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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-tert-leucine 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. How to cite Wang L, Zhu W, Gao Z, et al. Biosynthetic L-tert-leucine using Escherichia coli co-expressing a novel NADH-dependent leucine dehydrogenase and a formate dehydrogenase. Electron J Biotechnol 2020;47. https://doi.org/10.1016/j.ejbt.2020.07.001

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 Km and kcat 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.

Background l-2-aminobutyric acid (l-ABA) is an unnatural amino acid that is a key intermediate for the synthesis of several important pharmaceuticals. To make the biosynthesis of l-ABA environmental friendly and more suitable for the industrial-scale production. We expand the nature metabolic network of Escherichia coli using metabolic engineering approach for the production of l-ABA. Results In this study, Escherichia coli THR strain with a modified pathway for threonine-hyperproduction was engineered via deletion of the rhtA gene from the chromosome. To redirect carbon flux from 2-ketobutyrate (2-KB) to l-ABA, the ilvIH gene was deleted to block the l-isoleucine pathway. Furthermore, the ilvA gene from Escherichia coli W3110 and the leuDH gene from Thermoactinomyces intermedius were amplified and co-overexpressed. The promoter was altered to regulate the expression strength of ilvA* and leuDH. The final engineered strain E. coli THR ΔrhtAΔilvIH/Gap-ilvA*-Pbs-leuDH was able to produce 9.33 g/L of l-ABA with a yield of 0.19 g/L/h by fed-batch fermentation in a 5 L bioreactor. Conclusions This novel metabolically tailored strain offers a promising approach to fulfill industrial requirements for production of l-ABA. Electronic supplementary material The online version of this article (10.1186/s12934-019-1095-z) contains supplementary material, which is available to authorized users.

Thermostability is a key factor in the industrial and clinical application of enzymes, and understanding mechanisms of thermostability is valuable for molecular biology and enzyme engineering. In this chapter, we focus on the thermostability of leucine dehydrogenase (LDH, EC 1.4.1.9), an amino acid-metabolizing enzyme that is an NAD+-dependent oxidoreductase which catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH from Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has already been applied to quantify the concentration of BCAAs in biological specimens. However, the molecular mechanism of its thermostability had been unknown because no high-resolution structure was available. Here, we discuss the thermostability of GstLDH on the basis of its structure determined by cryo-electron microscopy. Sequence comparison with other structurally characterized LDHs (from Lysinibacillus sphaericus and Sporosarcina psychrophila) indicated that non-conserved residues in GstLDH, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric stability through intermolecular interactions between protomers. Furthermore, NAD+ binding to GstLDH increased the thermostability of the enzyme as additional intermolecular interactions formed on cofactor binding. This knowledge is important for further applications and development of amino acid metabolizing enzymes in industrial and clinical fields.

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, EsldhK72A, fdh and optimized expression through fine-tuning RBS intensity, so that the yield of E. coli BL21/pET28a-R3ivlA-EsldhK72A-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.

The general overview aimed to increase the current knowledge interactions between dehydrogenase enzymes and nanoparticles, and introduce dehydrogenases for industrial and health purposes. Nanoparticles (NPs) are particles constituting from 1 to 100 nm based on their size with a surrounding interfacial layer. Nanoparticle-Protein interactions include covalent and non-covalent attachments. Several dehydrogenase enzymes (e.g., alcohol dehydrogenase, lactate dehydrogenase, alanine dehydrogenase, glutamate dehydrogenase, leucine dehydrogenase, phenylalanine dehydrogenase, and malate dehydrogenase) are used for immobilization by nanoparticles. Such as magnetic nanoparticles and quantum dots, represent attractive model systems for biological enzyme assemblies and design of bioanalytical sensors. Further, bioconjugation of nanoparticles with dehydrogenase enzymes has broad applications in biocatalysis and nanomedicine for drug discovery. However, studies on the characterization of nanoparticle-enzyme complexes accept apparent that the anatomy and action of enzymes are afflicted by the chemistry of nanoparticle ligand, size, actual, and labeling methods. Moreover, the nanoparticle-protein conjugation revealed increased/decreased enzymatic activity due to nanoparticle features. Thus, this work reviewed the findings of nanoparticle-enzyme interactions for nanotechnology applications and conjugation techniques. We also highlight several challenges associated with the nanoparticle-enzyme interactions, including stability and reusability of the enzymes in nanoparticle-enzyme formation.

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.

