Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli

Liu, Weiming; Ma, Hongmin; Jian, Luo; Shen, Wenhe; Xu, Xing‐You; Li, Shuang; Hu, Yi; Huang, He

doi:10.1016/j.bej.2014.08.003

Cited by 44 publications

(33 citation statements)

References 34 publications

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“…The lowest conversion, about 1.8% even at 72th hour, was achieved without the addition of NADH, demonstrating that intracellular NADH amount in E. coli cells was not sufficient for preparative conversion. This result was in agreement with previous reports .…”

Section: Discussionsupporting

confidence: 94%

“…It was proved that the reaction rate became fast with the loss of the substrate concentration and the substrate concentration would be increased if the reaction time is extended. These phenomena could be explained by the inhibitory effect due to higher substrate concentration, so high concentration of substrate is improper for this type of reaction system . After the reaction reaches the maximum value, the l ‐ tert ‐leucine concentration had a small decrease in the four‐reaction system.…”

Section: Discussionmentioning

confidence: 99%

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A cold‐adapted leucine dehydrogenase from marine bacterium Alcanivorax dieselolei: Characterization and l‐tert‐leucine production

Jiang

Sun

et al. 2015

Engineering in Life Sciences

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l‐tert‐leucine, an intermediate in the synthesis of several chiral drugs, is mainly produced by bioconversion, in which leucine dehydrogenase (LeuDH) is the key enzyme. A novel leudh was obtained from the marine bacterium Alcanivorax dieselolei B‐5(T) by PCR. The gene encoded a novel cold‐adapted LeuDH that showed low similarity (less than 50%) to any known proteins; the highest similarity (42.6%) was found for LeuDH from Bacillus cereus. The cold‐adapted LeuDH showed optimal activity at 30℃ and pH 6.5, and was identified to be extremely cold‐adaptive, retaining over 90% activity in the temperature range of 0–37℃. The enzyme exhibited better stability in weak alkali environment (pH 6.0–8.5) than Thermoactinomyces intermedius LeuDH. The best substrate concentration was established, and LeuDH conversion rate in catalyzing trimethylpyruvic acid to l‐tert‐leucine was 54.6%. The cold activity and its ability to produce l‐tert‐leucine with excellent performance of enantiomers of choice make it a promising biocatalyst for industrial application under extreme conditions.

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Section: Discussionsupporting

confidence: 94%

Section: Discussionmentioning

confidence: 99%

A cold‐adapted leucine dehydrogenase from marine bacterium Alcanivorax dieselolei: Characterization and l‐tert‐leucine production

Jiang

Sun

et al. 2015

Engineering in Life Sciences

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“…In order to efficiently evaluate the stability of variants, Cu 2+ was used to promote oxidation of cysteine residues. The determination of substrate and l - tert -leucine levels was performed on an Agilent LC1260 high-performance liquid chromatography (HPLC) system as described by Liu et al (41). All assays were performed in triplicate.…”

Section: Methodsmentioning

confidence: 99%

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

Zheng

Yang

Zhou

et al. 2017

Appl Environ Microbiol

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NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10Cfdh) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10Cfdh, which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances.IMPORTANCE FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application.

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“…The results showed that the increasing of substrate concentration would in uence the initial catalytic rate and yield of L-tle synthesized by GDH-R3-LeuDH (Fig. 6e), for the substrate concentration is an important parameter in enzymatic reactions and it could in uence the full utilization of enzyme activity or result in substrate inhibition [4,35].…”

Section: Discussionmentioning

confidence: 99%

“…Due to the tremendous application in marketed drugs, chemical and enzymatic methods of producing Ltert-leucine (L-tle), which is widely used as a chiral building block in the synthesis of anti-tumor and antivirus drugs [1,2], such as atazanavir, telaprevir and boceprevir, have been developed rapidly. However, the disadvantages were signi cant and pervasive existed in most chiral l-tert chemical catalysis producing process, including energy-consuming, environmentally-unfriendly, and low conversion and enantioselectivity [3,4]. The greener biocatalysts, such as leucine dehydrogenase [5], branched chain aminotransferase [6], amidases [7], and proteases [8], penicillinyl enzymes [9], lipases [10], have been developed and applied in L-tle enzymatically producing process over the past few decades.…”

Section: Introductionmentioning

confidence: 99%

Construction and Characterization of a Novel Glucose Dehydrogenase-Leucine Dehydrogenase Bifunctional Enzyme for the Biosynthesis of L-Tert-Leucine

Liao

Zhang

Wang

et al. 2020

Preprint

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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 bifunctional 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 yeild of L-tle by GDH-R3-LeuDH was all enhanced by 2-fold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/d in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30℃, 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 bifunctional enzyme for the efficient biosynthesis of L-tle.

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Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli

Cited by 44 publications

References 34 publications

A cold‐adapted leucine dehydrogenase from marine bacterium Alcanivorax dieselolei: Characterization and l‐tert‐leucine production

A cold‐adapted leucine dehydrogenase from marine bacterium Alcanivorax dieselolei: Characterization and l‐tert‐leucine production

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

Construction and Characterization of a Novel Glucose Dehydrogenase-Leucine Dehydrogenase Bifunctional Enzyme for the Biosynthesis of L-Tert-Leucine

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