RESUMEN
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.
Asunto(s)
Bacillales/enzimología , Leucina-Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Bacillales/genética , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Concentración de Iones de Hidrógeno , Leucina-Deshidrogenasa/química , Leucina-Deshidrogenasa/genética , Leucina-Deshidrogenasa/aislamiento & purificación , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
OBJECTIVE: To make the previously developed biosynthesis of L-2-aminobutyric acid (L-ABA) more suitable for the industrial-scale production. RESULTS: 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. CONCLUSION: The recyclable biotransformation system is promising to fulfill industrial requirements for L-ABA production.