Among many genes encoding for amino acid dehydrogenase, a novel leucine dehydrogenase gene from Exiguobacterium sibiricum (EsiLeuDH) was isolated by using genome mining strategy. EsiLeuDH was overexpressed in Escherichia coli BL21 (DE3), followed by purification and characterization. The high thermostability of the enzyme confers its half-life up to 14.7 h at 50°C. Furthermore, the substrate specificity shows a broad spectrum, including many L-amino acids and aliphatic α-keto acids, especially some aryl α-keto acids. This enzyme coupled with recombinant formate dehydrogenase (FDH) was used to catalyze trimethylpyruvic acid (TMP) through reductive amination to generate enantiopure L-tert-leucine (L-Tle). In order to overcome the substrate inhibition effect, a fed-batch feeding strategy was adopted to transform up to 0.8 M of TMP to L-Tle, with an average conversion rate of 81% and L-Tle concentration of 65.6 g⋅L–1. This study provides a highly efficient biocatalyst for the synthesis of L-Tle and lays the foundation for large-scale production and application of chiral non-natural amino acids.

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 H2O2 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.

L-2-aminobutyric acid (L-ABA) as a precursor for the anticonvulsant and the antituberculotic is a key intermediate in the chemical and pharmaceutical industries. Recently, leucine dehydrogenase (LeuDH) with NAD+ regeneration was developed for L-ABA production on a large scale. Previously, the L-ABA yield was improved by optimizing conversion conditions, including cofactor regeneration and enzyme immobilization but not protein engineering on LeuDH due to lacking an applicable high-throughput screening (HTS) method. Recently, an HTS assay was developed by us, which enables researchers to engineer LeuDH in a relatively short period of time. Herein, a semirational engineering was performed on LeuDH to increase the catalytic efficiency of BcLeuDH. Firstly, the structure of wild-type (WT) BcLeuDH was modeled and seven potentially beneficial positions were selected for mutation. Five beneficial variants were then identified from the seven site-saturation mutagenesis (SSM) libraries by HTS and confirmed by rescreening via amino acid analyzer. The "best" variant M5 (WT + Q358N) showed 44.5-fold higher catalytic efficiency (k cat/K M) than BcLeuDH WT, which suggested that BcLeuDH M5 is an attractive candidate for L-ABA production on a large scale. Furthermore, the structure-functional relationship was investigated based on the docking and kinetic results.

Bacillus licheniformis has been regarded as an outstanding microbial cell factory for the production of biochemicals and enzymes. Due to lack of genetic tools to repress gene expression, metabolic engineering and gene function elucidation are limited in this microbe. In this study, an integrated CRISPR interference (CRISPRi) system was constructed in B. licheniformis. Several endogenous genes, including yvmC, cypX, alsD, pta, ldh, and essential gene rpsC, were severed as the targets to test this CRISPRi system, and the repression efficiencies were ranged from 45.02 to 94.00%. Moreover, the multiple genes were simultaneously repressed with high efficiency using this CRISPRi system. As a case study, the genes involved in by-product synthetic and L-valine degradation pathways were selected as the silence targets to redivert metabolic flux toward L-valine synthesis. Repression of acetolactate decarboxylase (alsD) and leucine dehydrogenase (bcd) led to 90.48% and 80.09 % increases in L-valine titer, respectively. Compared with the control strain DW9i△leuA (1.47 g/L and 1.79 g/L), the L-valine titers of combinatorial strain DW9i△leuA/pHYi-alsD-bcd were increased by 1.27-fold and 2.89-fold, respectively, in flask and bioreactor. Collectively, this work provides a feasible approach for multiplex metabolic engineering and functional genome studies of B. licheniformis.

Microaerobic cultivation conditions have been shown experimentally and theoretically to improve the performance of a number of bioproduction systems. However, under these conditions, the production of l-valine by Escherichia coli is decreased mainly because of a redox cofactor imbalance and a decreased l-glutamate supply. The synthesis of one mole of l-valine from one mole of glucose generates two moles of NADH via glycolysis but consumes a total of two moles of NADPH, one in the ketol-acid reductoisomerase (KARI) reaction and the other in the regeneration of l-glutamate as an amino group donor for the branched-chain amino acid aminotransferase (BCAT) reaction. The improvement of l-valine synthesis under oxygen deprivation may be due to solving these problems. Increased l-valine synthesis under oxygen deprivation conditions was previously shown in Corynebacterium glutamicum (Hasegawa et al., 2012). In this study, we have proposed the use of NADH-dependent leucine dehydrogenase (LeuDH; EC 1.4.1.9) Bcd from B. subtilis instead of the native NADPH-dependent pathway including aminotransferase encoded by ilvE to improve l-valine production in E. coli under microaerobic conditions. We have created l-valine-producing strains on the base of the aminotransferase B-deficient strain V1 (B-7 ΔilvBN ΔilvIH ΔilvGME::PL-ilvBNN17KDA) by introducing one chromosomal copy of the bcd gene or the ilvE gene. Evaluation of the l-valine production by the obtained strains under microaerobic and aerobic conditions revealed that leucine dehydrogenase Bcd had a higher potential for l-valine production under microaerobic conditions. The Bcd-possessing strain exhibited 2.2-fold higher l-valine accumulation (up to 9.1 g/L) and 2.0-fold higher yield (up to 35.3%) under microaerobic conditions than the IlvE-possessing strain. The obtained results could be interpreted as follows: an altering of redox cofactor balance in the l-valine biosynthesis pathway increased the production and yield by E. coli cells under microaerobic conditions. Thus, the effective synthesis of l-valine by means of "valine fermentation" was shown in E. coli. This methodology has the advantages of being an economical and environmentally friendly process.

