Racemization study on different N-acetylamino acids by a recombinant N-succinylamino acid racemase from Geobacillus kaustophilus CECT4264

• Published in: Process biochemistry (1991) Vol. 44; no. 8; pp. 835 - 841
• Main Authors: Pozo-Dengra, Joaquín, Martínez-Gómez, Ana Isabel, Martínez-Rodríguez, Sergio, Clemente-Jiménez, Josefa María, Rodríguez-Vico, Felipe, Heras-Vázquez, Francisco Javier Las
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01.08.2009
• Subjects: Amino acids; Enzyme characterization; Escherichia coli; Geobacillus; Geobacillus kaustophilus; N-Acetylamino acids; N-Acylamino acid racemase; N-Succinylamino acid racemase; Amino acids; N-Succinylamino acid racemase; Geobacillus kaustophilus; N-Acetylamino acids; N-Acylamino acid racemase; Enzyme characterization
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2009.03.020

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 N-succinylamino 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 °C and pH 8.0, using N-acetyl- d-methionine as substrate. GkNSAAR showed a gradual loss of activity at preincubation temperatures over 60 °C, suggesting that it is thermostable. As activity was greatly enhanced by Co 2+, Mn 2+ and Ni 2+ but inhibited by metal-chelating agents, it is considered a metalloenzyme. The Co 2+-dependent activity profile of the enzyme was studied with no detectable inhibition at higher metal ion concentrations. GkNSAAR showed activity towards both aliphatic and aromatic N-acetylamino acids such as N-acetyl-methionine and N-acetyl-phenylalanine, respectively, with k cat/ K m values ranging from 1 × 10 3 to 9 × 10 3 s −1 M −1. Kinetic parameters were better for N-acetyl- d-amino acids than for N-acetyl- l-specific ones.