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Leucine dehydrogenase (LeuDH) has emerged as the most promising biocatalyst for L-tert-leucine (L-Tle) production via asymmetric reduction in trimethylpyruvate (TMP). In this study, a new LeuDH named PbLeuDH from marine Pseudomonas balearica was heterologously over-expressed in Escherichia coli, followed by purification and characterization. PbLeuDH possessed a broad substrate scope, displaying activities toward numerous L-amino acids and α-keto acids. Notably, compared with those reported LeuDHs, PbLeuDH exhibited excellent catalytic efficiency for TMP with a Km value of 4.92 mM and a kcat/Km value of 24.49 s−1 mM−1. Subsequently, L-Tle efficient production was implemented from TMP by whole-cell biocatalysis using recombinant E. coli as a catalyst, which co-expressed PbLeuDH and glucose dehydrogenase (GDH). Ultimately, using a fed-batch feeding strategy, 273 mM (35.8 g L−1) L-Tle was achieved with a 96.1% yield and 2.39 g L−1 h−1 productivity. In summary, our research provides a competitive biocatalyst for L-Tle green biosynthesis and lays a solid foundation for the realization of large-scale L-Tle industrial production. [ABSTRACT FROM AUTHOR]

The asymmetric synthesis of chiral amino acids by leucine dehydrogenases has great potential for industrialization; however, the inhibitory effect of high‐concentration substrates limits its large‐scale application. Herein, using structure‐guided genome mining based on sequence‐structure prediction of substrate tolerance and specificity, a novel leucine dehydrogenase (LaLeuDH) from Labrenzia aggregate was identified and characterized, which exhibited the highest substrate tolerance and excellent activity to trimethyl pyruvate, even at 1.5 M concentration. Moreover, based on coenzyme binding structural information and sequence alignment, directed evolution of LaLeuDH was performed to increase affinity for NADH. The obtained variant D153 N/H191 N resulted in 25‐fold improved affinity for NADH, with 50‐fold enhanced catalytic efficiency (kcat/Km) of 40464.6 mM−1 s−1. Finally, through a combination of the above two strategies, as high as 1.5 M substrate could be completely converted in 18 h without coenzyme addition, demonstrating that this engineered enzyme has promising prospects for industrialization. [ABSTRACT FROM AUTHOR]

• Combining molecular dynamics, homology modeling, sequence analysis, and virtual saturation mutagenesis to identify key amino acid sites in PfleuDH, thereby enhancing its thermostability. • The combined mutant S220P/T347K exhibits higher thermostability, with 2.94 times longer t 1/2. • Through molecular dynamics and 3D modeling, this study conducted a mechanistic analysis of the t 1/2 and catalytic activity of the combined mutant S220P/T347K. LeuDH catalyzes the reversible deamination of L-leucine and certain branched-chain aliphatic amino acids, resulting in the corresponding α-keto acids. This process has received considerable attention in the food and pharmaceutical industries. Although LeuDH from Planifilum fimeticola shows higher catalytic efficiency, its lack of thermostability limits its widespread use in industrial applications. This study identified a mutant, S220P/T347K, with enhanced thermostability through rational design. The experimental results show that under optimal temperature conditions of 65 °C, the half-life (t 1/2) of the mutant is 2.94 times longer than that of the wild type, and it does not affect the catalytic activity. Molecular dynamics and 3D structural analysis revealed that the S220P/T347K mutant's increased thermostability mainly results from decreased flexibility, enhanced hydrophobicity, and a favorable electrostatic potential. Furthermore, the mutant's increased catalytic efficiency and specific activity may result from its tighter binding with small molecule substrates. This study is the first to use rational design to modify Pf leuDH, effectively enhancing its thermostability and catalytic activity. It provides a simple and effective method for strengthening LeuDH in industrial applications. [Display omitted] [ABSTRACT FROM AUTHOR]

