155 results about "Beta-Alanine" patented technology

The invention provides genetic engineering bacteria for producing beta-alanine. The genetic engineering bacteria are obtained by the following method of amplifying L-aspartic acid alpha-decarboxylase gene panD of a donor containing panD gene by PCY, cloning the panD to a plasmid capable of expressing a foreign gene at high efficiency to obtain a vector for abundant expression of the panD gene, and transforming the vector into a receiver to obtain the genetic engineering bacteria for producing beta-alanine. The genetic engineering bacteria and preparation and application thereof have the main advantages that (1) the engineering bacteria of beta-alanine can be synthesized by the preparation of Escherichia coli; (2) the engineering bacteria has high ability in synthesizing beta-alanine, and the primary study determines that the synthesis ability is up to 2.94 g/l and that the beta-alanine is unlikely be determined by an identical method using a starting strain; and (3) the Escherichia coli is improved by using genetic engineering technology to achieve definite breeding target and high efficiency.

The invention relates to a genetically engineered bacterium with a high pantothenic acid yield, a construction method of the bacterium, and application of the genetically engineered bacterium to preparation of D-pantothenic acid through microbial fermentation. The construction method comprises the following steps: (1) reinforcing a last step of an escherichia coli D-pantothenic acid synthesis wayto reinforce the utilization capacity of escherichia coli to extracellular beta-alanine; (2) reinforcing a pantoic acid synthetic way; (3) repairing an ilvG gene and weakening the feedback inhibitioneffect of byproducts on the pantoic acid synthetic way; and (4) weakening the flux of a valine synthetic way according to the phenotypic change of cells to obtain an escherichia coli genetically engineered strain with high yield of D-pantothenic acid. According to the invention, the expression of key enzymes PanB, PanC, PanE and IlvC in the D-pantothenic acid biogenic way is enhanced; the ilvG gene is repaired to weaken feedback regulation and control and reinforce the pantoic acid synthesis path, so that the extracellular accumulation of D-pantothenic acid and valine reaches 0.48 g/L and 0.51g/L respectively, a competitive branch is weakened by knocking out avtA and knocking down ilvE to obtain a plasmid-free high-yield bacterium, and the titer of D-pantothenic acid is increased from 0.48 g/L to 1.54 g/L.

The invention provides a method for synthesizing beta-alanine by biological catalysis, and belongs to the field of enzyme engineering. The method comprises the following steps: culturing escherichia coli microbe to obtain aspartase and L-aspartic acid-alpha-decarboxylase, mixing the enzyme-containing escherichia coli cells with an ammonium fumarate aqueous solution, performing an enzyme reaction to generate beta-alanine. The method of the invention is cheap and easily available in raw materials, simple in production process, safe, environment-friendly, mild in reaction condition, high in efficiency, low in production cost, and high in product quality, and has significant economic benefits and environmental protection benefits when compared with traditional methods.

The invention provides aminopeptidase for catalytic synthesis of carnosine, and a preparation method and application thereof. The aminopeptidase has the amino acid sequence shown by SEQ ID NO:2. For the preparation of biological enzymes, firstly, a gene engineering strain of the biological enzymes is built; the biological enzyme gene fragment obtained through full gene synthesis is subjected to linearized plasmid recombination by using restriction enzymes SalI. The built gene-engineered strain is suitable for high-density culture; the exoenzyme is biologically synthesized; solid-liquid separation is performed; ultrafiltration concentration is performed to obtain enzyme liquid; histidine and beta-alanine can be catalyzed in normal-temperature and normal-pressure water solution to efficiently synthesize carnosine; the reaction environment is friendly; the production cost is low; the conversion time is short; the process operation is simple; the reaction system impurity content is low; the post treatment is easy; the wide prospects of large-scale industrial application are realized.

The invention discloses a synthesis method for D-calcium pantothenate, which comprises: synthesizing keto lactone pantothenate with diethyl oxalate, isobutanal and formaldehyde, reducing said product, using carbamidine carbonate to resolution out D-pantothenic carbonate and L- pantothenic carbonate; preparing the product with D-pantoic lactone, beta-alanine both from D-pantothenic carbonate and calcium metal. This invention avoids the application of hypertoxic sodium cyanide, stabilizes product quality, and increases material utilization ratio.

