22 results about "Leucine dehydrogenase" patented technology

The invention belongs to the technical field of biology, and relates to an escherichia coli engineering strain for producing L-isoleucine through a methyl malic acid way as well as a construction method and application of the escherichia coli engineering strain. According to the invention, Escherichia coli BW25113 is used as a chassis strain, and a methyl malic acid synthase high-activity mutant gene cimA3.7, an isopropyl malic acid isomerase gene GsleuCD, a 3-isopropyl malic acid dehydrogenase gene AfleuB and a leucine dehydrogenase gene Lsleudh are over-expressed; a methyl malic acid absorption protein gene is over-expressed, and a branched chain amino acid transport system is modified to improve the exosome of the L-isoleucine; all methyl malic acid pathway related genes are integrated to a genome of a chassis strain, and a plasmid-free L-isoleucine production strain is constructed; the copy number of a key gene on a genome is optimized, and the yield of the engineering strain L-isoleucine is increased by introducing a non-oxidative glycolysis approach. The yield of the L-isoleucine after the engineering strain is fermented for 34 hours in a fermentation tank is 56.6 g / L.

The invention relates to the technical field of biology, in particular to an L-amino acid synthesis system and application. According to the L-amino acid synthesis system and application provided by the embodiment of the invention, in the presence of coenzyme NAD < + >, isopropanol is catalyzed by isopropanol dehydrogenase to generate acetone, and NAD < + > is reduced into coenzyme NADH; in the presence of coenzyme NADH, the leucine dehydrogenase mutant utilizes NH3 generated by ammonia water volatilization to perform asymmetric reductive amination on a prochiral alpha-keto acid compound, the NADH is oxidized into NAD +, the catalytic efficiency of asymmetric reductive amination of the leucine dehydrogenase mutant on the prochiral alpha-keto acid compound is improved, the circulating speed of the coenzyme NADH and NAD + is increased, and the catalytic activity of the leucine dehydrogenase mutant on the prochiral alpha-keto acid compound is improved. The catalytic efficiency of asymmetric reductive amination of the leucine dehydrogenase is favorably improved.

The invention discloses a leucine dehydrogenase mutant and application thereof.The mutant is obtained by replacing any one or more amino acid residues in the 36th site, the 59th site and the 109th site on the basis of a wild leucine dehydrogenase amino acid sequence, compared with the wild leucine dehydrogenase amino acid sequence, the enzyme activity of the mutant is improved by 5 times or above, the LeuDH mutant is guided into host bacteria, and the activity of the LeuDH mutant is improved by 5 times or above. The obtained recombinant bacterium is used for fermentation production of L-isoleucine, the yield of L-isoleucine reaches 30 g / L or above, compared with a wild type, the yield is remarkably improved, and powerful support is provided for fermentation production of an L-isoleucine product.

The application relates to a buffer reagent, a kit, a branched-chain alpha-keto acid or a branched-chain amino acid detection method in the technical field of biological detection. The buffer reagent comprises 15-25 mM, pH 8.5-9.5 Tris-HCl buffer solution, 10-50 g / L trehalose, 0.1-1.5 g / L Proclin 300, 0.1-0.5 g / L Triton X-100, 0.1-1.5 g / L sodium salt of EDTA, 1-10 kU / L ascorbic acid oxidase and 0.5-3 g / L bovine serum albumin. The buffer reagent is suitable for preparation of a reagent containing leucine dehydrogenase, the reagent containing the buffer reagent and the leucine dehydrogenase and the kit prepared by using the reagent can effectively eliminate interference problems in the process of being used for branched-chain alpha-keto acid, branched-chain amino acid detection, and has good correlation with mass spectrometry.

The present invention relates to a modified dehydrogenase, wherein one or more amino acid residues of the dehydrogenase are modified as compared to an unmodified dehydrogenase, and wherein the modified dehydrogenase has an increased ratio of leucine dehydrogenase or isoleucine dehydrogenase activity to valine dehydrogenase activity as compared to the unmodified dehydrogenase, and applications thereof.

The invention relates to the technical field of biology, in particular to a mutant of leucine dehydrogenase as well as a preparation method and application of the mutant. For the wild type leucine dehydrogenase, the mutant has one or more of D122F and E249K, and has one or more of M346A and K89R, and the mutant has one or more of D122F and E249K, and has one or more of M346A and K89R. Compared with the prior art, the invention provides the mutant of leucine dehydrogenase, the reaction rate of the mutant is obviously improved compared with that of wild type enzyme, and the mutant can be widely applied to detection and metabolism research of branched chain amino acid (BCAA) and branched chain alpha-keto acid.