L-tert-leucine (L-Tle) is widely used as vital chiral intermediate for pharmaceuticals and as chiral auxiliarie for organocatalysis. L-Tle is generally prepared via the asymmetric reduction of trimethylpyruvate (TMP) catalyzed by NAD+-dependent leucine dehydrogenase (LeuDH). To improve the catalytic efficiency and coenzyme affinity of LeuDH from Bacillus cereus, mutation libraries constructed by error-prone PCR and iterative saturation mutation were screened by two kinds of high-throughput methods. Compared with the wild type, the affinity of the selected mutant E24V/E116V for TMP and NADH increased by 7.7- and 2.8-fold, respectively. And the kcat/Km of E24V/E116V on TMP was 5.4-fold higher than that of the wild type. A coupled reaction comprising LeuDH with glucose dehydrogenase of Bacillus amyloliquefaciens resulted in substrate inhibition at high TMP concentrations (0.5 M), which was overcome by batch-feeding of the TMP substrate. The total turnover number and specific space-time conversion of 0.57 M substrate increased to 11,400 and 22.8 mmol·h−1·L−1·g−1, respectively. Key points: • The constructed new high-throughput screening strategy takes into account the two indicators of catalytic efficiency and coenzyme affinity. • A more efficient leucine dehydrogenase (LeuDH) mutant (E24V/E116V) was identified. • E24V/E116V has potential for the industrial synthesis of L-tert-leucine. [ABSTRACT FROM AUTHOR]

Background: Biosynthesis of l-tert-leucine (l-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of l-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. Results: In this work, a novel fusion enzyme (GDH–R3–LeuDH) for the efficient biosynthesis of l-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH–R3–LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of l-tle by GDH–R3–LeuDH was all enhanced by twofold. Finally, the space–time yield of l-tle catalyzing by GDH–R3–LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). Conclusions: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize l-tle and reach the highest space–time yield up to now. These results demonstrated the great potential of the GDH–R3–LeuDH fusion enzyme for the efficient biosynthesis of l-tle. [ABSTRACT FROM AUTHOR]

Background: L-tert-Leucine has been widely used in pharmaceutical, chemical, and other industries as a vital chiral intermediate. Compared with chemical methods, enzymatic methods to produce L-tert-leucine have unparalleled advantages. Previously, we found a novel leucine dehydrogenase from the halophilic thermophile Laceyella sacchari (LsLeuDH) that showed good thermostability and great potential for the synthesis of L-tertleucine in the preliminary study. Hence, we manage to use the LsLeuDH coupling with a formate dehydrogenase from Candida boidinii (CbFDH) in the biosynthesis of L-tert-leucine through reductive amination in the present study. Result: The double-plasmid recombinant strain exhibited higher conversion than the single-plasmid recombinant strain when resting cells cultivated in shake flask for 22 h were used. Under the optimized conditions, the double-plasmid recombinant E. coli BL21 (pETDute-FDH-LDH, pACYCDute-FDH) transformed 1 mol·L-1 trimethylpyruvate (TMP) completely into L-tert-leucine with greater than 99.9% ee within 8 h. Conclusions: The LsLeuDH showed great ability to biosynthesize L-tert-leucine. In addition, it provided a new option for the biosynthesis of L-tert-leucine. [ABSTRACT FROM AUTHOR]

• A novel gene of leucine dehydrogenase (Bc -LeuDH) was obtained from a thermophilic Bacillus coagulans NL01. • The recombinant leucine dehydrogenase (Bc -LeuDH) exhibited well-adapted at wide temperature and pH ranges. • The recombinant leucine dehydrogenase (Bc -LeuDH) exhibited excellent thermostability and well pH stability. L- Tert -leucine is the most representative unnatural amino acid and its production is valuable in industry. At present, l - tert -leucine is mainly produced by bioconversion, in which leucine dehydrogenase (LeuDH) plays a major role. In this study, a highly thermo- and pH-stable LeuDH from Bacillus coagulans NL01 (Bc -LeuDH) was reported and successfully expressed in Escherichia coli BL21(DE3). The enzyme was purified and its enzymatic properties were characterized. The specific activity of Bc -LeuDH at optimum condition (pH 8.0 and 50 °C) is 1337.97 U/mg, and the K m and k cat for sodium α-ketoisocaproate was 1.369 mM and 0.125 S -1, respectively. Furthermore, Bc -LeuDH possessed excellent thermostability that held nearly 80 % activity after 72 h incubation at temperature 50 °C and 55 °C. Notably, the half-life of Bc- LeuDH activity also was long for near 12 h at a high temperature of 70 °C, which was the longest in previous reports. Based on the sequence and structure analysis, the hydrophobic amino acid residues and hydrophobic patches are considered to have an important contribution to the thermostability of Bc -LeuDH. Furthermore, the Bc -LeuDH demonstrated better pH stability over a wide pH ranging from 7.0-10.0. These results indicate the potential industrial application of Bc -LeuDH in the future. [ABSTRACT FROM AUTHOR]