Leucine dehydrogenase (LDH) and formate dehydrogenase (FDH) were assembled together based on a high-affinity interaction between two different cohesins in a miniscaffoldin and corresponding dockerins in LDH and FDH. The miniscaffoldin with two enzymes was further absorbed by regenerated amorphous cellulose (RAC) to form a bifunctional enzyme complex (miniscaffoldin with LDH and FDH adsorbed by RAC, RSLF) in vitro. The enzymatic characteristics of the bifunctional enzyme complex and free enzymes mixture were systematically compared. The synthesis of L-tert-leucine by the RSLF and free enzyme mixture were compared under different concentrations of enzymes, coenzyme, and substrates. The initial L-tert-leucine production rate by RSLF was enhanced by 2-fold compared with that of the free enzyme mixture. Ninety-one grams per liter of L-tert-leucine with an enantiomeric purity of 99 % e.e. was obtained by RSLF multienzyme catalysis. The results indicated that the bifuntional enzyme complex based on cohesin-dockerin interaction has great potential in the synthesis of L-tert-leucine.

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 cat/K m) 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.

l-tert-leucine and its derivatives are useful as pharmaceutical active ingredients, in which leucine dehydrogenase (LeuDH) is the key enzyme in their enzymatic conversions. In the present study, a novel cold-adapted LeuDH, psleudh, was cloned from psychrotrophic bacteria Pseudoalteromonas sp. ANT178, which was isolated from Antarctic sea-ice. Bioinformatics analysis of the gene psleudh showed that the gene was 1209 bp in length and coded for a 42.6 kDa protein containing 402 amino acids. PsLeuDH had conserved Phe binding site and NAD+ binding site, and belonged to a member of the Glu/Leu/Phe/Val dehydrogenase family. Homology modeling analysis results suggested that PsLeuDH exhibited more glycine residues, reduced proline residues, and arginine residues, which might be responsible for its catalytic efficiency at low temperature. The recombinant PsLeuDH (rPsLeuDH) was purified a major band with the high specific activity of 275.13 U/mg using a Ni-NTA affinity chromatography. The optimum temperature and pH for rPsLeuDH activity were 30 &deg, C and pH 9.0, respectively. Importantly, rPsLeuDH retained at least 40% of its maximum activity even at 0 &deg, C. Moreover, the activity of rPsLeuDH was the highest in the presence of 2.0 M NaCl. Substrate specificity and kinetic studies of rPsLeuDH demonstrated that l-leucine was the most suitable substrate, and the catalytic activity at low temperatures was ensured by maintaining a high kcat value. The results of the current study would provide insight into Antarctic sea-ice bacterium LeuDH, and the unique properties of rPsLeuDH make it a promising candidate as a biocatalyst in medical and pharmaceutical industries.

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.

Background Optically pure d-lactic acid (≥ 99%) is an important precursor of polylactic acid. However, there are relatively few studies on d-lactic acid fermentation compared with the extensive investigation of l-lactic acid production. Most lactic acid producers are mesophilic organisms. Optically pure d-lactic acid produced at high temperature not only could reduce the costs of sterilization but also could inhibit the growth of other bacteria, such as l-lactic acid producers. Results Thermophilic Bacillus coagulans is an excellent producer of l-lactic acid with capable of growing at 50 °C. In our previous study, the roles of two l-lactic acid dehydrogenases have been demonstrated in B. coagulans DSM1. In this study, the function of another annotated possible l-lactate dehydrogenase gene (ldhL3) was verified to be leucine dehydrogenase with an activity of 0.16 units (μmol/min) per mg protein. Furthermore, the activity of native d-lactate dehydrogenase was too low to support efficient d-lactic acid production, even under the control of strong promoter. Finally, an engineered B. coagulans D-DSM1 strain with the capacity for efficient production of d-lactic acid was constructed by deletion of two l-lactate dehydrogenases genes (ldhL1 and ldhL2) and insertion of the d-lactate dehydrogenase gene (LdldhD) from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 at the position of ldhL1. Conclusions This genetically engineered strain produced only d-lactic acid under non-sterilized condition, and finally 145 g/L of d-lactic acid was produced with an optical purity of 99.9% and a high yield of 0.98 g/g. This is the highest optically pure d-lactic acid titer produced by a thermophilic strain. Electronic supplementary material The online version of this article (10.1186/s12934-017-0827-1) contains supplementary material, which is available to authorized users.