This invention provides a leucine dehydrogenase mutant, a valine-producing strain comprising this leucine dehydrogenase mutant, and its applications. The invention designs a leucine dehydrogenase mutant with the following mutations: tyrosine (Tyr) at position 48 is mutated to serine (Ser), histidine (His) at position 127 is mutated to arginine (Arg), tyrosine (Tyr) at position 186 is mutated to phenylalanine (Phe), isoleucine (Ile) at position 256 is mutated to valine (Val), and lysine (Lys) at position 308 is mutated to arginine (Arg). These mutations are used to construct a valine-producing strain. The new strain is plasmid-free, has no growth defects, requires no induction, and exhibits advantages such as good genetic stability and high fermentation yield. It is an excellent strain capable of stably producing valine. This strain efficiently synthesizes valine de novo using glucose as a substrate, significantly increasing valine yield.

The invention relates to the technical field of agricultural biology, in particular to a method for increasing the yield of strain L-leucine, a mutant and application. According to the technical scheme, the relative activity of the leucine dehydrogenase mutants AsleudhM65L and AsleudhM65L / N69D for the activity of KIC / KIV is remarkably improved compared with that of wild leucine dehydrogenase, and after the encoding genes of the AsleudhM65L and AsleudhM65L / N69D are expressed in a recombinant strain, the yield of L-leucine is greatly improved, and the yield of L-valine is reduced.

This invention discloses a method for the enzymatic synthesis of L-aminobutyric acid (GABA), comprising the following steps: using threonine and ammonium formate as substrates, a three-enzyme combined catalytic reaction is carried out using leucine dehydrogenase, threonine deaminase, and formate dehydrogenase at a temperature of 40-55°C and a pH of 7.0-9.0 to obtain L-aminobutyric acid. The leucine dehydrogenase is the leucine dehydrogenase with the amino acid sequence shown in SEQ ID NO:1 or a mutant thereof. This invention can rapidly convert threonine to L-aminobutyric acid, yielding L-aminobutyric acid with high optical purity, thus improving economic efficiency.

The present invention discloses leucine dehydrogenase mutants and their applications in the synthesis of non-natural α-amino acids, belonging to the technical fields of enzyme engineering and microbial engineering. The kinetic parameters were determined using phenylglyoxylic acid as a substrate. The catalytic efficiency (k cat / K m ) of the double mutant T143G / L49G was 16.84 mM ‑1 ·s ‑1 , which was 2.8 times higher than that of the wild type. At high substrate concentrations, it could effectively catalyze the synthesis of L-phenylglycine from phenylglyoxylic acid, and the conversion rate could reach 99%. The kinetic parameters were determined using 2-oxobutyric acid as a substrate. The catalytic efficiency (k cat / K m ) of the double mutant T143L / V303L was 34.08 mM ‑1 ·s ‑1 , which was 13.4 times higher than that of the wild type. At high substrate concentrations, it could still effectively catalyze the synthesis of L-2-aminobutyric acid from 2-oxobutyric acid, and the conversion rate could reach 99%. On the basis of successfully improving the catalytic efficiency (k cat / K m ), the double mutants T143G / L49G and T143L / V303L in the present invention still maintained the good temperature stability of the wild-type strain and had more practical industrial application value.

The invention relates to the technical field of biology, in particular to a coenzyme circulation system and application. According to the coenzyme circulation system and application provided by the embodiment of the invention, in the presence of coenzyme NAD < + >, ammonium formate is catalyzed by formate dehydrogenase to generate NH3, and NAD < + > is reduced into coenzyme NADH; in the presence of coenzyme NADH, the leucine dehydrogenase mutant utilizes generated NH3 to carry out asymmetric reductive amination on a prochiral alpha-keto acid compound, the NADH is oxidized into NAD +, the catalytic efficiency of asymmetric reductive amination of the leucine dehydrogenase mutant on the prochiral alpha-keto acid compound is improved, the circulating speed of the coenzyme NADH and the NAD + is increased, and the catalytic activity of the leucine dehydrogenase mutant on the prochiral alpha-keto acid compound is improved. The catalytic efficiency of asymmetric reductive amination of the leucine dehydrogenase is favorably improved.

