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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

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.

In this study, Escherichia coli BL21 (DE3) was used as the host to construct 2 recombinant E. coli strains that co-expressed leucine dehydrogenase (LDH, Bacillus cereus)/formate dehydrogenase (FDH, Ancylobacter aquaticus), or leucine dehydrogenase (LDH, Bacillus cereus)/alcohol dehydrogenase (ADH, Rhodococcus), respectively. L-2-aminobutyric acid was then synthesized by L-threonine deaminase (L-TD) with LDH-FDH or LDH-ADH by coupling with two different NADH regeneration systems. LDH-FDH process and LDH-ADH process were optimized and compared with each other. The optimum reaction pH of LDH-FDH process was 7.5, and the optimum reaction temperature was 35 °C. After 28 h, the concentration of L-2-aminobutyric acid was 161.8 g/L with a yield of 97%, when adding L-threonine in batches for controlling 2-ketobutyric acid concentration less than 15 g/L and using 50 g/L ammonium formate, 0.3 g/L NAD+, 10% LDH-FDH crude enzyme solution (V/V) and 7 500 U/L L-TD. The optimum reaction pH of LDH-ADH process was 8.0, and the optimum reaction temperature was 35 °C. After 24 h, the concentration of L-2-aminobutyric acid was 119.6 g/L with a yield of 98%, when adding L-threonine and isopropanol (1.2 times of L-threonine) in batches for controlling 2-ketobutyric acid concentration less than 15 g/L, removing acetone in time and using 0.3 g/L NAD⁺, 10% LDH-ADH crude enzyme solution (V/V) and 7 500 U/L L-TD. The process and results used in this paper provide a reference for the industrialization of L-2-aminobutyric acid.文中以大肠杆菌 BL21(DE3) 为宿主，构建两株分别共表达亮氨酸脱氢酶 (LDH，来源蜡样芽孢杆菌)/甲酸脱氢酶 (FDH，来源水生弯杆菌) 和亮氨酸脱氢酶 (LDH，来源蜡样芽孢杆菌)/醇脱氢酶 (ADH，来源红球菌)的重组大肠杆菌。通过偶联两种不同NADH 再生体系，以L-苏氨酸为起始原料，利用苏氨酸脱氨酶 (L-TD) 与LDH-FDH或LDH-ADH 一锅法合成L-2-氨基丁酸，并对LDH-FDH 工艺和LDH-ADH 工艺进行对比优化。LDH-FDH 工艺的最适反应pH 为7.5，最适反应温度为35 ℃，通过加入50 g/L 甲酸铵、0.3 g/L NAD⁺、10% LDH-FDH 粗酶液 (V/V)和7 500 U/L 的L-TD 酶液，对L-苏氨酸进行分批补加，以便控制2-丁酮酸浓度小于15 g/L，反应28 h，实现了L-2-氨基丁酸的产量为161.8 g/L，产率97%。LDH-ADH 工艺的最适pH 为8.0，最适反应温度为35 ℃，通过加入0.3 g/LNAD+、10% LDH-ADH 粗酶液 (V/V) 及7 500 U/L 的L-TD 酶液，分批补加L-苏氨酸及1.2 倍摩尔量异丙醇，以便控制2-丁酮酸浓度小于15 g/L，且每生成约40 g/L 的L-2-氨基丁酸，抽真空去除丙酮，反应24 h，实现了L-2-氨基丁酸的产量为119.6 g/L，产率98%。文中所采用的工艺及结果可为L-2-氨基丁酸的工业化提供一定的参考依据。.

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.L-2-氨基丁酸 (L-ABA) 是一种重要的化工原材料和手性医药中间体，为了实现L-ABA 的高效生产， 本研究在大肠杆菌Escherichia coli BL21 (DE3) 中分别表达大肠杆菌来源的苏氨酸脱氨酶 (Threonine deaminase，TD)、苏云金芽孢杆菌来源的亮氨酸脱氢酶 (Leucine dehydrogenase，LDH) 和博伊丁假丝酵母来源的甲酸脱氢酶 (Formate dehydrogenase，FDH)，构建体外级联酶催化反应实现L-苏氨酸向L-ABA 的转化，体系中TD、LDH 和 FDH 添加最适比例为 1∶1∶0.2。为了简化生产工艺，将 3 种酶在一株菌 E. coli 3FT+L 中共表达并实现上述配比，在30 L 发酵罐中用E. coli 3FT+L 全细胞转化12 h，L-ABA 的产量为68.5 g/L，底物L-苏氨酸的摩尔转化率达到99.0%。该工艺路线绿色高效，为未来大规模生产L-ABA 提供借鉴。.