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-rbs 4 leudh-3fdh A10C was finally regulated to 2:1, which was the optimal one determined by enzyme-catalyzed synthesis. The catalyst activity of E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C 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.

In recent years, the reductive amination of ketones in the presence of amine dehydrogenases emerged as an attractive synthetic strategy for the enantioselective preparation of amines starting from ketones, an ammonia source, a reducing reagent and a cofactor, which is recycled in situ by means of a second enzyme. Current challenges in this field consists of providing a broad synthetic platform as well as process development including enzyme immobilization. In this contribution these issues are addressed. Utilizing the amine dehydrogenase EsLeuDH-DM as a mutant of the leucine dehydrogenase from Exigobacterium sibiricum, a range of aryl-substituted ketones were tested as substrates revealing a broad substrate tolerance. Kinetics as well as inhibition effects were also studied and the suitability of this method for synthetic purpose was demonstrated with acetophenone as a model substrate. Even at an elevated substrate concentration of 50 mM, excellent conversion was achieved. In addition, the impact of water-miscible co-solvents was examined, and good activities were found when using DMSO of up to 30% (v/v). Furthermore, a successful immobilization of the EsLeuDH-DM was demonstrated utilizing a hydrophobic support and a support for covalent binding, respectively, as a carrier. (C) 2017 Elsevier Ltd. All rights reserved.

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.

Enantiopure l -tert-leucine ( l -Tle) was synthesized through reductive amination of trimethylpyruvate catalyzed by cell-free extracts of recombinant Escherichia coli coexpressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH). The leudh gene from Lysinibacillus sphaericus CGMCC 1.1677 encoding LeuDH was cloned and coexpressed with NAD+-dependent FDH from Candida boidinii for NADH regeneration. The batch reaction conditions for the synthesis of l -Tle were systematically optimized. Two substrate feeding modes (intermittent and continuous) were addressed to alleviate substrate inhibition and thus improve the space-time yield. The continuous feeding process was conveniently performed in water at an overall substrate concentration up to 1.5 M, with both conversion and ee of >99% and space-time yield of 786 g L−1 d−1, respectively. Furthermore, the preparation was successfully scaled up to a 1 L scale, demonstrating the developed procedure showed a great industrial potential for the production of enantiopure l -Tle.

Immobilization of enzymes onto nanoparticles and retention of their structure and activity, which may be related to the orientation of enzymes on nanoparticles, remain a challenge. Here, we developed a novel enzyme-orientation strategy to enhance the activity of formate dehydrogenase immobilized on polydopamine-coated iron oxide nanoparticles via site-directed mutation. Seven mutants were constructed based on homology modeling of formate dehydrogenase and immobilized on polydopamine-coated iron oxide nanoparticles to investigate the influence of these mutations on immobilization. The immobilized mutant C242A/C275V/C363V/K389C demonstrated the highest immobilization yield and retained 90% of its initial activity, which was about 3-fold higher than that of wild-type formate dehydrogenase. Moreover, co-immobilization of formate dehydrogenase and leucine dehydrogenase was performed for the synthesis of l-tert-leucine. The catalytic efficiency of the co-immobilized mutant C242A/C275V/C363V/K389C and leucine dehydrogenase increased by more than 4-fold compared to that of co-immobilized wild-type formate dehydrogenase and leucine dehydrogenase.

A leucine dehydrogenase from Exiguobacterium sibiricum ( Es LeuDH) was discovered by genome mining approach. The Es LeuDH was overexpressed in Escherichia coli BL21, purified to homogeneity and characterized. This enzyme showed good thermostability with a half-life of 3.1 h at 60 °C. Furthermore, Es LeuDH has a broad spectrum of substrate specificity, showing activities toward many aliphatic α -keto acids and L -amino acids, in addition to some aryl α -keto acids and aryl α -amino acids, such as α -oxobenzeneacetic and l -phenylglycine. The Es LeuDH was successfully coexpressed with Bacillus megaterium glucose dehydrogenase ( Bm GDH) in Escherichia coli BL21 for the production of l - tert -leucine. By using the coexpressed whole cells, a decagram preparation of l - tert -leucine was performed at a substrate concentration of 0.6 M (78.1 g L −1 ) in 1 L scale with 99% conversion after 5.5 h, resulting in 80.1% yield and > 99% ee (enantiomeric excess).