Whole-cell catalysis with co-expression of two or more enzymes in a single host as a simple low-cost biosynthesis method has been widely studied and applied but hardly with regulation of multi-enzyme expression. Here we developed an efficient whole-cell catalyst for biosynthesis of l-phenylglycine (l-Phg) from benzoylformic acid through co-expression of leucine dehydrogenase from Bacillus cereus (BcLeuDH) and NAD+-dependent mutant formate dehydrogenase from Candida boidinii (CbFDHA10C) in Escherichia coli with tunable multi-enzyme-coordinate expression system. By co-expressing one to four copies of CbFDHA10C and optimization of the RBS sequence of BcLeuDH in the expression system, the ratio of BcLeuDH to CbFDH in E. coli BL21/pETDuet-rbs4leudh-3fdhA10C was finally regulated to 2:1, which was the optimal one determined by enzyme-catalyzed synthesis. The catalyst activity of E. coli BL21/pETDuet-rbs4leudh-3fdhA10C was 28.4 mg L−1 min−1 g−1 dry cell weight for l-Phg production using whole-cell transformation, it’s was 3.7 times higher than that of engineered E. coli without enzyme expression regulation. Under optimum conditions (pH 8.0 and 35 °C), 60 g L−1 benzoylformic acid was completely converted to pure chiral l-Phg in 4.5 h with 10 g L−1 dry cells and 50.4 g L−1 ammonium formate, and with enantiomeric excess > 99.9%. This multi-enzyme-coordinate expression system strategy significantly improved l-Phg productivity and demonstrated a novel low-cost method for enantiopure l-Phg production. [ABSTRACT FROM AUTHOR]

This study reports the application of peptide linker in the construction of bi-functional formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH) enzymatic complex for efficient cofactor regeneration and L-tert leucine (L-tle) biotransformation. Seven FDH-LeuDH fusion enzymes with different peptide linker were successfully developed and displayed both parental enzyme activities. The incorporation order of FDH and LeuDH was investigated by predicting three-dimensional structures of LeuDH-FDH and FDH-LeuDH models using the I-TASSER server. The enzymatic characterization showed that insertion of rigid peptide linker obtained better activity and thermal stability in comparison with flexible peptide linker. The production rate of fusion enzymatic complex with suitable flexible peptide linker was increased by 1.2 times compared with free enzyme mixture. Moreover, structural analysis of FDH and LeuDH suggested the secondary structure of the N-, C-terminal domain and their relative positions to functional domains was also greatly relevant to the catalytic properties of the fusion enzymatic complex. The results show that rigid peptide linker could ensure the independent folding of moieties and stabilized enzyme structure, while the flexible peptide linker was likely to bring enzyme moieties in close proximity for superior cofactor channeling. [ABSTRACT FROM AUTHOR]

