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Chiral compounds are important drug intermediates that play a critical role in human life. Herein, we report a facile method to prepare multi-enzyme nano-devices with high catalytic activity and stability. The self-assemble molecular binders SpyCatcher and SpyTag were fused with leucine dehydrogenase and glucose dehydrogenase to produce sc-LeuDH (SpyCatcher-fused leucine dehydrogenase) and GDH-st (SpyTag-fused glucose dehydrogenase), respectively. After assembling, the cross-linked enzymes LeuDH-GDH were formed. The crosslinking enzyme has good pH stability and temperature stability. The coenzyme cycle constant of LeuDH-GDH was always higher than that of free double enzymes. The yield of l-tert-leucine synthesis by LeuDH-GDH was 0.47 times higher than that by free LeuDH and GDH. To further improve the enzyme performance, the cross-linked LeuDH-GDH was immobilized on zeolite imidazolate framework-8 (ZIF-8) via bionic mineralization, forming LeuDH-GDH @ZIF-8. The created co-immobilized enzymes showed even better pH stability and temperature stability than the cross-linked enzymes, and LeuDH-GDH@ZIF-8 retains 70% relative conversion rate in the first four reuses. In addition, the yield of LeuDH-GDH@ZIF-8 was 0.62 times higher than that of LeuDH-GDH, and 1.38 times higher than that of free double enzyme system. This work provides a novel method for developing multi-enzyme nano-device, and the ease of operation of this method is appealing for the construction of other multi-enzymes @MOF systems for the applications in the kinds of complex environment. [ABSTRACT FROM AUTHOR]

This study reported the cloning, expression, and characterization of a new salt-tolerant leucine dehydrogenase (PrLeuDH) from Pseudoalteromonas rubra DSM 6842. A codon-optimized 1038 bp gene encoding PrLeuDH was successfully expressed on pET-22b(+) in E. coli BL21(DE3). The purified recombinant PrLeuDH showed a single band of about 38.7 kDa on SDS-PAGE. It exhibited the maximum activity at 40 °C and pH 10.5, while kept high activities in the range of 25–45 °C and pH 9.5–12. The Km value and turnover number kcat for leucine of PrLeuDH were 2.23 ± 0.12 mM and 35.39 ± 0.05 s−1, respectively, resulting in a catalytic efficiency kcat/Km of 15.87 s−1/mM. Importantly, PrLeuDH remained 92.1 ± 2.67% active in the presence of 4.0 M NaCl. The study provides the first in-depth understanding of LeuDH from marine Pseudoalteromonas rubra, meanwhile the unique properties of high activity at low temperature and high salt tolerance make it a promising biocatalyst for the synthesis of non-protein amino acids and α-ketoacids under special conditions in pharmaceutical industry. [ABSTRACT FROM AUTHOR]

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

Abstract 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.

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

Leucine dehydrogenase (LeuDH, EC 1.4.1.9) can reversibly catalyze the oxidative deamination of l-leucine and some other specific α-amino acids to form the corresponding α-ketoacids. This reaction has great significance in the field of food additives and the pharmaceutical industry. The LeuDH from Exiguobacterium sibiricum ( Es LeuDH) has high catalytic efficiency but limited thermal stability, hindering its widespread industrial application. In this study, a mutant N5F/I12L/A352Y of Es LeuDH (referred to as M2) was developed with enhanced thermal stability and catalytic activity through rational modification. The M2 mutant exhibits a half-life at 60 °C ( t 1/2 (60 °C)) of 975.7 min and a specific activity of 69.6 U mg -1 , which is 5.4 and 2.1 times higher than those of Es LeuDH, respectively. This research may facilitate the utilization of Es LeuDH at elevated temperatures, enhancing its potential for industrial applications. The findings offer a practical and efficient approach for optimizing LeuDH and other industrial enzymes.

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

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

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

Leucine dehydrogenase (LDH) is a NAD+-dependent oxidoreductase, which can selectively catalyze α-keto acids to obtain α-amino acids and their derivatives. It plays a key role in the biosynthesis of L-tert-leucine (L-Tle). As a non-naturally chiral amino acid, L-Tle can be used as an animal feed additive, nutrition fortifier, which is a perspective and important building block in the pharmaceutical, cosmetic, and food additive industry. In this study, four hypothetical leucine dehydrogenases were discovered by using genome mining technology, using the highly active leucine dehydrogenase LsLeuDH as a probe. These four leucine dehydrogenases were expressed in Escherichia coli BL21(DE3), respectively, and purified to homogeneity and characterized. Compared with the other enzymes, the specific activity of PfLeuDH also shows stronger advantage. In addition, the highly selective biosynthesis of L-Tle from trimethylpyruvic acid (TMP) was successfully carried out by whole-cell catalysis using engineered E. coli cells as biocatalyst, which can efficiently coexpress leucine dehydrogenase and formate dehydrogenase. One hundred-millimolar TMP was catalyzed for 25 h, and the yield and space-time yield of L-Tle reached 87.38% (e.e. >99.99%) and 10.90 g L–1 day–1. In short, this research has initially achieved the biosynthesis of L-Tle, laying a solid foundation for the realization of low-cost and large-scale biosynthesis of L-Tle.