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 &plusmn, 0.17 g&middot, L&minus, 1 by strain &Delta, LtbR-acetohydroxyacid isomeroreductase (AHAIR)M/ABNCME, and the concentrations of the by-products (l-valine and l-alanine) increased, compared to the strain &Delta, LtbR/ABNCE. Strain &Delta, LtbR-AHAIRMLeuDH/ABNCMLDH accumulated 22.87&plusmn, 0.31 g&middot, 1 l-leucine, but showed a drastically low l-valine accumulation (from 8.06 &plusmn, 0.35 g&middot, 1 to 2.72 &plusmn, 0.11 g&middot, 1), in comparison to strain &Delta, LtbR-AHAIRM/ABNCME, which indicated that LeuDH has much specificity for l-leucine synthesis but not for l-valine synthesis. Subsequently, the resultant strain &Delta, LtbR-AHAIRMLeuDHRocG/ABNCMLDH accumulated 23.31 &plusmn, 0.24 g&middot, 1 l-leucine with a glucose conversion efficiency of 0.191 g&middot, g&minus, 1.

To reduce the unpleasant odor during 1-deoxynojirimycin (DNJ) production, the genes of leucine dehydrogenase (bcd) and phosphate butryltransferase (ptb) were deleted from Bacillus amyloliquefaciens HZ-12, and the concentrations of branched-chain short fatty acids (BCFAs) and DNJ were compared.By knockout of the ptb gene, 1.01 g BCFAs kgHZ-12Δptb had decreased BCFAs formation but also maintained the stable DNJ yield, which contributed to producing DNJ-rich products with decreased unpleasant smell.

l-tert-Leucine (l-Tle) and its derivatives are extensively used as crucial building blocks for chiral auxiliaries, pharmaceutically active ingredients, and ligands. Combining with formate dehydrogenase (FDH) for regenerating the expensive coenzyme NADH, leucine dehydrogenase (LeuDH) is continually used for synthesizing l-Tle from α-keto acid. A multilevel factorial experimental design was executed for research of this system. In this work, an efficient optimization method for improving the productivity of l-Tle was developed. And the mathematical model between different fermentation conditions and l-Tle yield was also determined in the form of the equation by using uniform design and regression analysis. The multivariate regression equation was conveniently implemented in water, with a space time yield of 505.9 g L−1 day−1 and an enantiomeric excess value of >99 %. These results demonstrated that this method might become an ideal protocol for industrial production of chiral compounds and unnatural amino acids such as chiral drug intermediates.

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 340 kDa. 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 40 min, 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 Km values for l -leucine and NAD+ at 20 °C were 0.65 and 0.015 mM, 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.

A potential thermotolerant L-leucine dehydrogenase from Laceyella sacchari (Ls-LeuDH) was over-expressed in E. coli, purified and characterized.Ls-LeuDH had excellent thermostability with a specific activity of 183 U/mg at pH 10.5 and 25 °C. It retained a high activity in 200 mM carbonate buffer from pH 9.5 to 11. The optimal temperature for Ls-LeuDH was 60 °C.It is the first time that a thermostable and highly active LeuDH originating from L. sacchari has been characterized. It may be useful for medical and pharmaceutical applications.

Two types of extracellular proteases with molecular mass of 50.0 and 44.8 kDa were found in H10 enzymes partially purified from Bacillus cereus H10. Further identification using liquid chromatography-tandem mass spectrometry, the enzyme with 50.0 kDa was identified as being similar to leucine dehydrogenase; while the enzyme with 44.8 kDa might be a novel keratinolytic enzyme with little similarity to other proteins. To maximize the keratinolytic and proteolytic abilities in the H10 enzymes, a combination of response surface methodology and sequential quadratic programming technique was used to study the hydrolytic pH and temperature. Results showed that the H10 enzymes could produce optimal proteolytic and keratinolytic activities at a hydrolysis temperature of 59°C at pH 7.57. Testing the protease activity on various protein substrates and temperatures indicated that the H10 enzymes showed high thermal stability and were very effective in porcine hair.

