51 results about "Alanine racemase" patented technology

The invention discloses a strain of engineering bacteria producing DL-alanine. Lactic dehydrogenase, pyruvate formate lyase, alcohol dehydrogenase, acetic acid kinase, fumaric acid reductase, alanine racemase and methyl glyoxal synthetase of the strain of engineering bacteria producing the DL-alanine are inactivated; and exogenous L-alanine dehydrogenase gene and alanine racemase gene are integrated on the chromosome of the engineering bacteria. According to the invention, pyroracemic acid, an intermediate product of the glycolysis is converted to L-alanine by integrating the exogenous L-alanine dehydrogenase gene into the chromosome of the engineering bacteria; and an exogenous alanine racemase gene is further integrated into the chromosome, and part of the L-alanine is converted into D-alanine. Then producing the DL-alanine from raw material sugar in one step is realized, the production period of the DL-alanine is decreased and the productivity of the DL-alanine is enhanced.

The invention relates to a recombinant bacillus subtilis for expressing cellobiose-2-epimerase based on a D-alanine defective screening marker, as well as a construction method and an application of the recombinant bacillus subtilis, belonging to the technical field of genetic engineering of enzyme. According to the invention, bacillus subtilis 1A751 is taken as an original strain, D-alanine racemase genes (dal) on the chromosome of the bacillus subtilis 1A751 strain are knocked out, so that D-alanine defective bacillus subtilis 1A751 (dal<->) is obtained; by taking cellobiose-2-epimerase (CE enzyme) from thermoanaerobacter tengcongensis as a parent, fusing a P43 promoter on the upstream of the parent to establish P43-TsCE, establishing P43-TsCE to plasmid pUB110 to obtain pUB-P43-TsCE, and replacing antibiotics resistance genes kanamycin and bleomycin on the plasmid pUB-P43-TsCE with (dal), so as to construct pUB-P43-TsCE-dal; and transforming pUB-P43-TsCE-dal into host 1A751 (dal<->), and thus obtaining the recombinant bacillus subtilis for expressing cellobiose-2-epimerase, wherein the preservation number is CCTCC NO: M 2015582. The total enzyme activity of fermentation liquor reaches 7U/mL, and thus the recombinant bacillus subtilis has an important industrial application value.

The invention relates to a method for preparing a chiral compound based on splitting by adopting a biological enzyme preparation, in particular to a method for preparing an L-tertiary leucine compound by a two enzyme system. The method prepares the chiral L-tertiary leucine compound of the formula III by using the reaction of an N-acylating tertiary leucine compound of the formula II in water or a buffer solvent at 15 to 60 DEG C in the presence of the two enzyme system. The biological enzyme is a mixed enzyme of an acylating amino acid racemase (alanine racemase) and a hydrolase; and the preparation reaction equation is as follows, wherein X is any group of OH, NH2, NR1R2 and OR3 (wherein R1, R2, R3 are a C1-C5 straight chain, a branch chain and a one, two or three-halogen substituted alkane respectively). The invention can be implemented at room temperature and ensures simple operation and no pollution, while remarkably lowering the production cost.

The invention relates to a construction method and a fermenting method of antibiotic-resistance-free recombinant bacillus subtilis for expressing glutamate decarboxylase and belongs to the technical field of bioengineering. The construction method includes taking bacillus subtilis WB600 as an original strain, and knocking out a D-alanine racemase gene on a chromosome of the bacillus subtilis WB600 so as to obtain D-alanine deficient WB600 (dal); fusing an optimized gad gene with an antibiotic-resistance-free expression vector pUB110 (a antibiotics resistance gene is replaced by the D-alanine racemase gene) through an overlap extension PCR technology to obtain a polymer, transforming the polymer into competence of the bacillus subtilis WB600, and enabling the polymer to recombine in a host so as to obtain a recombinant plasmid Pub-HpaII-P43-gad-dal, which is named as bacillus subtilis SK44.001 with the preservation number being CCTCC NO:M 2016774. The fermentation liquor enzyme activity of the antibiotic-resistance-free recombinant bacillus subtilis can be up to 8.6 U/mL, and accordingly the antibiotic-resistance-free recombinant bacillus subtilis has significant industrial application value.

