in enzyme resolution processes.

The present invention introduces a heterologous formate-tetrahydrofolate ligase gene into a microorganism having a D-pantoic acid synthetic metabolic pathway, and assimilates one molecule of formate to generate one molecule of 5,10-methylenetetrahydrofolate, and uses the microorganism to produce a new process of ketopantoate and / or D-pantoic acid from formate and glucose.

The application discloses preparation and application of ketopantoate hydroxymethyltransferase mutants for D-pantoic acid production and belongs to the technical field of genetic engineering and enzyme engineering. By means of genetic engineering and enzyme engineering, the application mutates ketopantoate hydroxymethyltransferase of Escherichia coli, expresses the obtained single mutation and double mutation in an improved chassis cell, and can significantly improve the D-pantoic acid yield of the Escherichia coli, thereby creating conditions for expanding industrial application.

The present application relates to the technical field of biological catalysis, and discloses application of L-pantoic acid lactone dehydrogenase and a mutant thereof in preparation of D-pantoic acid lactone, wherein the L-pantoic acid lactone dehydrogenase and the mutant thereof with improved activity are screened, and ketopantoic acid lactone reductase is co-expressed to catalyze production of D-pantoic acid lactone.The present application adopts a method for co-expressing L-pantoic acid lactone dehydrogenase mutants and ketopantoic acid lactone reductase to catalyze synthesis of D-pantoic acid lactone, and has the advantages of simple process, high enzymatic reaction selectivity, no by-product, simple product extraction and refining, and the like.

The invention provides an engineering bacterium for producing D-pantothenic acid as well as a construction method and application of the engineering bacterium. The engineering bacterium expresses an acetolactate synthase large subunit gene ilv2, an acetolactate synthase small subunit gene ilv6, a keto acid reductoisomerase gene ilvC, a dihydroxy acid dehydratase gene ilvD, a hydroxymethyltransferase gene ecm31, a keto pantoic acid reductase gene panE, an L-aspartic acid decarboxylase gene panD and a pantothenic acid synthase gene panC. According to the method, mitochondria is selected as a targeting compartment, and modular combination optimization approach positioning is adopted, so that the yield of D-pantothenic acid is greatly increased, compared with the yield of an original strain without spatial tissue optimization, the yield is increased by more than one time, the metabolic flux bottleneck in a traditional cytoplasm synthesis mode is broken through, and efficient synthesis of D-pantothenic acid is realized.

A 3-methyl-2-oxobutanoate hydroxymethyltransferase variant, a microorganism having enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase, a composition for producing pantothenic acid and / or pantoic acid comprising the microorganism, and a method for preparing pantothenic acid and / or pantoic acid comprising culturing the microorganism are provided.

The application discloses a conjugated polyketide reductase mutant, which is obtained by single-point mutation or multi-point combined mutation of the 64th or 206th site of the amino acid sequence shown in SEQ ID NO. 1. The mutant of the conjugated polyketide reductase can improve the enzyme activity by more than 25% compared with the wild type. The application also provides a coding gene, a recombinant vector containing the coding gene, a co-expression engineering bacterium and application. The conjugated polyketide reductase mutant, glucose dehydrogenase and L-panthenolide dehydrogenase are used to construct a three-enzyme co-expression engineering bacterium as a biological catalyst to catalyze the synthesis of optically pure D-panthenolide from racemic panthenolide, improve the catalytic efficiency of multi-enzyme cascade reaction, reduce the hydrolysis loss of intermediate product KPL, and significantly improve the yield of DPL. The yield of the obtained DPL can be up to 98%, and the optical purity can be up to 99.9%.

The invention discloses an acetohydroxy acid reductoisomerase mutant and application thereof. The invention provides an acetohydroxy acid reductoisomerase mutant. The amino acid sequence of the acetohydroxy acid reductoisomerase mutant is shown as SEQ ID NO: 1. The invention also provides a method for improving the enzyme activity of the wild-type acetohydroxy acid reductoisomerase. The method comprises the following step: mutating the 105th amino acid residue of the wild-type acetohydroxy acid reductoisomerase from F into L. The present invention is useful in the preparation of products of synthetic pathways in which acetohydroxy acid reductoisomerase is involved, such as branched chain amino acids or downstream products thereof, pantoic acids or downstream products thereof, and pantothenic acids or downstream products thereof.

This invention relates to the field of genetic engineering technology, and discloses an engineered bacterium that stably produces high levels of D-pantothenic acid, its construction method, and its applications. This invention utilizes metabolic engineering techniques to modify key genes in its metabolic pathway. Specifically, by enhancing the expression of the ysaA gene, the methylenetetrahydrofolate (MTF) biosynthesis pathway is modified, increasing the MTF level, which leads to an increase in the level of ketopantothenic acid, a key intermediate in the pantothenic acid biosynthesis pathway, thereby increasing the yield of D-pantothenic acid. Simultaneously, knocking out the spoIVB and / or spoIIIE genes in the genome reduces the energy consumption and material waste associated with spore formation, promotes the generation of secondary metabolites, and further increases the synthesis of D-pantothenic acid. Additionally, knocking out the prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ), and prophage6 (yobB-yobO) regions in the genome reduces genomic redundancy, improves the metabolic efficiency and stability of the strain, and thus effectively increases the synthesis of D-pantothenic acid.