The invention provides a peptoid as well as a preparation method and application thereof. The peptoid comprises the following subunits: ethylenediamine (I), piperonylamine (II), beta-alanine (III), 1-naphthylamine (IV) and cysteine (V). The peptoid is simple in synthesis, has strong combining capacity with alpha-synuclein, can effectively screen the serum of PD patients and able-bodied persons through alpha-synuclein in the serum, and provides a novel liquid biopsy method and concept for diagnosis and monitoring of the Parkinson's disease.

The invention belongs to the biotechnical field, and in particular relates to a method for preparing beta-alanine by a coupled enzymatic reaction. The method comprises the following steps: by using ammonium fumarate as a raw material, mixing bacterial cells respectively comprising aspartase and aspartic acid-alpah-decarboxylase or a crude enzyme liquor of the two enzymes with an ammonium fumarate aqueous liquor, wherein pH of the ammonium fumarate aqueous liquor is 7.0-7.5; performing enzymatic reaction at 25-55 DEG C; obtaining high purity beta-alanine by separating converted products by isoelectric point crystallization or a method combining isoelectric point crystallization or ion exchange resin. The beta-alanine is prepared by coupled enzymatic reaction, and the method has the advantages of wide source of raw materials, low cost, simplicity and convenience in operation, short enzymatic conversion time, low production cost and the like.

The invention discloses a recombinant escherichia coli strain (Escheerichiacoli) AHB-36 for producing beta-alanine, the preservation number is CCTCC M 2013629, the recombinant escherichia coli strain is a deficiency aspartic acid amino lyase gene and contains an aspartic acid-1-decarboxylase gene. The invention further aims at providing a construction method of the recombinant escherichia coli strain. The recombinant escherichia coli strain can be used for knocking off the aspartic acid amino lyase gene and preventing the escherichia coli from pyrolyzing the aspartic acid into fumaric acid, so that the aspartic acid is completely applied to production of beta-alanine, and meanwhile aspartic acid-1-decarboxylase is expressed in the recombinant escherichia coli strain, and thus the yield of beta-alanine is further increased.

The invention provides new genetic engineering bacteria used for producing beta-alanine, having strong synthetic ability and having higher enzyme activity, construction, expression and purification of the high-production engineering bacteria, and methods for synthesis of beta-alanine respectively through whole cell transformation and fermentation liquid direct transformation. A synthetic method comprises the steps: an L-aspartic-alpha-alanine decarboxylase (PanD) gene of lactobacillus plantarum is obtained by a gene engineering method and has the gene sequence number of NC-004567.2, the gene is constructed into a high-efficiency expression vector, then the high-efficiency expression vector is transformed into recipient bacteria, and thus the genetic engineering bacteria for producing beta-alanine are obtained. The genetic engineering bacteria are subjected to fermentation culture, 80 g/L of a substrate can be transformed by whole cells, and the beta-alanine content reaches 59.7 g/L; and the substrate is directly added into a fermentation liquid, the transformation concentration can reach 10 g/L, the beta-alanine content reaches 6.8 g/L, and the synthetic ability is higher than that of conventional reported genetic engineering bacteria.

The invention discloses a synthetic method of beta-alanine. The method comprises the steps: a) preparation of a substrate: filling 5g/L-80g/L L-aspartic acid substrate solutions into an enzyme reactor, and regulating pH into 5.0-8.5 by using alkalizers; b) enzymic catalytic reaction: adding enzyme-containing bacterium solutions into the substrate solutions for performing catalytic reaction, adding a proper amount of L-aspartic acid in batch during reaction to regulate pH of a reactant to 5.0-9.0, and performing fermentation culture, condensation and pulverization on the enzyme-containing bacterium solutions by utilizing engineering bacteria of high-yield L-aspartic acid alpha-decarboxylase; and c) filtration, decoloring and crystallizing treatment of the reactant in the enzyme reactor to obtain beta-alanine. L-aspartic acid is further catalyzed by using the enzyme-containing bacterium solutions and converted into beta-alanine, and the beta-alanine is mild in production condition, simple in process, low in environment pollution, high in quality of products and convenient to extract at the downstream; the purity reaches 99.0 percent, and the conversion rate of beta-alanine reaches 99.5 percent.

