72 results about "Pantoic acid" patented technology

The invention discloses a thermostable conjugated polyketoreductase mutant, which is obtained by performing single-point mutation or multi-point combined mutation on the 25th site, the 92th site, the 214th site or the 256th site of an amino acid sequence as shown in SEQ ID NO.1. The invention further discloses a preparation method of the thermostable conjugated polyketoreductase mutant. The initial enzyme activity of the mutant is high, and the residual enzyme activity is gt after incubation at 50 DEG C; the Tm value can be increased by more than 9 DEG C compared with that of an original strain, the 50 DEG C half-life period t1 / 2 is increased by more than 377.8 times, and the thermal stability is obviously improved. The invention also provides a coding gene, a recombinant vector containing the coding gene and a co-expression engineering bacterium. The thermostable conjugated polyketoreductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase are used for constructing a three-enzyme co-expression engineering bacterium as a biocatalyst, racemization pantolactone is catalyzed to synthesize DPL, for a 1M substrate, the yield of the DPL reaches 97.5% within 36 hours, KPL hydrolysis is avoided, the reaction time is shorter, the product concentration is higher, and the thermostable conjugated polyketoreductase mutant has great market application value.

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 recombinant bacterium for fermentation production of D-panthenol and application of the recombinant bacterium. The invention relates to the technical field of biology, and provides a recombinant bacterium for fermentation production of D-panthenol, which is obtained by expressing tyrosine decarboxylase and D-pantothenic acid synthetase in a chassis host bacterium, the chassis host bacteria are escherichia coli capable of synthesizing D-pantoic acid and L-homoserine by using glucose. Compared with an existing chemical synthesis method, pure biosynthesis from glucose to D-panthenol is achieved, the method has the advantages of being environmentally friendly, low in cost and the like, and a good foundation is laid for large-scale industrial production of D-panthenol.

The invention relates to the technical field of genetic engineering, and discloses an engineering bacterium capable of stably producing D-pantothenic acid at high yield as well as a construction method and application of the engineering bacterium. According to the invention, key genes in a metabolic pathway are modified through a metabolic engineering technology, and particularly, the level of MTF is improved by enhancing expression of a ysaA gene and modifying a biosynthetic pathway of methylenetetrahydrofolic acid (MTF), so that the level of ketopantoic acid serving as a key intermediate in the biosynthetic pathway of pantothenic acid is improved, and the yield of D-pantothenic acid is improved. Meanwhile, spoIVB or / and spoIIIE genes for producing spores in the genome are knocked out, so that energy consumption and material waste caused by spore formation are reduced, generation of secondary metabolites is promoted, and synthesis of D-pantothenic acid is increased; besides, the regions of prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ) and prophage6 (yobB-yobO) in the genome are knocked out, so that the redundancy of the genome is reduced, the metabolic efficiency and stability of the strain are improved, and the synthesis of D-pantothenic acid is effectively increased.

The invention relates to a preparation method of D-panthenol. The preparation method comprises the following steps: (1) carrying out mixed reaction on DL-pantoic acid lactone and D-pantoic acid lactone hydrolase, extracting, and separating a water phase to obtain D-pantoic acid; (2) mixing the D-pantoic acid with a compound enzyme auxiliary agent for reaction to obtain D-pantoic acid lactone; (3) mixing the D-pantoic acid lactone with beta aminopropanol for reaction to obtain D-panthenol crude product slurry; (4) purifying the D-panthenol crude product slurry to obtain D-panthenol; the compound enzyme auxiliary agent comprises esterase, carbonyl reductase, alcohol dehydrogenase and alcohol nitrile enzyme. According to the method disclosed by the invention, the esterase, the carbonyl reductase, the alcohol dehydrogenase and the alcohol nitrile enzyme act synergistically to catalyze an esterification reaction in D-pantoic acid molecules and promote the D-pantoic acid molecules to form a lactone structure, so that side reactions are reduced, the efficiency of the esterification reaction of the D-pantoic acid is improved, and finally, the yield and the purity of the D-panthenol are improved.

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 discloses a bagasse microcrystalline cellulose immobilized enzyme polysulfone membrane packed bed reactor, and belongs to the technical field of biology. According to the immobilized D-pantoic acid lactone hydrolase packed bed reactor, commercial immobilized enzyme is not adopted, the enzyme activity is effectively improved through the physical adsorption effect or weak chemical adsorption effect of the enzyme and the adsorbent, the enzyme is not prone to leaking out in the reaction, and additional environmental pollution is avoided. In addition, the proenzyme reaction device and the external circulation quasi-immobilized enzyme reaction device can react simultaneously, all existing process parameters are not changed, and the reaction speed is greatly increased. After the reactor is repeatedly used for 10 times, the hydrolysis rate is kept at 28% or above, and after the immobilized enzyme is stored at 4 DEG C for 180 days, the initial enzyme activity is kept at 90% or above. The production cost is greatly reduced, and repeated disassembly and assembly are not needed, so that the disassembly and assembly time is saved, the production efficiency is improved, and good economic and social benefits are generated.