Perindopril is an effective antihypertensive drug in strong demand used to treat hypertension. l -norvaline is a vital intermediate of Perindopril production mainly produced by chemical synthesis with low purity. We developed an environmentally friendly method to produce l -norvaline with high purity based on a desymmetrization process. d -Norvaline was oxidized to the corresponding keto acid by d -amino acid oxidase from the substrate dl -norvaline. Asymmetric hydrogenation of the keto acid to l -norvaline was carried out by leucine dehydrogenase with concomitant oxidation of NADH to NAD + . A NADH regeneration system was introduced by overexpressing a formate dehydrogenase. The unwanted H 2 O 2 by-product generated during d -norvaline oxidation was removed by adding catalase. A total of 54.09 g/L of l -norvaline was achieved, with an enantiomeric excess over 99% under optimal conditions, with a 96.7% conversion rate. Our desymmetrization method provides an environmental friendly strategy for the production of enantiomerically pure l -norvaline in the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

L -Phosphinothricin (L -PPT) is the effective constituent in racemic PPT (a high-efficiency and broad-spectrum herbicide), and the exploitation of green and sustainable synthesis route for L -PPT has always been the focus in pesticide industry. In recent years, "one-pot, two-step" enzyme-mediated cascade strategy is a mainstream pathway to obtain L -PPT. Herein, Rg DAAO and Bs LeuDH were applied to expand "one-pot, two-step" process. Notably, a NADH-dependent leucine dehydrogenase from Bacillus subtilis (Bs LeuDH) was firstly characterized and attempted to generate L -PPT, achieving an excellent enantioselectivity (99.9% ee). Meanwhile, a formate dehydrogenase from Pichia pastoris (Pp FDH) was utilized to implement NADH cofactor regeneration and only CO 2 was by-product. Sufficient amount of the corresponding keto acid precursor PPO was obtained by oxidation of D -PPT relying on a D -amino acid oxidase from Rhodotorula gracilis (Rg DAAO) with content conversion (46.1%). L -PPT was ultimately prepared from racemized PPT via oxidative deamination catalyzed by Rg DAAO and reductive amination catalyzed by Bs LeuDH, achieving 80.3% overall yield and > 99.9% ee value. [Display omitted] • A "one-pot, two-step" cascade process catalyzed by Rg DAAO and Bs LeuDH was explored to prepare L -phosphinothricin (L -PPT) starting from racemic PPT. • A novel NADH-dependent leucine dehydrogenase (Bs LeuDH) coupled with Pp FDH was applied to the synthesis of L -PPT for the first time. • The cascade yielded 80.3% L -PPT with 99.9% ee in a 12 h bioconversion of 0.1 M rac-PPT. [ABSTRACT FROM AUTHOR]

l- tert-Leucine and its derivatives are used as synthetic building blocks for pharmaceutical active ingredients, chiral auxiliaries, and ligands. Leucine dehydrogenase (LeuDH) is frequently used to prepare l- tert-leucine from the α-keto acid precursor trimethylpyruvate (TMP). In this study, a high-throughput screening method for the l- tert-leucine synthesis reaction based on a spectrophotometric approach was developed. Directed evolution strategy was applied to engineer LeuDH from Lysinibacillus sphaericus for improved efficiency of l- tert-leucine synthesis. After two rounds of random mutagenesis, the specific activity of LeuDH on the substrate TMP was enhanced by more than two-fold, compared with that of the wild-type enzyme, while the activity towards its natural substrate, leucine, decreased. The catalytic efficiencies ( k/ K) of the best mutant enzyme, H6, on substrates TMP and NADH were all enhanced by more than five-fold as compared with that of the wild-type enzyme. The efficiency of l- tert-leucine synthesis by mutant H6 was significantly improved. A productivity of 1170 g/l/day was achieved for the mutant enzyme H6, compared with 666 g/l/day for the wild-type enzyme. [ABSTRACT FROM AUTHOR]

In the present paper, the reductive amination ofα-ketoisocaproic acid to L-leucine was monitored using a new modified electrode based on gold nanoparticles (AuNPs) and leucine dehydrogenase (LeuDH). The modified electrode was prepared by self-assembling of AuNPs with thiocytosine followed by the immobilization of the enzyme. The enzyme-modified electrode response was investigated by chronoamperometry for indirect quantification of ammonium ion () involved in the reaction. This reaction is catalyzed by LeuDH and takes place in the presence of ammonium chloride, as nitrogen source, and nicotinamide adenine dinucleotide (NADH), as cofactor. The enzyme-modified electrode was also used for in situ electrochemical NADH regeneration. After one step NADH regeneration the conversion of ammonium ion was improved. The modified electrode has good sensitivity (0.91 × 10−3A/M) and reproducibility with an RSD of 8.4% (n = 4). The experimental determination of ammonium ion was confirmed by standard Berthelot spectrophotometric method. [ABSTRACT FROM PUBLISHER]