The invention relates to genetic engineering bacteria capable of producing pantothenic acid at high yield without addition of beta-alanine, a construction method of the genetic engineering bacteria, and application of the genetic engineering bacteria in preparation of D-pantothenic acid by microbial fermentation. According to the invention, (1), the final step of an escherichia coli D-pantothenicacid synthesis pathway is enhanced, and the utilizing ability of escherichia coli to extracellular beta-alanine, (2), a pantoic acid synthesis pathway is enhanced, (3), ilvG gene is repaired, and thefeedback inhibition effect of by-products on a pantoic acid synthesis pathway is weakened, (4), according to the change in cell phenotype, flux of a valine synthesis pathway is weakened, (5), a CRISPRi technique is used to screen metabolic modification sites of TCA cycle, a PPP pathway and a by-product metabolic pathway, according to the result, an isoleucine synthesis pathway is blocked, and thecompetition of 2-butanoic acid for reaction of acetolactate synthesized from pyruvic acid under catalysis of acetolactate synthase is relieved, and (6), aspartate decarboxylase from other strains is subjected to heterologous expression to obtain a genetical engineering strain capable of producing the pantothenic acid at high yield without addition of the beta-alanine. By combined expression of panB and panC which are derived from corynebacterium glutamicum and panD derived from bacillus subtilis together on pTrc99A plasmids, 1.2g/L of D- pantothenic acid is obtained without adding the beta-alanine.

The invention discloses a synthesis method for L-Carnosine. In the prior art, the imidazole ring of the L-Histidine can take part in the reaction and has side-reactions, which decreases the yields of the products and makes the L-Carnosine assume the toxicity at the same time. In this invention, the o-phthalic anhydride is reacted with beta-alanine to produce the o-phthalic beta-alanine, the o-phthalic beta-alanine is made an acyl chlorination by the chloro-substitution reagent to synthesize into the o-phthalic beta-alanyl chloride; L-Carnosine is reacted with the edittrialkyl chlorosilane or hexamethyldisilazane to produce the trialky silane protection compound which is condensed with the o-phthalic beta-alanyl chloride to produce the hydrochloride; the hydrochloride is made a de-protection by the absolute alcohol, and then is neutralized by the basic solution to produce a product of neutralization; the hydrazine hydrate is used for a hydrazinolysis to the product of neutralization, then the L-Carnosine is precipitated from the absolute alcohol. The invention protects the imidazole ring on the L-Carnosine, avoids that the imidazole ring on the L-Carnosine makes side-reactions with other substances so as to obtain the pure L-Histidine, has low side-effects and high total yields as well as contents.

The invention discloses a method for constructing an engineering bacterium capable of producing beta-alanine and a method for producing beta-alanine by adopting the engineering bacterium, belonging to the field of biotechnologies. The invention provides L-aspartate alpha-decarboxylase gene of a bacillus tequilensis strain, as shown in SEQ ID NO.1, and the engineering bacterium comprising the PanD gene. The invention further provides a constructing method of the engineering bacterium capable of producing the L-aspartate alpha-decarboxylase, L-aspartate alpha-decarboxylase obtained through expression and secretion is adopted for transforming L-aspartic acid, and thus beta-alanine is produced. According to the invention, L-aspartate alpha-decarboxylase derived from bacillus tequilensis is adopted for transforming L-aspartic acid for producing beta-alanine for the first time at home and abroad, after the enzyme is expressed in escherichia coli, the generated L-aspartate alpha-decarboxylase has high enzyme activity, the thallus does not need to be crushed and can be directly used for carrying out transformation, L-aspartic acid of 180g/L can be transformed to the maximum, the yield of beta-alanine can achieve 119.8g/L, and the industrial application has the advantages.