Our group has developed a stereospecific enzymatic method, which is very efficient for the in vitro synthesis of l-[(15)N]serine, l-[(15)N]methionine and l-[(15)N]glutamic acid. These amino acids were prepared from the corresponding alpha -ketoacids in the suitable enzymatic systems. The bacterial NAD-dependent amino acid dehydrogenases alanin dehydrogenase, leucin dehydrogenase and glutamate dehydrogenase were used as catalysts. Glucose dehydrogenase was used for the regeneration of NADH and (15)NH(4)Cl as isotopically labelled material at 99 at.% (15)N. All reactions are inexpensive and easy to perform on a synthetically useful scale (1-10g) giving high yields of l-amino acids. The (15)N isotope content was determined by mass spectrometry.

We have developed an efficient stereospecific enzymatic synthesis of L-[15N]-valine, L-[15N]-leucine, L-[15N]-norvaline, L-[15N]-norleucine and L-[15N]-isoleucine from the corresponding α-keto acids by coupling the reactions catalysed by leucine dehydrogenase and glucose dehydrogenase/galactose mutarotase. Giving high yields of L-amino acids, the procedure is economical and easy to perform and to monitor at a synthetically useful scale (1–10 g). Copyright © 2008 John Wiley & Sons, Ltd.

L-tert-Leucine is an unnatural amino acid that is a key intermediate for the synthesis of several important drugs. The L-tert-Leucine synthesis can be performed continuously by the collaboration of leucine dehydrogenase and formate dehydrogenase. In this study, recombinant strains of Escherichia coli expressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH), respectively, and the strain co-expressing the two enzymes were constructed. The activity for the two enzymes of the cell extraction from different recombinant strains was determined. L-tert-Leucine was successfully synthesized by the recombinant strains, and the yield in different conditions was compared. The production of L-tert-Leucine was the highest when cell extraction of strains containing pLeuDH and pFDH, 1 mL cells extract could produce 4.5 mg L-tert-Leucine, while 1 mL whole cells could only produce 1.05 mg L-tert-Leucine. The yield of L-tert-Leucine was 3.375 mg/mL cell extraction of the strain containing pLeuDHFDH when NAD was added, while the yield fell to 1.635 mg/mL when the whole cell was used.

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.

The α and β subunits of group II chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1, were produced in Escherichia coli . Thermococcus KS-1 α and β chaperonins were purified from a crude cell extract by heat treatment and subsequent chromatographic purification in the presence and absence of Mg 2+ to produce hexadecameric and monomeric form, respectively. The monomeric α and β subunits were able to form homo-hexadecamers in the presence of Mg 2+ . In the absence of ATP, the α and β homo-hexadecamers arrested the refolding of guanidine hydrochloride-denatured Bacillus stearothermophilus leucine dehydrogenase (LeuDH) and Thermus flavus malate dehydrogenase (MDH), which were released by the addition of ATP at 50–65 °C. In the presence of ATP, the α and β homo-hexadecamers facilitated the refolding of LeuDH and MDH. The α homo-hexadecamer showed greater complex stability and greater ability to facilitate the refolding of enzymes than the β homo-hexadecamer. On the other hand, both the α and β monomers facilitated the refolding of the proteins in the absence of ATP. Thermococcus KS-1 chaperonin homo-hexadecamers and monomers could both therefore be used as molecular tools in biotechnology.

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.

Through comparison with the high-resolution structure of Clostridium symbiosum glutamate dehydrogenase, the different substrate specificities of the homologous enzymes phenylalanine dehydrogenase and leucine dehydrogenase were attributed to two residues, glycine 124 and leucine 307, in Bacillus sphaericus phenylalanine dehydrogenase, which are replaced with alanine and valine in leucine dehydrogenases [Britton, K.L., Baker, P.J., Engel, P.C., Rice, D.W. & Stillman, T.J. (1993) J. Mol. Biol.234, 938–945]. As predicted, making these substitutions in phenylalanine dehydrogenase decreased the specific activity towards aromatic substrates and enhanced the activity towards some aliphatic amino acids in standard assays with fixed concentrations of both substrates [Seah, S.Y.K., Britton, K.L., Baker, P.J., Rice, D.W., Asano, Y. & Engel, P.C. (1995) FEBS Lett.370, 93–96]. This study did not, however, distinguish effects on affinity from those on maximum catalytic rate. A fuller kinetic characterization of the single- and double-mutant enzymes now reveals that the extent of the shift in specificity was underestimated in the earlier study. The maximum catalytic rates for aromatic substrates are reduced for all the mutants, but, in addition, the apparent Km values are higher for the single-mutant G124A and double-mutant G124A/L307V compared with the wild-type enzyme. Conversely, specificity constants (kcat/Km) for the nonpolar aliphatic amino acids and the corresponding 2-oxoacids for the mutants are all markedly higher than for the wild type, with up to a 40-fold increase for l-norvaline and a 100-fold increase for its 2-oxoacid in the double mutant. In some cases a favourable change in Km was found to outweigh a smaller negative change in kcat. These results emphasize the risk of misjudging the outcome of protein engineering experiments through too superficial an analysis. Overall, however, the success of the predictions from molecular modelling indicates the usefulness of this strategy for engineering new specificities, even in advance of more detailed 3D structural information.

