Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli |
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Institution: | 1. Laboratory of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China;2. College of Food Science and Technology, Nanchang University, Nanchang 214122, China;1. Institute of Chemical Technology and Engineering, Faculty of Chemical Technology, Poznan University of Technology, Berdychowo 4, PL-60965 Poznan, Poland;2. Department of Biotechnology and Biomedicine, DTU Bioengineering, Technical University of Denmark, Soltofts Plads 227, DK-2800 Kgs. Lyngby, Denmark |
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Abstract: | 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. |
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Keywords: | Reductive amination Biocatalysis Biotransformation Bioprocess design Optimization |
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