The invention provides a synthetic method of urethane artificial antigen. The urethane artificial antigen is prepared by the following steps of: 1, preparation of beta-alanine and ethyl chloroformate; 2, synthesis of hapten; 3, purification of hapten; 4, preparation of 1-(3-dimethylamino propyl)-3-ethyl carbodiimide solution; 5, synthesis of artificial antigen; 6, dialysis and detection of artificial antigen; and 7, calculation of quantity of different used carrier proteins. According to the synthetic method, the shielding effect of the protein to the hapten obtained by chemical synthesis is low, therefore, high-titer polyclonal antibody can be generated in an immune animal, and the molecular weight can be detected during synthesizing the artificial antigen, and the coupling ratio can be calculated, and the quantity of used raw materials for synthesizing is precise.

The invention discloses L-aspartate-alpha-decarboxylase with improved thermal stability and belongs to the technical field of gene engineering. Three enzyme mutants including K49R, G369A and K221R areobtained through site-directed mutation of L-aspartate-alpha-decarboxylase from tribolium castaneum, recombinant plasmid of the mutants is transformed into escherichia coli BL21, and a substrate L-aspartic acid is catalyzed to produce beta-alanine after expression separation and purification. At the treatment temperature of 50 DEG C, K49R can improve the thermal stability by 14% as compared withwild enzyme, G369A can improve the thermal stability by 20% as compared with wild enzyme, and K221R can improve the thermal stability by 23% as compared with wild enzyme, wherein K221 can make the enzyme activity reach about 320 U/g and has the enzyme activity improved by 23% as compared with the wild enzyme, and the discovery has important study value for industrial preparation of beta-alanine.

The invention belongs to the technical field of bioengineering, and relates to carnosine hydrolase and a mutant thereof, a recombinant expression vector containing the enzyme and a mutant gene, and arecombinant expression transformant, a preparation method of the recombinant enzyme, a method for immobilizing the recombinant enzyme, and a method for preparing levo form L-carnosine by using the recombinant enzyme through reverse hydrolysis reaction. Compared with the prior art, the carnosine hydrolase disclosed by the invention is high in activity and high in thermal stability, and the enzyme is used for catalyzing beta-alanine and L-histidine to prepare L-carnosine through direct condensation, so that the steps of protection and deprotection in a conventional chemical method of synthesizing peptides are avoided; the process is simple, and the conditions are mild and environmentally friendly. Therefore, the carnosine hydrolase has an extremely good application prospect in industrial production of the L-carnosine.

The invention provides pharmaceutical compositions having improved effects and synergistic efficacy against pain, and provides methods for their use to treat pain, wherein the composition comprises: a sulfur-containing amino acid; a carnitine compound; and at least one compound that is a L-citrulline compound or a beta-alanine compound.

The invention discloses a preparation method of indium tin oxide composite powder, belonging to the technical field of preparation of indium tin oxide composite powder. The method comprises the following steps: preparation of mixed salt liquid: putting an InCl3 liquid and an SnCl4 liquid into a reaction kettle according to the In2O3:SnO2 weight ratio of 9:1, and uniformly mixing; addition of dispersing agents: adding polyvinylpyrrolidone, beta-alanine and ethanediol according to the weight ratio of 2:1:3, and adding more than 20% excessive of urea; coprecipitation reaction: gradually heating to 60-90 DEG C, keeping the temperature while continuously stirring until the In<3+> in the reaction solution is completely precipitated, carrying out ultrasonic treatment for 1 hour, continuing stirring, and aging to obtain an indium tin precipitate; and washing, drying, grinding, screening and carrying out high-temperature calcination to obtain the high-sintering-activity nano indium tin oxide composite powder. The indium tin oxide composite powder prepared by the method has the advantages of less hard aggregation and favorable flowability, and is easy for pressure molding.

The invention discloses a beta-alanine producing strain and a preparation method and application thereof. The beta-alanine producing strain (Escherichia coli) has a preservation number of CGMCC NO: 17830. The beta-alanine producing strain constructed by the invention can realize the effective accumulation of the beta-alanine in the fermentation liquor in the fermentation process without adding aspartic acid in the fermentation process, and improves the yield of the beta-alanine and the sugar conversion rate. The yield of the beta-alanine is up to 50g/L, the sugar-acid conversion rate is up to 40%, and the producing strain has the application potential of large-scale production.