The invention provides a strain for producing high-yield DL-alanine and an application thereof, and particularly provides an engineering strain for producing D-alanine racemase. The strain is esherichia coli and carries an expression vector for expressing Bacillus subtilis D-alanine racemase. The expression strain can be effectively used for producing the DL-alanine at a low cost, and has a higher application value.

The invention provides methods of screening a microbial host cell for a property of interest in a microfluidic device, the method comprising the steps of: a) transforming a d-alanine racemase-deficient microbial host cell with a polynucleotide construct comprising: i) one or more polynucleotide region providing the property of interest when present in the host cell, and ii) at least one polynucleotide region complementing the d-alanine racemase deficiency when present in the host cell; and b) screening the transformed host cell for the property of interest in the microfluidic device in the absence of externally provided d-alanine.

The invention discloses a method for preparing DL-alanine and D-alanine. Fumaric acid and ammonia react through aspartase in a coupling reactor to synthesize L-aspartic acid; the synthesized L-aspartic acid reacts with L-aspartate dacarboxylase to obtain L-alanine; L-alanine undergoes racemization through alanine racemase to obtain DL-alanine; and DL-alanine concentrated mother liquor undergoes splitting through L-alanine aminotransferase in a fermentation cylinder so as to obtain D-alanine. Production of DL-alanine or D-alanine requires no extraction of intermediate products. The final product is obtained directly by one step. Thus, usage of sulfuric acid required for electroextraction of L-aspartic acid and the like, labor for each extraction process, energy consumption and production loss such as dissipation in actual production process are reduced. Meanwhile, DL-alanine mother liquor is comprehensively utilized for production of D-alanine, and overall yield and economic benefit are raised.

The invention discloses a cryoprotectant. The cryoprotectant comprises the following components: dimethyl sulfoxide in volume percentage of 1-5%, 20-80mmol/L trehalose, 5-15ug/mL catalase, 10-40ug/mLZ-valine-alanine racemase-ASP (methyl ester)-methyl fluoride ketone, 0.1-3ug/mL insulin and 0.01-10mg/L selenium. The cryoprotectant disclosed by the invention is serum-free, so that the pollution ofpathogenic bacteria can be avoided; the content of dimethyl sulfoxide (DMSO) is less, so that the cytotoxicity is reduced; the cryoprotectant disclosed by the invention is prepared from pure chemicalsubstances, the components are stable and the batch-to-batch stability is high; after the cells cryopreserved with the cryoprotectant disclosed by the invention revive, the cell viability is obviouslyenhanced. The invention also discloses an application of the cryoprotectant in cryopreservation of umbilical cord mesenchymal stem cells. The invention also discloses a cryopreservation method of theumbilical cord mesenchymal stem cells.

The invention discloses animal waste metagenome-derived alanine racemase as well as preparation and application thereof. The amino acid sequence is shown as SEQ ID NO.2, and after the alanine racemase is treated by a buffer solution with the pH value of 9.0-12.0 for 1 hour, the residual enzyme activity is 90% or above; when the alanine racemase is tolerant for 1 hour under the conditions of 30 DEG C, 37 DEG C, 40 DEG C and 45 DEG C, 90% of enzyme activity is still kept; the optimal cofactor concentration is 10 mumol/L, and 77% of relative activity is still maintained after the reaction is carried out for 10 min under the condition that no exogenous cofactor is added. Km and Vmax of the enzyme are 14.81 mmol/L and 89.06 mumol/L/min respectively under the conditions that the pH is 12.0 and the temperature is 40 DEG C NaCl, and the enzyme has thermal stability and alkali resistance, still has higher activity without adding exogenous cofactors, and has potential application value in the aspects of synthesizing D-amino acid by an enzyme method and screening novel antibacterial drugs.

