Racemization study on different N-acetylamino acids by a recombinant N-succinylamino acid racemase from Geobacillus kaustophilus CECT4264
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Pozo Dengra, Joaquín; Martínez Gómez, Ana Isabel; Martínez Rodríguez, Sergio; Clemente Jiménez, Josefa María; Rodríguez Vico, Felipe; [et al.]Date
2009Abstract
N-Succinylamino acid racemase (NSAAR) with N-acylamino acid racemase (NAAAR) activity together with a D- or L-aminoacylase allows the total transformation of N-acetylamino acid racemic mixtures into optically pure D- or L-amino acids, respectively. In this work we have cloned and expressed the Nsuccinylamino acid racemase gene from the thermophilic Bacillus-related species Geobacillus kaustophilus CECT4264 in Escherichia coli BL21 (DE3). G. kaustophilus NSAAR (GkNSAAR) was purified in a one-step procedure by immobilized cobalt affinity chromatography and showed an apparent molecular mass of 43 kDa in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 150 kDa, suggesting that the native enzyme is a homotetramer. Optimum reaction conditions for the purified enzyme were 55 8C and pH 8.0, using N-acetyl-Dmethionine
as substrate. GkNSAAR showed a gradual loss of activity at preincubation temperatures over 60 8C, suggesting that it is thermo...
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Amino acids
N-Acetylamino acids
N-Succinylamino acid racemase
N-Acylamino acid racemase
Enzyme characterization
Geobacillus kaustophilus