The present invention discloses a method for high-throughput screening of leucine dehydrogenase and a leucine dehydrogenase. Using the method of the present invention, protein engineering modification is carried out on leucine dehydrogenase BtLDH, and a high-throughput screening method is used to culture recombinant strains and strain mutants containing leucine dehydrogenase BtLDH in 96-well plates, and subculture induction is carried out. Cells containing leucine dehydrogenase BtLDH of Bacillus thuringiensis are centrifuged and collected to transform 2-ketobutyric acid to produce L-2-aminobutyric acid, and after color development with 2,4-dinitrofluorobenzene (DNFB), the absorbance value is read at 500 nm, and the strains with large absorbance values are verified in shake flasks. Each leucine dehydrogenase is successfully screened within a short time. Among them, the optimal mutant A262S produces 9.22 g / L of L-2-aminobutyric acid, and the yield of L-2-aminobutyric acid is increased by 3.04 g / L compared with the wild type.

This application relates to the field of biotechnology, and more particularly to a leucine dehydrogenase mutant and its applications. The leucine dehydrogenase mutant and its applications provided in this application are obtained by screening wild-type leucine dehydrogenase for mutations, resulting in a leucine dehydrogenase mutant with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. This mutant exhibits improved catalytic efficiency and thermal stability for the asymmetric reductive amination of prochiral α-keto acid compounds, which is beneficial for improving the synthesis efficiency of L-amino acids.

The invention belongs to the technical field of biology, and particularly relates to application of leucine dehydrogenase and a mutant thereof in asymmetric synthesis of an L-phenylglycine compound. The amino acid sequence of the leucinase mutant M2-1 is as shown in SEQ ID NO.2, the amino acid sequence of the leucinase mutant M2-2 is as shown in SEQ ID NO.3, the amino acid sequence of the leucinase mutant M2-3 is as shown in SEQ ID NO.4, and the amino acid sequence of the leucinase mutant M2-4 is as shown in SEQ ID NO.5. The leucine dehydrogenase and the leucine dehydrogenase mutant show significantly improved catalytic activity and strict stereoselectivity on benzoyl formic acid derivatives, and have practical industrial application value.

The present application relates to a kind of multi-enzyme synergistic expression recombinant genetically engineered bacteria, and its application in the biological catalysis preparation L-2-amino butyric acid.The present application constructs leucine dehydrogenase gene and formic acid dehydrogenase gene co-expression system, and is transformed into expression bacteria to obtain recombinant genetically engineered bacteria, with the live cells of the recombinant genetically engineered bacteria fermented and cultivated as catalyst applied in the catalytic reaction system of preparation L-2-amino butyric acid, production efficiency is high, when L-threonine feed amount is 300g / L, reaction 12h, conversion rate reaches more than 99%, effectively improve the ability of catalytic high concentration substrate to generate L-2-amino butyric acid.The present application uses whole cell as catalyst, avoids the purification of enzyme, avoids broken cell, and operation is simple;Two enzymes in one cell, can reduce steric hindrance, speed up the reaction speed;With deionized water as reaction medium to form reaction system, reaction product is easy to purify;Substrate concentration is high in the reaction process, saves production cost and time.

The invention relates to a buffer reagent, a kit and a detection method of branched chain alpha-ketonic acid or branched chain amino acid in the technical field of biological detection. The buffer reagent comprises a Tris-HCl buffer solution with the concentration of 15 to 25 mM and the pH value of 8.5 to 9.5, trehalose with the concentration of 10 to 50 g / L, Proclin300 with the concentration of 0.1 to 1.5 g / L, Triton X-100 with the concentration of 0.1 to 0.5 g / L, EDTA (Ethylene Diamine Tetraacetic Acid) sodium salt with the concentration of 0.1 to 1.5 g / L, ascorbic acid oxidase with the concentration of 1 to 10 kU / L and bovine serum albumin with the concentration of 0.5 to 3 g / L. The buffer reagent is suitable for preparation of a reagent containing leucine dehydrogenase, and the reagent containing the buffer reagent and the leucine dehydrogenase and a kit prepared by using the reagent can effectively eliminate the interference problem in the detection process of branched chain alpha-keto acid and branched chain amino acid, and have good correlation with mass spectrometry.