Highlights: [•] An EsLeuDH with relatively high activity and thermal stability was discovered. [•] The EsLeuDH was coexpressed with BmGDH to avoid the usage of external cofactor. [•] Enantiopure l-tert-leucine was obtained at a yield of 80% in a 1-L reaction system. [•] The space-time-yield reached 944.7gL−1 d−1, higher than the results reported. [ABSTRACT FROM AUTHOR]

Leucine plays an important role in protein synthesis, brain functions, building muscle mass, and helping the body when it undergoes stress. Here, we report a new amperometric bienzyme screen-printed biosensor for the determination of leucine, by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and leucine dehydrogenase (LDH) on a screen-printed electrode with NADP and p-hydroxybenzoate as the cofactors. The detection principle of the sensor is that LDH catalyzes the specific dehydrogenation of leucine by using NADP as a cofactor. The product, NADPH, triggers the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a change in electron concentration at the working carbon electrode, which is detected by the potentiostat. The sensor shows a linear detection range between 10 and 600 μM with a detection limit of 2 μM. The response is reproducible and has a fast measuring time of 5-10 s after the addition of a given concentration of leucine. [ABSTRACT FROM AUTHOR]

Abstract: Leucine dehydrogenase (LeuDH, l-leucine: NAD+ oxidoreductase, deaminating, EC 1.4.1.9) was screened in six psychrophilic bacteria, and the highest levels of enzyme activity were found in Sporosarcina psychrophila DSM 3. As the first LeuDH from a psychrophilic bacterium, the enzyme was purified to homogeneity and characterized. The protein had an octameric structure with identical 43-kDa subunits, giving a total molecular mass of about 340kDa. The enzyme exhibited the highest activity at 50°C and exhibited one-tenth of that activity even at temperatures as low as 0°C. The enzyme lost no activity with incubation at temperatures lower than 40°C for 40min, but there was marked loss of activity with incubations at temperatures higher than 50°C. The optimum pHs were 11 for deamination of l-leucine and 9 for amination of 4-methyl-2-oxopentanoate. The K m values for l-leucine and NAD+ at 20°C were 0.65 and 0.015mM, respectively. The catalytic properties of S. psychrophila LeuDH were similar to those of LeuDHs from Lysinibacillus sphaericus and Geobacillus stearothermophilus, except for its lower optimal reaction temperature and thermostability at low temperatures. Crystal structural analysis of S. psychrophila LeuDH showed the total structure to be similar to that of the L. sphaericus enzyme, except minor alterations reduced the hydrophobic interactions and hydrogen bonds within and between subunits. [Copyright &y& Elsevier]

Abstract: A high-sensitive l-leucine biosensors consisting of l-leucine dehydrogenase (LeuDH) immobilized membrane on the edge of a fiber was developed and demonstrated using a high-intensity ultra violet light-emitting diodes (UV-LED) excitation system (λ =340nm), an optical fiber probe and a photomultiplier tube. The biosensors were fabricated by attaching l-leucine dehydrogenase immobilized polytetrafluoroethylene (PTFE) membrane with 2-methacryloyloxyethyl phosphorylcholine polymer (PMEH) polymer to the optical fiber. A l-leucine concentration in an aqueous solution was measured by immersing the sensor probes in the phosphate buffer which contained nicotinamide adenine dinucleotide (NAD+) solution. The fluorescent signals of nicotinamide adenine dinucleotide (NADH), produced by enzymatic reactions between NAD+ and an installation of l-leucine solution, were then guided to the photomultiplier tube and a signal detector. The fluorescent intensities were related linearly to the l-leucine concentration from 5μM to 10mM. The calibration ranges were acceptable for clinical applications of maple syrup urine disease (MSUD) determinations. High-sensitive and low power consumption of a UV-LED biosensing system allow to be contributed to their enhance quality of life for daily self-care of MSUD patients. [Copyright &y& Elsevier]

