31 results about "Aspartate decarboxylase" patented technology

The invention relates to a genetically engineered bacterium for high producing beta-alanine, co-culture preparation of D-pantothenic acid, a construction method of the genetically engineered bacterium and application of the genetically engineered bacterium to co-culture preparation of the D-pantothenic acid. According to the invention, original promoters of panD, aspC and ppc genes on a genome are replaced with a pTrc99A-derived Trc promoter and a ribosomebinding site (RBS) sequence so as to enhance synthesis of the beta-alanine, and genes pykA and pykF are knocked out to block consumption of phosphoenolpyruvate (PEP) and modify a glucose uptake path of escherichia coli, and thus, a non-phosphotransferase system (non-PTS) transport system is enhanced to block a PTS transport system, and synthesized precursor PEP is accumulated; on the basis, heterologous aspartate decarboxylase genes panD and aspC of E.coli W3110 are introduced to enhance the enzyme activity of key enzymes, so that supply and conversion of beta-alanine precursors are enhanced; gdhA genes of E.coli W3110 are introduced to enhance cyclic regeneration of coenzymes NADP / DNAPH, and finally, the titer of the beta-alanine is increased from 0 to 2.48g / L. The strain and a previous D-pantothenic acid producing strain DPA 21 / pBCST3 undergo construction of a primary co-culture system; the inoculation ratio is optimized; and when the inoculation ratio of the two strains is 1: 1, the co-culture strain can produce 3.08 g / L of D-pantothenic acid in a same fermentation medium.

The invention provides a method for preparing L-aspartic acid and L-alanine by using maleic anhydride. The method comprises the following steps: heating the maleic anhydride as the raw material to obtain a maleic acid solution through hydration reaction; adding maleate isomerase and L-aspartase; after filtering and concentrating obtained liquid of enzymatic reaction to deaminize, regulating the pH value until the isoelectric point of the L-aspartic acid is equal to 2.8 by using the maleic acid solution as an acidifying agent; crystallizing to separate out the L-aspartic acid; and converting the L-aspartic acid into the L-alanine by further using aspartate decarboxylase. The L-aspartic acid is prepared through one-step reaction by a two-enzyme method, the process is simple and convenient, the conversion ratio is high, production of malic acid as a by-product can be controlled effectively, and the quality of the product is good; and maleic acid is used as the acidifying agent to prepare the L-aspartic acid in an isoelectric point crystallization manner, crystallization mother liquor can be used as an enzyme reaction substrate and then is recycled, the discharge amount of waste water is greatly reduced, and the method conforms to the idea of environmental protection. In addition, the L-aspartic acid is further converted into the L-alanine by an enzymic method, so that products obtained by a whole process are diversified; and the market competitiveness is high.

The invention discloses L-aspartate beta-decarboxylase mutants and an application thereof, and belongs to the technical field of biological engineering. The catalytic enzyme activity of the L-aspartate beta-decarboxylase mutants N35D and A179E provided by the invention are improved by 238% and 244% respectively under the pH of 7.0, the remaining enzyme activity of the mutants N35D and A179E treated for 12 h under the pH of 4.5 is 83.9% and 85.5%, respectively, and compared with a control wild-type enzyme having residual enzyme activity of 24.8%, the mutants provided by the invention have significantly-improved acid stability, and therefore the L-aspartate beta-decarboxylase mutants N35D and A179E provided by the invention have better enzymatic properties.

The invention relates to the field of bioengineering, in particular to L-aspartate-alpha-decarboxylase. The L-aspartate-alpha-decarboxylase has an amino acid sequence and a gene sequence shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in SEQUENCE LISTING. The gene is synthesized in vitro after codon optimization based on a gene sequence encoding cysteinesulfinate decarboxylase in genome of Myzus persicae. The gene is cloned into an expression vector and transformed into Escherichia coli to construct a high-expression engineering strain. After the engineering strain is cultured and induced, it is found that the enzyme has activity of L-aspartate-alpha-decarboxylase, and the enzyme can convert L-aspartic acid into beta-alanine under proper conditions, overcome a defect that prokaryotic-derived L-aspartate-alpha-decarboxylase in the prior art is easy to inactivate, and can be used for industrial production of beta-alanine under conditions of small enzyme dosage and low cost.

The invention discloses an enzyme catalyst and a preparation method and application thereof in production of 3-aminopropionic acid. The preparation method comprises the following steps: (1) culturingan L-aspartate decarboxylase strain, and performing centrifuging to obtain thalli; (2) re-suspending the thalli obtained in the step (1) with water to obtain a resuspension, adding a surfactant into the resuspension, and performing stirring and centrifuging to obtain a supernatant; and (3) adding metal ions into the supernatant obtained in the step (2), performing mixing, adding a ligand solution,and performing stirring, washing and centrifuging to obtain a precipitate, namely the enzyme catalyst. Compared with the prior art, the method simplifies the operation process of extracting enzyme from cells, prepares the nano-material immobilized enzyme catalyst in a two-step rapid synthesis form, is simple and convenient in preparation process, and has low requirement on equipment; and the method realizes recycling and good repeated utilization rate of L-aspartate decarboxylase.