The invention provides a leucine dehydrogenase mutant and application thereof, and belongs to the technical field of bioengineering. The leucine dehydrogenase mutant provided by the invention can maintain higher catalytic stability in a wider pH value and temperature range, and the adaptability and durability of the leucine dehydrogenase mutant in an industrial reaction environment are remarkably improved. The improvement directly improves the efficiency and reliability of the process for catalytically synthesizing L-2-aminobutyric acid by using the enzyme, so that the reaction process is easier to control, and the cost loss caused by enzyme inactivation is potentially reduced. Finally, the mutant provides a catalyst with excellent performance for green, efficient and large-scale production of L-2-aminobutyric acid with high optical purity, so that a preparation process of chiral amino acid is simplified; and moreover, the method has important significance in reducing the production cost of key intermediates of downstream chiral drugs (such as certain antiepileptic drugs and antituberculosis drugs) and improving the product quality, and shows a good industrial application prospect.

The present invention discloses a leucine dehydrogenase mutant and its application in the preparation of L-phenylglycine. The leucine dehydrogenase mutant is: using the wild-type leucine dehydrogenase MsLDH shown in SEQ ID NO.1 as a template, aspartic acid D at the 332nd position is mutated to glutamic acid E, glycine G at the 333rd position is mutated to glutamic acid E, and leucine L at the 334th position is mutated to arginine R to obtain the mutant MsLDH-EER, and the amino acid sequence is as shown in SEQ ID NO.2. The present invention obtains a leucine dehydrogenase mutant MsLeuDH with improved catalytic activity and pH stability through molecular modification. Compared with the wild-type MsLeuDH, the conversion rate is 68.0% at 4 h under a substrate concentration of 200 mM. According to the mutant MsLDH-EER provided by the present invention, the conversion rate reaches 99.5% at 3 h, and the e.e. value of the product is greater than 99%.

The present application relates to the field of biotechnology, and specifically to a mutant of leucine dehydrogenase, a preparation method thereof, and an application thereof. For wild-type leucine dehydrogenase, the mutant has one or more of D122F and E249K, and one or more of M346A and K89R. Compared to conventional technologies, the present application provides a mutant of leucine dehydrogenase, which has a significantly improved reaction rate relative to the wild-type enzyme and can be widely used in the detection and metabolism research of branched-chain amino acids (BCAAs) and branched-chain α-keto acids.

In a method for detecting phenylglyoxylic acid (PGA) according to the present disclosure, at least the presence of phenylglyoxylic acid in a sample is detected by measuring the decrease in a coenzyme, which occurs in association with the conversion of phenylglyoxylic acid in the sample into L-2-phenylglycine in the presence of the coenzyme and ammonium ions (NH4 +) using leucine dehydrogenase (LeuDH) that catalyzes a reverse reaction of a dehydrogenation reaction. The measurement of the decrease in the coenzyme may be performed by measuring an absorbance caused by the coenzyme. It is also possible to measure the amount of decrease in the coenzyme by measuring the change in the absorbance and to quantify the phenylglyoxylic acid in the sample on the basis of the measured amount of decrease.

The invention belongs to the technical field of biology, and particularly relates to leucine dehydrogenase and application thereof in synthesis of L-phenylglycine compounds. The amino acid sequence of the leucine dehydrogenase BmLeuDH is as shown in SEQ ID NO. 1, and the amino acid sequence of the leucine dehydrogenase BcLeuDH is as shown in SEQ ID NO. 3. As a biocatalyst, the leucine dehydrogenase disclosed by the invention can stereoselectively catalyze benzoylformic acid and derivatives thereof to prepare corresponding L-phenylglycine compounds, shows a wide substrate spectrum and strict stereoselectivity, and particularly shows excellent catalytic activity on benzoylformic acid. According to the invention, an L-phenylglycine compound synthetase tool library is enriched, and a green and efficient biocatalyst is provided for synthesis of the L-phenylglycine compound, especially L-phenylglycine.

The invention provides a leucine dehydrogenase mutant with the capability of catalytically synthesizing D-amino acid as well as a preparation method and application of the leucine dehydrogenase mutant, and belongs to the technical field of protease. The invention relates to a method for changing chiral selectivity of leucine dehydrogenase, which comprises the following steps of: replacing corresponding active center amino acid sites in the leucine dehydrogenase with active center amino acid sites of diaminopimelate dehydrogenase and active center amino acid sites of mutants of the diaminopimelate dehydrogenase; the amino acid sequence of the obtained leucine dehydrogenase mutant is as shown in SEQ ID NO: 4. The leucine dehydrogenase mutant can catalyze various D-amino acids (D-tertiary leucine, D-leucine and D-phenylglycine), shows excellent D-amino acid enzyme catalytic activity, and provides a powerful guarantee and a solid foundation for application of the leucine dehydrogenase mutant in the field of chiral amino acid synthesis.