Abstract：
:N-Succinyl-amino acid racemase (NSAAR), long referred to as N-acyl- or N-acetyl-amino acid racemase, is an enolase superfamily member whose biotechnological potential was discovered decades ago, due to its use in the industrial dynamic kinetic resolution methodology first known as "Acylase Process". In previous works, an extended and enhanced substrate spectrum of the NSAAR from Geobacillus kaustophilus CECT4264 toward different N-substituted amino acids was reported. In this work, we describe the cloning, purification, and characterization of the NSAAR from Geobacillus stearothermophilus CECT49 (GstNSAAR). The enzyme has been extensively characterized, showing a higher preference toward N-formyl-amino acids than to N-acetyl-amino acids, thus confirming that the use of the former substrates is more appropriate for a biotechnological application of the enzyme. The enzyme showed an apparent thermal denaturation midpoint of 77.0 ± 0.1 °C and an apparent molecular mass of 184 ± 5 kDa, suggesting a tetrameric species. Optimal parameters for the enzyme activity were pH 8.0 and 55-65 °C, with Co(2+) as the most effective cofactor. Mutagenesis and binding experiments confirmed K166, D191, E216, D241, and K265 as key residues in the activity of GstNSAAR, but not indispensable for substrate binding.
journal_name
Mol Biotechnoljournal_title
Molecular biotechnologyauthors
Soriano-Maldonado P,Andújar-Sánchez M,Clemente-Jiménez JM,Rodríguez-Vico F,Las Heras-Vázquez FJ,Martínez-Rodríguez Sdoi
10.1007/s12033-015-9839-4subject
Has Abstractpub_date
2015-05-01 00:00:00pages
454-65issue
5eissn
1073-6085issn
1559-0305journal_volume
57pub_type
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