The utility model discloses a thallus recovery device for pantoic acid lactone hydrolysate, and belongs to the field of recovery equipment. A thallus recovery device for pantoic acid lactone hydrolysate comprises a biological hydrolysis tank, a self-cleaning filter and a clear liquid storage tank, and a water inlet and a pressure gauge are arranged at the top end of the self-cleaning filter; the two sides of the upper end of the self-cleaning filter are respectively provided with a material return port and a plurality of filtering outlets, the two sides of the lower end of the self-cleaning filter are respectively provided with a material inlet and a vent hole, the bottom end of the self-cleaning filter is provided with a residual liquid port, and the residual liquid port is connected with a thallus recovery tank. According to the device, a plate frame filter is replaced by a self-cleaning filter, so that the automation is high, the labor cost is saved, the automation of production is realized, the working efficiency is improved, the equipment structure is simple, the production cost is reduced, the operation is simple, and the continuity and safety of production are ensured.

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.

The invention relates to brevibacterium abyssinus R19 capable of producing D-pantoic acid lactone hydrolase and application of the brevibacterium abyssinus R19, and belongs to the technical field of marine microorganisms. The preservation number of the strain is CCTCC (China Center For Type Culture Collection) M 20242906. The brevibacterium abyssinus R19 is put into a liquid seed culture medium, the culture medium is cultured for 18-24 h at the temperature of 30-37 DEG C in a shaking table at the constant temperature, then a culture solution is taken and inoculated into a liquid fermentation culture medium according to the inoculum size volume ratio of 5-10%, culture is conducted for 40-50 h at the constant temperature of 25-30 DEG C in the shaking table, centrifugation is conducted, and supernate, namely crude D-pantoic acid lactone hydrolase is obtained. D-pantoic acid lactone can be subjected to enzymolysis to obtain D-pantoic acid by utilizing the crude enzyme liquid.

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 dynamic regulation and control system, a pantoic acid production strain and a preparation method and application of the pantoic acid production strain. The dynamic regulation and control system comprises a LuxI module, a LuxR module, a tetR module and a tetracycline regulation and control module, wherein the LuxI module comprises a LuxI gene and a promoter M1-93, the LuxR module comprises a LuxR gene and a promoter lpp, the tetR module comprises a tetR gene and a promoter pLux, and the tetracycline regulation and control module comprises a promoter Pteto for down-regulating gltA expression. Experiments prove that the dynamic regulation and control system has an obvious positive effect in the aspect of controlling the biomass accumulation of the pantoic acid producing microorganisms, not only is the problem of overhigh biomass accumulation of pantoic acid engineering bacteria in industrial production solved, but also the yield of pantoic acid is increased, and a foundation is laid for realizing industrial microbial fermentation production of pantoic acid. The method has an important application value.

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 invention provides a method for recovering DL-pantoic acid lactone from DL-panthenol raffinate, which comprises the following steps: 1) mixing the DL-panthenol raffinate, water and an anionic surfactant to form a uniform stable phase; the anionic surfactant is selected from sodium dodecyl benzene sulfonate; 2) heating the uniform stable phase, preserving heat, and hydrolyzing to obtain a hydrolysate; 3) cooling the hydrolysate, and performing solvent extraction to obtain an extraction phase and a raffinate phase; and 4) carrying out evaporative crystallization on the extraction phase to obtain the DL-pantoic acid lactone. According to the method, the DL-pantoic acid lactone can be recycled from the DL-panthenol residual liquid, the recycling efficiency is high, the pH requirement range is mild, corrosion to equipment is small, reaction steps are reduced, the production cost is reduced, and the recycled DL-pantoic acid lactone can be used for synthesizing DL-calcium pantothenate and DL-panthenol. The hydrolysis rate is 64 to 99.5 percent, and the purity of the DL-pantoic acid lactone is more than 98.5 percent.

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 present invention relates to a continuous crystallization process for calcium D-pantothenate, belonging to the technical field of chemical synthesis. The process comprises the following steps: S1, pre-cooling the calcium aminopropionate methanol solution and the D-pantolactone methanol solution respectively; S2, after the pre-cooling is completed, adding the two raw materials into a kettle A in a continuous feeding manner. The kettle A adopts a double cooling method of external circulation cooling and the jacket of the reaction kettle to control the reaction temperature in the kettle A at -10 to -5°C. After the liquid level in the kettle rises to 80%, continuous overflow discharging starts to a kettle B; S3, the kettle B adopts a double cooling method of external circulation cooling and the jacket of the reaction kettle to reduce the temperature of the material to -15 to -10°C. After the liquid level in the kettle B rises to 80%, continuous overflow discharging starts to a kettle C; S4, controlling the reaction temperature in the kettle C at -18 to -12°C. After the liquid level in the kettle C rises to 80%, continuous overflow discharging starts to a kettle D. After the liquid level is stable, centrifugation is carried out to obtain the wet product of calcium D-pantothenate.

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 invention discloses preparation and application of a ketone pantoic acid hydroxymethyltransferase mutant for D-pantothenic acid production, and belongs to the technical field of gene engineering and enzyme engineering. The escherichia coli ketopantoic acid hydroxymethyltransferase is mutated by means of genetic engineering and enzyme engineering, and the obtained single mutation and double mutation are expressed in improved chassis cells, so that the yield of the escherichia coli D-pantothenic acid can be remarkably increased, and conditions are created for expanding industrial application of the escherichia coli D-pantothenic acid.

The invention belongs to the field of biotechnology and relates to a method for preparing D-pantoic acid by enzymatic conversion. The method utilizes a D-pantoic acid lactone hydrolase (D-Lac) mutant to catalyze the splitting of a DL-pantoic acid lactone substrate to generate D-pantoic acid and L-pantoic acid lactone, and after the reaction is completed, the conversion product is extracted and separated to obtain D-pantoic acid and L-pantoic acid lactone. The method has the advantages of high enzyme catalysis efficiency, short conversion time, simple operation, low production cost, and the like.