The invention belongs to the field of biological medicine, and particularly relates to an engineering bacterium for producing D-pantoic acid lactone hydrolase and a culture and whole-cell immobilization method thereof, the engineering bacterium takes E.coli BL21 (DE3) as a host and pET28a (+) as a carrier, and the D-pantoic acid lactone hydrolase with a nucleotide sequence shown as SEQ ID NO.1 is expressed. The engineering bacterium can efficiently secrete the D-pantoic acid lactone hydrolase, and the engineering bacterium can be used for pilot-scale high-density fermentation culture, so that the industrial production of the D-pantoic acid lactone hydrolase becomes possible. According to the fermentation culture method disclosed by the invention, the supply of a carbon source and a nitrogen source is controlled by screening a fermentation culture medium and controlling the replenishing time, replenishing rate and dosage of glycerol and ammonia water, and the supply of oxygen is controlled by adjusting the stirring rotating speed and ventilation volume, so that the high-density growth of thalli and the efficient expression of target protease are realized. The invention further discloses an immobilization method of the D-pantoic acid lactone hydrolase, and the stability of the D-pantoic acid lactone hydrolase is improved by 32.12%. The production cost is greatly reduced, and the method has practical industrial production value.

Provided are: a polypeptide having activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase; a microorganism having increased activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase; a composition for producing pantothenic acid and / or pantoic acid, comprising the polypeptide and / or the microorganism, and a pantothenic acid and / or pantoic acid production method comprising a step for culturing the microorganism.

The invention discloses klebsiella pneumoniae for efficiently producing D-pantothenic acid as well as a construction method and application of the klebsiella pneumoniae, and the specific construction method comprises the following steps: by taking wild klebsiella pneumoniae as an original strain, overexpressing a ketopantoic acid hydroxymethyltransferase gene panB, a pantothenic acid synthetase gene panC and a ketopantoic acid reductase gene panE from escherichia coli; the invention relates to related genes for weakening the expression of a valine-pyruvate transaminase gene ilvE and inactivating byproduct synthesis in a host, and the related genes comprise an alpha-acetolactate decarboxylase gene budA, an acetoin reductase gene budC, glycerol dehydrogenase gldA, a pyruvate oxidase gene poxB, a pyruvate formate lyase activating enzyme gene pflB, an ethanol dehydrogenase gene adhE and a lactic dehydrogenase gene ldhA. The constructed klebsiella pneumoniae engineering bacterium can efficiently synthesize D-pantothenic acid by fermenting glucose, and has great application potential and economic value.

The invention discloses a method for recovering D-calcium pantothenate mother liquor. The recovery method comprises the following steps: S1, carrying out pretreatment, precipitation treatment and hydrolysis treatment on D-calcium pantothenate mother liquor to obtain a reaction solution 1; s2, carrying out lactonization reaction on a mixed solution of the reaction solution 1 and a solvent to obtain a reaction solution 2; and S3, carrying out solid-liquid separation on the reaction liquid 2 to obtain a solid, namely beta-alanine, and crystallizing the obtained liquid to obtain the D-pantoic acid lactone. According to the recovery method, high-purity beta-alanine and D-pantoic acid lactone can be obtained, and meanwhile, the recovery rate of the beta-alanine and the D-pantoic acid lactone is effectively increased; in the whole process, a large amount of acid is not needed for neutralization reaction, extraction operation is not needed, the input of organic solvents is reduced, and the production cost is effectively reduced.

Provided are a genetically engineered bacterium with a high yield of D-pantothenic acid based on one-carbon unit supply, a construction method therefor, and the use of said genetically engineered bacterium in the preparation of D-pantothenic acid by microbial fermentation. By enhancing a glycine cleavage system, the supply of a one-carbon cofactor in Escherichia coli organisms is enhanced, so that the carbon flux flows to the synthesis of D-pantothenic acid. In order to continuously enhance the carbon flux, genes related to a THF synthesis pathway are enhanced to regulate a serine synthesis pathway; and by combining the enhancement of the glycine cleavage system and the expression of key genes of a pantoic acid synthesis pathway, a plasmid-free and antibiotic-free engineered bacterial strain used for high-yield production of D-pantothenic acid is finally obtained. Finally, the titer of D-pantothenic acid in shake flask culture is increased by 31.7% relative to that of the original strain and reaches 6.85 g / L, and after fermentation in a 5 L fermentation tank for 92 hours, the D-pantothenic acid yield can reach 112.62 g / L.