The invention belongs to the technical field of biology and particularly relates to a method for preparing DL-alanine with fumaric acid as raw material through multi-enzyme coupling. The preparation method uses the fumaric acid as raw materials, bacteria cells containing aspartase, aspartic acid-beta-decarboxylase and alanine racemase or crude enzyme of the aspartase, the aspartic acid-beta-decarboxylase and the alanine racemase is mixed with a fumaric acid water solution of a certain concentration, enzymatic reaction is carried out at the temperature of 25-55 DEG C, under the pH 6-8, converted products are separated though an isoelectric point crystallization method or a method combining isoelectric point crystallization and ion exchange resin, and high-purity DL-alanine is obtained. According to the method, the DL-alanine is prepared efficiently by adopting the multi-enzyme coupling technology, the method has the advantages of being wide in raw material source, simple in operation of production process, short in enzymatic conversion time, low in production cost and the like.

The invention discloses a method for preparing DL-serine by a one-pot enzyme method. The method comprises the following steps: (1) taking formaldehyde and glycine as substrates, in the presence of coenzyme, catalytically converting one molecule of formaldehyde and one molecule of glycine into D-serine by a recombinant D-threonine aldolase; and (2) converting the D-serine to DL-serine under the action of recombinant alanine racemase. The invention has the advantages of simplicity in operation, short production period, low production cost, high yield, small emission of three wastes, suitabilityfor large-scale industrial production and the like.

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for preparing D-alanine by a microbial fermentation method. The method comprises the following steps: firstly, constructing a genetically engineered bacterium for producing D-alanine, taking corynebacterium glutamicum ATCC 13032 as a starting strain of the genetically engineered bacterium, and knocking out an alanine racemase gene alr and L-alanine transaminase alaT; allowing meso-diaminopimelate dehydrogenase coding gene to be subjected to overexpression; knocking out an iolR repressor protein gene and simultaneously performing genome integration expression on a glucokinase gene glk1; and integrating edd and eda genes from Escherichia coli to obtain the gene. The strain uses glucose as a substrate to produce D-type amino acid through a direct fermentation method, the cost is low, the technology is simple, and the yield of D-alanine in a 5L fermentation tank can reach 50-60g/L. Besides, efficient synthesis of the D-type amino acid by a fermentation method belongs to creative research, and by means of the method, the fermentation method can be infinitely expanded, and various D-type amino acids can be efficiently synthesized at low cost.

The invention provides a method for synthesizing D-biphenyl alanine. The method comprises the following steps: 1) adding a solvent to a reaction kettle; during stirring, sequentially adding diethyl acetamidomalonate, metallic sodium and 4-bromomethyl biphenyl; after conducting a reaction, reacting a condensation product, which is obtained through cooling crystallization, and hydrochloric acid while conducting stirring, and conducting cooling crystallization so as to obtain N-acetyl-DL-biphenyl alanine; and 2) dissolving the N-acetyl-DL-biphenyl alanine in water; adjusting pH to 6.5-8.0 by virtue of ammonia water; adding catalyzing enzymes; and after a reaction, conducting centrifuging, rinsing with water and drying so as to obtain the D-biphenyl alanine, wherein the added catalyzing enzymes include L-aminoacylase and L-alanine racemase, and the total addition of the catalyzing enzymes is 0.1-0.15% of the mass of the N-acetyl-DL-biphenyl alanine. The method provided by the invention has the advantages that the method has fewer steps, and the method is mild in reaction, free from high temperature and high pressure, highly environment-friendly, high in yield and the like.

The invention provides alanine racemase encoding polynucleotides, which are capable of conferring increased nematode resistance in a plant. Specifically, the invention relates to methods of producing transgenic plants with increased nematode resistance, expression vectors comprising polynucleotides encoding alanine racemase, and transgenic plants and seeds generated thereof.