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 k cat 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.

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.

Leucine dehydrogenase ( l -leucine: NAD+ oxidoreductase, deaminating, EC 1.4.1.9) was purified to homogeneity from the crude extract of an alkaliphilic halophile, Natronobacterium magadii MS-3, with a yield of 16%. The enzyme had a molecular mass of about 330 kDa and consisted of six subunits identical in molecular mass (55 kDa). The enzyme required a high concentration of salt for stability and activity. It retained the full activity after heating at 50 °C for 1 h and about 50% activity after being kept at 30 °C for 2 months in the presence of 2.5 M NaCl. The enzyme required NAD+ as a coenzyme and showed maximum activity in the presence of more than 3 M salt, as CsCl, RbCl, NaCl, or KCl. In addition to l -leucine, l -valine and l -isoleucine were also good substrates in the oxidative deamination. In the reductive amination, 2-keto analogs of branched-chain amino acids were substrates. The Michaelis constants were 0.69 mM for l -leucine, 0.48 mM for NAD+, 4.0 mM for 2-ketoisocaproate, 220 mM for ammonia, and 0.02 mM for NADH in the presence of 4 M NaCl. The Km for l -leucine depended on the concentration of salt and increased with decreasing salt concentration. The N. magadii enzyme was unique in its halophilicity among leucine dehydrogenases studied so far.

Homology-based modeling of phenylalanine dehydrogenases (PheDHs) from various sources, using the structures of homologous enzymes Clostridium symbiosum glutamate dehydrogenase and Bacillus sphaericus leucine dehydrogenase as a guide, revealed that an asparagine residue at position 145 of B. sphaericus PheDH was replaced by valine or alanine in PheDHs from other sources. This difference was proposed to be the basis for the poor discrimination by the B. sphaericus enzyme between the substrates L-phenylalanine and L-tyrosine. Residue 145 of this enzyme was altered, by site-specific mutagenesis, to hydrophobic residues alanine, valine, leucine, and isoleucine, respectively. The resultant mutants showed a high discrimination, above 50-fold, between L-phenylalanine and L-tyrosine. This higher specificity toward L-phenylalanine was due to K(m) values for L-phenylalanine lowered more than 20-fold compared to the values for L-tyrosine. The greater specificity for L-phenylalanine in the wild-type Bacillus badius enzyme, which has a valine residue in the corresponding position, was also found to be largely due to a lower K(m) for this substrate. Activities were also measured with a range of six amino acids with aliphatic, nonpolar side chains, and with the corresponding oxoacids, and in all cases the specificity constants for these substrates were increased in the mutant enzymes. As with phenylalanine, these increases are mainly attributable to large decreases in K(m) values.

We created a novel chimeric amine dehydrogenase (AmDH) via domain shuffling of two parent AmDHs (‘L- and F-AmDH’), which in turn had been generated from leucine and phenylalanine DH, respectively. Unlike the parent proteins, the chimeric AmDH (‘cFL-AmDH’) catalyzes the amination of acetophenone to (R)-methylbenzylamine and adamantylmethylketone to adamantylethylamine.

A strategy for scaffold-free self-assembly of multiple oligomeric enzymes was developed by exploiting enzyme oligomerization and protein–protein interaction properties, and was tested both in vitro and in vivo. Octameric leucine dehydrogenase and dimeric formate dehydrogenase were fused to a PDZ (PSD95/Dlg1/zo-1) domain and its ligand, respectively. The fusion proteins self-assembled into extended supramolecular interaction networks. Scanning-electron and atomic-force microscopy showed that the assemblies assumed two-dimensional layer-like structures. A fluorescence complementation assay indicated that the assemblies were localized to the poles of cells. Moreover, both in vitro and in vivo assemblies showed higher NAD(H) recycling efficiency and structural stability than did unassembled structures when applied to a coenzyme recycling system. This work provides a novel method for developing artificial multienzyme supramolecular devices and for compartmentalizing metabolic enzyme cascades in living cells.