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.

Formate dehydrogenase (FDH) is a d -2‑hydroxy acid dehydrogenase, and catalyzes the oxidation of formate to carbon dioxide, coupled with reduction of NAD+ to NADH that plays a key role in the process of NADH regeneration. In order to obtain high activity formate dehydrogenase mutants, the formate dehydrogenase CbFDHC23S was used as the parent to conduct two rounds of directed evolution, and a mutant M2 was obtained which specific activity was about 4 times more than the parent and was more suitable for coenzyme regeneration under physiological conditions. Then, the molecular mechanism of temperature characteristic and catalytic efficiency change was preliminarily elucidated by computer-aided method. Finally, an engineered E. coli strain was established to co-express formate dehydrogenase and l -leucine dehydrogenase and enantioselectively transform phenylglyoxylic acid to give l - phenylglycine (e.e. >99%), the yield and space-time yield of l - phenylglycine can reach 90.46% and 82.07 g·L−1·d−1. This study laid a theoretical foundation for the green biosynthesis of food additives such as chiral alcohols and amino acid derivatives catalyzed by FDH coupling to enhance the regeneration capacity of NADH, reduce the regeneration cost of NADH, and achieve high efficiency and low cost.

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.

Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD

Different co-expression strategies to express leucine dehydrogenase and glucose dehydrogenase in E. coli were done to observe the effect of expression of different enzyme. A recombinant strain with two high enzyme activities was built for efficiently asymmetric synthesis of L-tert-leucine.The leucine dehydrogenase (ldh) from Bacillus cereus and glucose dehydrogenase (gdh) from Bacillus sp. were co-expressed by three different strategies, including co-expressing two genes in single vector, co-expressing two genes in two vectors and expressing fusion protein. The catalytic efficiencies of recombinant strains with different enzyme activity ratio in different modes of biocatalyst were compared to produce L-tert-leucine from its corresponding α-keto acids.Different co-expression strategies displayed a slight impact on leucine dehydrogenase expression, whereas, a greater impact on glucose dehydrogenase. All the activity of leucine dehydrogenase was normally expressed, but the fusion proteins lost the activity of glucose dehydrogenase. Besides, the activity of glucose dehydrogenase was also totally inhibited when the 6-histidine tag was fused at C termini, which indicated the additional 6-histidine tag considerately depressed the glucose dehydrogenase activity. After optimization of expression, three recombinant strains exhibiting high enzyme activity and different enzyme activity ratio were used to synthesis L-tert-leucine in the mode of cell-free extracts and whole-cell. Result displayed a great influence on the catalytic efficiencies resulted from the mode of catalyst and enzyme activity. When the cell-free crude culture broth of E. coli BL21/pET28a-L-SD-AS-G coexpressing two genes in single vector was used as biocatalyst, 15 g/L cell loading and 0.1 mmol/L NAD+ were enough to completely transform 0.5 mol/L trimethylpyruvate into L-tert-leucine.The recombinant strain with high activities of leucine dehydrogenase and glucose dehydrogenase was achieved by co-expressing two genes in single vector without histidine tag in E. coli and L-tert-leucine was efficiently produced with this recombinant strain.

To make the previously developed biosynthesis of L-2-aminobutyric acid (L-ABA) more suitable for the industrial-scale production.A recyclable biotransformation system was developed based on immobilized enzyme technology. The conversion yield of L-threonine (at 90 g l(-1)) reached 99.9 % and the theoretical yield of L-ABA reached more than 90 % using the optimized biotransformation system by the individual immobilization of threonine deaminase and the co-immobilization of L leucine dehydrogenase and formate dehydrogenase. 90 g L-threonine l(-1) was converted to 73.9 g L-ABA l(-1)95 % theoretical yield, within 120-145 min in 30 batch transformation experiments.The recyclable biotransformation system is promising to fulfill industrial requirements for L-ABA production.

In the present paper, the reductive amination of α-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 × 10−3 A/M) and reproducibility with an RSD of 8.4% (n = 4). The experimental determination of ammonium ion was confirmed by standard Berthelot sp...

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.

A strategy for constructing a reusable multi-enzyme supramolecular device was developed by reprogramming protein-protein interactions and disulfide bond locking. The resultant multi-enzyme supramolecular device demonstrated good reusability, and approximately 80% of its initial catalytic activity was retained even after eight cycles of reuse.

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.