A simple and rapid screening method for amino acid dehydrogenase (e.g., leucine dehydrogenase, LDH) has been developed. It relies on a competitive relationship between a non-fluorescent Cu(II)–calcein complex and amino acid (e.g., l -2-aminobutyric acid, l -ABA). When ABA was introduced to a Cu(II)–calcein solution, it bound with the Cu(II) ions and this released calcein from the complex, which was detected as strong fluorescence. The principle of this high-throughput screening method was validated by screening an LDH mutant library. Compared with other methods, this method provided much quicker l -ABA detection and screening for leucine dehydrogenase mutations. [ABSTRACT FROM AUTHOR]

α-Amino acids and α-keto acids are versatile building blocks for the synthesis of several commercially valuable products in the food, agricultural, and pharmaceutical industries. In this study, a novel transamination-like reaction catalyzed by leucine dehydrogenase was successfully constructed for the efficient enzymatic co-synthesis of α-amino acids and α-keto acids. In this reaction mode, the α-keto acid substrate was reduced and the α-amino acid substrate was oxidized simultaneously by the enzyme, without the need for an additional coenzyme regeneration system. The thermodynamically unfavorable oxidation reaction was driven by the reduction reaction. The efficiency of the biocatalytic reaction was evaluated using 12 different substrate combinations, and a significant variation was observed in substrate conversion, which was subsequently explained by the differences in enzyme kinetics parameters. The reaction with the selected model substrates 2-oxobutanoic acid and L-leucine reached 90.3% conversion with a high total turnover number of 9.0 × 106 under the optimal reaction conditions. Furthermore, complete conversion was achieved by adjusting the ratio of addition of the two substrates. The constructed reaction mode can be applied to other amino acid dehydrogenases in future studies to synthesize a wider range of valuable products. [ABSTRACT FROM AUTHOR]

To characterize foot odor, we analyzed its components by sensory tests, isolated microorganisms that produce it, and evaluated the mechanism of the occurrence of foot odor. As a result, foot odor was found to be derived from isovaleric acid, which is produced when Staphylococcus epidermidis, a resident species of the normal cutaneous microbial flora, degrades leucine present in sweat. In addition, Bacillus subtilis was detected in the plantar skin of subjects with strong foot odor, and this species was shown to be closely associated with increased foot odor. Therefore, we screened various naturally occurring substances and fragrant agents that inhibit microbial production of foot odor without disturbing the normal microbial flora of the human skin. As a result, we identified citral, citronellal, and geraniol as fragrant agents that inhibit the generation of isovaleric acid at low concentrations. [ABSTRACT FROM AUTHOR]

Abstract: Five different immobilized NAD+ derivatives were employed to compare the behavior of four amino acid dehydrogenases chromatographed using kinetic-based enzyme capture strategies (KBECS): S6-, N6-, N1-, 8′-azo-, and pyrophosphate-linked immobilized NAD+. The amino acid dehydrogenases were NAD+-dependent phenylalanine (EC 1.4.1.20), alanine (EC 1.4.1.1), and leucine (EC 1.4.1.9) dehydrogenases from various microbial species and NAD(P)+-dependent glutamate dehydrogenase from bovine liver (GDH; EC 1.4.1.3). KBECS for bovine heart l-lactate dehydrogenase (EC 1.1.1.27) and yeast alcohol dehydrogenase (EC 1.1.1.1) were also applied to assist in a preliminary assessment of the immobilized cofactor derivatives. Results confirm that the majority of the enzymes studied retained affinity for NAD+ immobilized through an N6 linkage, as opposed to an N1 linkage, replacement of the nitrogen with sulfur to produce an S6 linkage, or attachment of the cofactor through the C8 position or the pyrophosphate group of the cofactor. The one exception to this was the dual-cofactor-specific GDH from bovine liver, which showed no affinity for N6-linked NAD+ but was biospecifically adsorbed to S6-linked NAD+ derivatives in the presence of its soluble KBEC ligand. The molecular basis for this is discussed together with the implications for future development and application of KBECS. [Copyright &y& Elsevier]