In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH. The S380A mutant shows much lower activity than wild-type GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards L-methionine, L-norleucine and L-norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH. Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used L-aspartate and L-leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher K(m ) values for glutamate/2-oxoglutarate at pH 7.0, but a similar k(cat)/K(m) value and lower K(m) at pH 8.0, and a nearly 22-fold lower S(0.5) (substrate concentration giving half-saturation under conditions where Michaelis-Menten kinetics does not apply) at pH 9.0. Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100-1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially L-norleucine and L-methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported. At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate k(cat) and K(m) separately, but for reductive amination the additional mutations have no significant effect on k(cat) at pH 7, and the increase in catalytic efficiency is entirely attributable to the measured decrease in K(m). At pH 8 the enhancement of catalytic efficiency with the novel substrates was much more striking (e.g. for norleucine approximately 2000-fold compared with wild-type or the K89L mutant), but it was not established whether this is also exclusively due to more favourable Michaelis constants.

X-ray crystallographic studies revealed that various amino acid dehydrogenases fold into two domains in each subunit, a substrate-binding domain and an NAD(P)+-binding domain (Baker, P. J., Turnbull, A. P., Sedelnikova, S. E., Stillman, T. J., and Rice, D. W. (1995) Structure 3, 693–705). To elucidate the function and folding process of these two domains, we have genetically constructed a fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus consisting of an N-terminal polypeptide fragment corresponding to the substrate-binding domain including an N-terminus, and a C-terminal fragment corresponding to the NAD+-binding domain. The two peptide fragments were expressed in separate host cells and purified. When both fragments were mixed, the leucine dehydrogenase activity with a specific activity of 1.4% of that of the wild-type enzyme appeared. This suggests that both peptide fragments mutually recognize each other, associate and fold correctly to be catalytically active, although the activity is low. However, the fragmentary form of enzyme produced catalyzed the oxidative deamination of l -leucine, l -isoleucine, and l -valine with broad substrate specificity compared to that of the wild-type enzyme. The fragmentary enzyme retained more than 75% of the initial activity after heating at 50°C for 60 min. The fragmentary enzyme was more stable on heating than separate peptide fragments. These results suggest that the two domains of leucine dehydrogenase probably fold independently, and the two peptide fragments interact and associate with each other to form a functional active site.

The catabolism of branched chain amino acids, especially valine, appears to play an important role in furnishing building blocks for macrolide and polyether antibiotic biosyntheses. To determine the active site residues of ValDH, we previously cloned, partially characterized, and identified the active site (lysine) of Streptomyces albus ValDH. Here we report further characterization of S. albus ValDH. The molecular weight of S. albus ValDH was determined to be 38 kDa by SDS-PAGE and 67 kDa by gel filtration chromatography indicating that the enzyme is composed of two identical subunits. Optimal pHs were 10.5 and 8.0 for dehydrogenase activity with valine and for reductive amination activity with α-ketoisovaleric acid, respectively. Several chemical reagents, which modify amino-acid side chains, inhibited the enzyme activity. To examine the role played by the residue for enzyme specificity, we constructed mutant ValDH by substituting alanine for glycine at position 124 by site-directed mutagenesis. This residue was chosen because it has been considered to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). The Ala-124–Gly mutant enzyme displayed lower activities toward aliphatic amino acids, but higher activities toward L-phenylalanine, L-tyrosine, and L-methionine compared to the wild type enzyme suggesting that Ala-124 is involved in substrate binding in S. albus ValDH.

A tri-enzyme sensor was developed for the flow-injection determination of branched chain amino acids ( L -valine, L -leucine and L -isoleucine). Leucine dehydrogenase, NADH oxidase and peroxidase were coimmobilized covalently on tresylate-hydrophilic vinyl polymer beads and packed into transparent PTFE tube (20 cm×1.0 i.d.), which was used as flow cell. The calibration graph was linear for 30 nM–5 μM; the detection limit (signal-to-noise=3) was 10 nM. The sampling rate was 25 h −1 without carryover. The sensor was stable for two weeks. The sensor system was applied to the determination of branched chain amino acid in plasma.

The l -leucine dehydrogenase gene from Bacillus cereus (DSM 626) was cloned from a partial genomic library and sequenced. The open reading frame has 1101 bp and codes for a protein of 39.9 kDa. The deduced amino acid sequence of the LeuDH from B. cereus shares 70–80% identity with LeuDH's from the thermophilic strains B. stearothermophilus and Thermoactinomyces intermedius . The active protein was overexpressed in Escherichia coli to yield approximately 30% of the total soluble protein.

NAD + and NADP + were retained in continuously operated enzyme membrane reactors by nanofiltration membranes with retention rates between 0.86–0.98. For lower molecular weight substrates and products, retention rates below 0.35 were found. For the reductive amination of trimethylpyruvate to l - tert -leucine, the reactor was stably operated for 10 days. The total turnover number could be increased 3.4 times to values higher than 7,900 by this approach.