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

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

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.

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

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

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

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

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

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 / K ) 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. [ABSTRACT FROM AUTHOR]

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

Objectives: 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. Results: By knockout of the ptb gene, 1.01 g BCFAs kg was produced from fermented soybean by HZ-12Δ ptb. This was a 56% decrease compared with that of HZ-12 (2.27 g BCFAs kg). Moreover, no significant difference was found in the DNJ concentration (0.7 g kg). After further deletion of the bcd gene from HZ-12Δ ptb, no BCFAs was detected in fermented soybeans with HZ-12Δ ptbΔ bcd, while the DNJ yield decreased by 26% compared with HZ-12. Conclusions: HZ-12Δ ptb had decreased BCFAs formation but also maintained the stable DNJ yield, which contributed to producing DNJ-rich products with decreased unpleasant smell. [ABSTRACT FROM AUTHOR]

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

L-2-aminobutyric acid (L-2-ABA) is a chiral precursor for the synthesis of anti-epileptic drug levetiracetam and anti-tuberculosis drug ethambutol. Asymmetric synthesis of L-2-ABA by leucine dehydrogenases has been widely developed. However, the limitations of natural enzymes, such as poor stability, low catalytic efficiency, and inhibition of high-concentration substrates, limit large-scale applications. Herein, by directed screening of a metagenomic library from unnatural amino acid-enriched environments, a robust leucine dehydrogenase, TvLeuDH, was identified, which exhibited high substrate tolerance and excellent enzymatic activity towards 2-oxobutyric acid. In addition, TvLeuDH has strong affinity for NADH. Subsequently, a three-enzyme co-expression system containing L-threonine deaminase, TvLeuDH, and glucose dehydrogenase was established. By optimizing reaction conditions, 1.5 M L-threonine could be converted to L-2-ABA with a 99% molar conversion rate and a space-time yield of 51.5 g·L -1 ·h -1 . In this process, no external coenzyme was added. The robustness of TvLeuDH allowed the reaction to be performed without the addition of extra salt as the buffer, demonstrating the simplest reaction system currently reported. These unique properties for the efficient and environmentally friendly production of chiral amino acids make TvLeuDH a particularly promising candidate for industrial applications, which reveals the great potential of directed metagenomics for industrial biotechnology., (© 2023 Wiley-VCH GmbH.)

L-2-aminobutyrate (L-ABA) is an important chiral drug intermediate with a key role in modern medicinal chemistry. Here, we describe the development of an efficient method for the asymmetric synthesis of L-ABA in a tri-enzymatic cascade in Escherichia coli BL21 (DE3) using a cost-effective L-Thr. Low activity of leucine dehydrogenase from Bacillus thuringiensis (BtLDH) and unbalanced expression of enzymes in the cascade were major challenges. Mechanism-based protein engineering generated the optimal triple variant BtLDH M3 (A262S/V296C/P150M) with 20.7-fold increased specific activity and 9.6-fold increased k cat /K m compared with the wild type. Optimizing plasmids with different copy numbers regulated enzymatic expression, thereby increasing the activity ratio (0.3 : 1:0.6) of these enzymes in vivo close to the optimal ratio (0.4 : 1 : 1) in vitro. Importing the optimal triple mutant BtLDH M3 into our constructed pathway in vivo and optimization of transformation conditions achieved one-pot conversion of L-Thr to 130.2 g/L L-ABA, with 95 % conversion, 99 % e.e. and 10.9 g L -1  h -1 productivity (the highest to date) in 12 h on a 500 mL scale. These results describe a potential biosynthesis approach for the industrial production of L-ABA., (© 2023 Wiley-VCH GmbH.)

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 °C and pH 9.0, respectively. Importantly, rPsLeuDH retained at least 40% of its maximum activity even at 0 °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.

Enzymatic asymmetric synthesis of chiral amino acids has great industrial potential. However, the low catalytic efficiency of high-concentration substrates limits their industrial application. Herein, using a combination of substrate catalytic efficiency prediction based on "open to closed" conformational change and substrate specificity prediction, a novel leucine dehydrogenase (TsLeuDH), with high substrate catalytic efficiency toward benzoylformic acid (BFA) for producing l-phenylglycine (l-Phg), was directly identified from 4695 putative leucine dehydrogenases in a public database. The specific activity of TsLeuDH was determined to be as high as 4253.8 U mg -1 . Through reaction process optimization, a high-concentration substrate (0.7 m) was efficiently and completely converted within 90 min in a single batch, without any external coenzyme addition. Moreover, a continuous flow-feeding approach was designed using gradient control of the feed rate to reduce substrate accumulation. Finally, the highest overall substrate concentration of up to 1.2 m BFA could be aminated to l-Phg with conversion of >99% in 3 h, demonstrating that this new combination of enzyme process development is promising for large-scale application of l-Phg., (© 2023 Wiley-VCH GmbH.)

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. [ABSTRACT FROM AUTHOR]

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

Objective: A potential thermotolerant l-leucine dehydrogenase from Laceyella sacchari ( Ls-LeuDH) was over-expressed in E. coli, purified and characterized. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

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