The changes in intracellular pool of branched-chain amino acids (BCAA) regulate different physiological processes in bacteria. Up to date, the only available photometric test for measuring BCAA concentration was adapted for blood and plasma samples in diagnostic purposes. We have modified this method for use on bacterial cells, and tested its applicability on several model organisms: Lactococcus lactis, Bacillus subtilis and Escherichia coli. [Copyright &y& Elsevier]

The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward l-phenylalanine and phenylpyruvate with 1.6 and 7.8% of kcat values of the wild-type enzyme for the preferred substrates, l-leucine and its keto-analog, respectively. Indeed, the residue A113, corresponding to G114 in PheDH, affects the volume of the side-chain binding pocket and has a critical role in discrimination of the bulkiness of the side chain. Another two sets of mutants, substituting L40 and V294 of LeuDH with the corresponding residues predicted in GluDH and LysDH, were also constructed and characterized. Emergence of GluDH and LysDH activities in L40K/V294S and L40D/V294S mutants, respectively, indicates that the two corresponding residues in the active site of amino acid dehydrogenases are important for discrimination of the hydrophobicity/polarity of the aliphatic substrate side chain. All these results demonstrate that the substrate specificities of the amino acid dehydrogenases can be altered by protein engineering. The engineered dehydrogenases are expected to be used for production and detection of natural and non-natural amino acids. [Copyright &y& Elsevier]

The structural gene (leudh) coding for leucine dehydrogenase from Bacillus sphaericus IFO 3525 was cloned into Escherichia coli cells and sequenced. The open reading frame coded for a protein of 39.8 kDa. The deduced amino acid sequence of the leucine dehydrogenase from B. sphaericus showed 76–79% identity with those of leucine dehydrogenases from other sources. About 16% of the amino acid residues of the deduced amino acid sequence were different from the sequence obtained by X-ray analysis of the B. sphaericus enzyme. The recombinant enzyme was purified to homogeneity with a 79% yield. The enzyme was a homooctamer (340 kDa) and showed the activity of 71.7 μmol·min−1·mg−1) of protein. The mutant enzymes, in which more than six amino acid residues were deleted from the C-terminal of the enzyme, showed no activity. The mutant enzyme with deletion of four amino acid residues from the C-terminal of the enzyme was a dimer and showed 4.5% of the activity of the native enzyme. The dimeric enzyme was more unstable than the native enzyme, and the Km values for l-leucine and NAD+ increased. These results suggest that the Asn-Ile-Leu-Asn residues of the C-terminal region of the enzyme play an important role in the subunit interaction of the enzyme. [Copyright &y& Elsevier]

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of l-leucine with α-ketoglutarate results in formation of α-ketoisocaproate, which is reductively aminated back to l-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between α-ketoiso[1-14C]valerate and l-isoleucine. Due to the presence of glutamate dehydrogenase substrates (α-ketoglutarate, ammonia, and NADH) and l-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure. [Copyright &y& Elsevier]