A biologic synthesis method for beta-alanine includes providing the microbes able to generate aminating enzyme, adding them to the seed culture medium containing acrylic acid and the fermenting culture medium containing acrylic acid to culture relative aminating enzyme, adding them to the aqueous solution of ammonia containing acrylic acid, synthesizing beta-alanine under the action of enzyme, removing ammonia, and purifying. Its advantages are high efficiency, no pollution and high purity.

The invention discloses a mass spectrometry probe for thrombin activity detection, a method for preparing the mass spectrometry probe and application thereof, and belongs to the field of medicine screening and evaluation. The mass spectrometry probe comprises polypeptides and piperazines compounds. Amino acid sequences of the polypeptides are Phe-Pro-Arg-beta-Ala, and the piperazines compounds aremodified on beta-alanine of the polypeptides. The mass spectrometry probe, the method and the application have the advantages that the polypeptides Phe-Pro-Arg-beta-Ala capable of being specificallyrecognized by thrombin are linked with the small-molecular piperazines compounds with high mass spectrometry response to obtain the mass spectrometry probe, accordingly, the mass spectrometry probe can be specifically recognized by the thrombin, enzyme digestion can be carried out on the mass spectrometry probe by the thrombin, the mass spectrometry probe is extremely high in mass spectrometry response and mass spectrometry detection accuracy, the activity of the thrombin or the inhibitory activity of thrombin inhibitors can be accurately reflected by the mass spectrometry probe, and the massspectrometry probe is quite applicable to screening compounds with thrombin inhibitory activity from complicated systems such as traditional Chinese medicines.

The invention discloses recombinant bacteria for producing beta-alanine as well as a construction method and application of the recombinant bacteria. The construction method for the recombinant bacteria disclosed by the invention comprises the following steps: knocking out fadR gene, fabF gene, fabH gene, iclR gene and sucA gene of recipient bacteria; introducing aspC gene or a gene cluster, panDgene, alkL gene and gdh gene into the recipient bacteria; enhancing expression of fadL gene, fadD gene, sthA gene, an atoSC gene cluster and an aceBA gene cluster in the recipient bacteria, wherein the recipient bacteria are bacteria or fungi containing the fadR gene, the fabF gene, the fabH gene, the iclR gene and the sucA gene. Experiments prove that the conversion rate of the beta-alanine produced by using the recombinant bacteria disclosed by the invention with fatty acid as a raw material is 60.87 percent and shows that the recombinant bacteria disclosed by the invention can be used for preparing the beta-alanine.

The invention discloses a method of preparing beta-alanine by means of a microchannel reactor. The method comprises the steps of (1) mixing well acrylic acid and ammonia water by a premixer of the microchannel reactor, allowing the mixture to enter a thermostatic reactor of the microchannel reactor and react to obtain discharge that includes beta-alanine; (2) decoloring the discharge of the step (1), filtering, concentrating, crystallizing, and drying to obtain refined beta-alanine. The microchannel reactor is used herein as a reaction device; adding additives to inhibit side reactions is notrequired; conversion rate of the acrylic acid is higher than 99%; product yield reaches 85% and above.

The invention discloses a panD mutant gene with a nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.3. According to SEQ ID No.1 of the panD mutant gene, the 34th base of the panD gene is transformed from A into G; according to SEQ ID No.3 of the panD mutant gene, the 50th base of the panD gene is transformed from T into C. The invention also discloses a genetically engineered bacterium comprising the mutant gene and application of the genetically engineered bacterium in catalytic production of beta-alanine. The genetically engineered bacterium comprising the panD mutant gene can efficiently transform L-aspartic acid to prepare the beta-alanine through a biological method; the yields of the beta-alanine reach about 42.81 g/L and 19.46 g/L respectively, which are increased by almost 4.8times and 2.2 times when being compared with the yield of the beta-alanine of a parent strain.