A high-throughput screening method for an L-aspartate beta-decarboxylase producing strain is disclosed. The method includes 1) subjecting microorganisms in a sample to enrichment culture and primary separation by adopting an enrichment culture medium, 2) inoculating single colonies obtained through separation from a plane plate to a 96-micropore plate primary screening semisolid medium and performing primary screening to obtain L-aspartate beta-decarboxylase producing strains, 3) inoculating the strains obtained through primary screening to a 24-prore plate, performing liquid culturing, and performing secondary screening to obtain the L-aspartate beta-decarboxylase producing strain with high yield by combining paper chromatography, and 4) subjecting the high-yield strain obtained through secondary screening to fermentation in a shake flask, and determining enzyme activity by combining an oxidation coloration process. The method is suitable for high-throughput screening of the L-aspartate beta-decarboxylase producing strain. The strain obtained through screening is alkali-resistant, thus avoiding tedious pH value controlling steps in a cell production processes. References are provided for high-throughput screening of producing strains bio-enzymes of the same kind.

The invention discloses an immobilizing method of L-aspartic acid-alpha-decarboxylase, and belongs to the technical field of biological engineering, TiO2 modified by a poly amino acid is used as a carrier, the L-aspartic acid-alpha-decarboxylase is bonded onto the surface of the carrier, and the poly amino acid is gamma-polyglutamic acid or epsilon-polylysine. Immobilization studies on the L-aspartic acid-alpha-decarboxylase by respective use of the modified carrier and an unmodified carrier find that the enzyme carrier content of the modified carrier is about 8 times of that of the unmodified carrier.

The invention relates to a panD mutant gene, encoded protein, a vector, an engineering bacterium and an application of the encoded protein. Series recombinant escherichia coli, for high-yield production of L-aspartate-[alpha]-decarboxylase (PanD), is obtained by changing codon base of the panD gene through random mutation of error-prone PCR. With the application of gene engineering bacterium for high-yield production of the PanD, the efficient transformation of the L-aspartate is achieved, so that [beta]-alanine can be prepared by virtue of a biological method, wherein PanD enzyme activity of recombinant escherichia coli numbered as 6-85 can reach 250U / L or above and [beta]-Ala yield can reach about 8.5g / L, nearly 4 times and 3 times above that of a parent strain.

The invention provides a preparation method of beta-alanine. The method comprises the following steps of: preparing a beta-alanine product from a reactant containing fumaric acid and ammonia water inthe presence of a catalyst, wherein the catalyst comprises a catalytic composition comprising aspartase and L-aspartate-alpha-decarboxylase, and fumaric acid is added during the reaction, and the total molar amount of the added fumaric acid is equal to the amount obtained by subtracting the initial molar amount of fumaric acid in the reactant from the initial molar amount of ammonia water in the reactant.

The present invention relates to a strain with high production of aspartate decarboxylase and a method for producing alanine, which belongs to the field of biotechnology. The strain is classified and named as Bacillus tequila PanD37, which was published on February 2, 2015. Preserved in China General Microorganism Culture Collection Management Center, preservation number: CGMCC No.10506. The Bacillus tequila producing L-aspartic acid α-decarboxylase provided by the present invention is reported for the first time. The bacterial strain PanD37 has good genetic stability and low cultivation cost, and the produced L-aspartic acid α-decarboxylase enzyme With high activity, the fermentation broth of this strain can be used to directly convert β-alanine. No need to add surfactants, organic solvents and other membrane-permeable agents during the catalytic synthesis, avoiding the introduction of exogenous impurities, which is conducive to extraction and separation operations, and has advantages in industrial applications .

The invention discloses a L-aspartic acid β-decarboxylase mutant and application thereof, belonging to the technical field of bioengineering. For the L-aspartic acid β-decarboxylase mutants N35D and A179E provided by the present invention, the catalytic enzyme activity is increased by 238% and 244% respectively under the condition of pH 7.0. After 12 hours of treatment at pH 4.5, 83.9% and 85.5% of the remaining enzyme activity remained, compared with 24.8% of the remaining enzyme activity of the wild-type enzyme in the control, the acid stability of the mutant was significantly improved. Therefore, the L-aspartic acid β-decarboxylase mutants N35D and A179E provided by the present invention have better enzymatic properties.

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 discloses a method for preparing L-2-aminobutyric acid in tandem with two enzymes, belonging to the field of bioengineering. The present invention obtains L-glutamic acid mutase and L-aspartic acid-β-decarboxylase by respectively cultivating recombinant Escherichia coli expressing L-glutamic acid mutase and L-aspartic acid-β-decarboxylase decarboxylase. The two enzymes are added to the reaction system with a certain mass ratio, and L-glutamic acid is used as a substrate to carry out an enzyme reaction to prepare L-2-aminobutyric acid. When the amount of L-aspartic acid-β-decarboxylase 2mg / mL, reacted for 24h, converted into 8.5mmol / L L-2 aminobutyric acid, and the molar conversion rate was 85.00%. Compared with the chemical production method, this method has a safe production process and no environmental pollution. Compared with the multi-enzyme synthesis system using threonine as the substrate, the substrate is cheaper and the process is simpler.

A high enzyme activity aspartate decarboxylase mutant can be obtained by a directed evolution method, the high enzyme activity aspartate decarboxylase mutant can efficiently catalyze decarboxylation of aspartic acid to produce beta-alanine, and by optimization of the reaction process, the aspartic acid substrate concentration can reach 250g / L. After 24 hours of conversion, the conversion rate canreach above 98.3%, and the high enzyme activity aspartate decarboxylase mutant has industrial application prospects.

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.