• A novel HTS method was established combined with disrupting cells by lysozyme. • Lys72Ala was determined to possess high catalytic activity and 2-OBA tolerance. • The expression of multi-enzyme cascade was regulated by fine-tuning RBS intensity. • 121 g L-2-ABA in 5 L fermenter was produced with a productivity of 5.04 g·L-1·h−1. • It was a highest yield of L-2-ABA currently reported by single-cell biotransformation. Leucine dehydrogenase (LDH) is widely used in the preparation of L-2-aminobutyric acid (L-2-ABA), however its wide application is limited by 2-ketobutyric acid (2-OBA) inhibition. Firstly, a novel high-throughput screening method of LDH was established, specific enzyme activity and 2-OBA tolerance of Lys72Ala mutant were 33.3% higher than those of the wild type. Subsequently, we constructed a single cell comprised of ivlA , Esldh K72A , fdh and optimized expression through fine-tuning RBS intensity, so that the yield of E. coli BL21/pET28a-R3 ivlA-Esldh K72A -fdh was 2.6 times higher than that of the original strain. As a result, 150 g L-threonine was transformed to 121 g L-2-ABA in 5 L fermenter with 95% molar conversion rate, and a productivity of 5.04 g·L-1·h−1, which is the highest productivity of L-2-ABA currently reported by single-cell biotransformation. In summary, our research provided a green synthesis for L-2-ABA, which has potential for industrial production of drug precursors. [ABSTRACT FROM AUTHOR]

• A novel HTS method was established combined with disrupting cells by lysozyme. • Lys72Ala was determined to possess high catalytic activity and 2-OBA tolerance. • The expression of multi-enzyme cascade was regulated by fine-tuning RBS intensity. • 121 g L-2-ABA in 5 L fermenter was produced with a productivity of 5.04 g·L-1·h−1. • It was a highest yield of L-2-ABA currently reported by single-cell biotransformation. Leucine dehydrogenase (LDH) is widely used in the preparation of L-2-aminobutyric acid (L-2-ABA), however its wide application is limited by 2-ketobutyric acid (2-OBA) inhibition. Firstly, a novel high-throughput screening method of LDH was established, specific enzyme activity and 2-OBA tolerance of Lys72Ala mutant were 33.3% higher than those of the wild type. Subsequently, we constructed a single cell comprised of ivlA , Esldh K72A , fdh and optimized expression through fine-tuning RBS intensity, so that the yield of E. coli BL21/pET28a-R3 ivlA-Esldh K72A -fdh was 2.6 times higher than that of the original strain. As a result, 150 g L-threonine was transformed to 121 g L-2-ABA in 5 L fermenter with 95% molar conversion rate, and a productivity of 5.04 g·L-1·h−1, which is the highest productivity of L-2-ABA currently reported by single-cell biotransformation. In summary, our research provided a green synthesis for L-2-ABA, which has potential for industrial production of drug precursors. [ABSTRACT FROM AUTHOR]

The production of l-leucine was improved by the disruption of ltbR encoding transcriptional regulator and overexpression of the key genes (leuAilvBNCE) of the l-leucine biosynthesis pathway in Corynebacterium glutamicum XQ-9. In order to improve l-leucine production, we rationally engineered C. glutamicum to enhance l-leucine production, by improving the redox flux. On the basis of this, we manipulated the redox state of the cells by mutating the coenzyme-binding domains of acetohydroxyacid isomeroreductase encoded by ilvC, inserting NAD-specific leucine dehydrogenase, encoded by leuDH from Lysinibacillus sphaericus, and glutamate dehydrogenase encoded by rocG from Bacillus subtilis, instead of endogenous branched-chain amino acid transaminase and glutamate dehydrogenase, respectively. The yield of l-leucine reached 22.62 ± 0.17 g·L−1 by strain ΔLtbR-acetohydroxyacid isomeroreductase (AHAIR)M/ABNCME, and the concentrations of the by-products (l-valine and l-alanine) increased, compared to the strain ΔLtbR/ABNCE. Strain ΔLtbR-AHAIRMLeuDH/ABNCMLDH accumulated 22.87±0.31 g·L−1l-leucine, but showed a drastically low l-valine accumulation (from 8.06 ± 0.35 g·L−1 to 2.72 ± 0.11 g·L−1), in comparison to strain ΔLtbR-AHAIRM/ABNCME, which indicated that LeuDH has much specificity for l-leucine synthesis but not for l-valine synthesis. Subsequently, the resultant strain ΔLtbR-AHAIRMLeuDHRocG/ABNCMLDH accumulated 23.31 ± 0.24 g·L−1l-leucine with a glucose conversion efficiency of 0.191 g·g−1. [ABSTRACT FROM AUTHOR]