title: Discovery of a mandelonitrile hydrolase from Bradyrhizobium japonicum USDA110 by rational genome mining.
abstract: A mandelonitrile hydrolase bll6402 from Bradyrhizobium japonicum USDA110 was predicted by rational genome mining, i.e. combining traditional genome mining with functional analysis of the genetic organization of the putative nitrilase gene within the chromosome of microorganisms. This putative gene was cloned and over-expressed in Escherichia coli, and the encoded protein was purified to give a nitrilase with a molecular mass of about 37kDa. The molecular weight of the holoenzyme was about 455kDa, suggesting that nitrilase bll6402 self-aggregated to the active form with native structure being 12 subunits of identical size. This nitrilase was most active toward mandelonitrile with V(max) and K(m) for mandelonitrile being 44.7U/mg and 0.26mM, respectively. The k(cat) and overall catalytic efficiency k(cat)/K(m) were 27.0s(-1) and 1.04x10(5)M(-1)s(-1), indicating that nitrilase bll6402 is very active for the hydrolysis of mandelonitrile to mandelic acid. Nitrilase bll6402 also effectively hydrolyzed several mandelonitrile derivatives.
authors.affiliation: Department of Chemistry, Southern Methodist University, Dallas, TX 75275, USA.
authors.forename: Dunming
authors.initials: D
authors.lastname: Zhu
authors.forename: Chandrani
authors.initials: C
authors.lastname: Mukherjee
authors.forename: Edward R
authors.initials: ER
authors.lastname: Biehl
authors.forename: Ling
authors.initials: L
authors.lastname: Hua
mesh_terms: D000619:Aminohydrolases / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020369:Bradyrhizobium / Q000201:enzymology*; D003001:Cloning, Molecular; D017931:DNA Primers; D005784:Gene Amplification; D016680:Genome, Bacterial; D007700:Kinetics; D016366:Open Reading Frames
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D000619:Aminohydrolases; C522996:mandelonitrile hydrolase, Bradyrhizobium japonicum; C021867:nitrilase