Gene cloning and characterization of arylamine N-acetyltransferase from Bacillus cereus strain 10-L-2

摘要

Bacillus cereus strain 10-L-2 synthesizes two arylamine N-acetyltransferases(Nat-a and Nat-b)with broad substrate specificities toward aniline and its derivatives.In southern blot analysis using probes encoding the NH2-terminus of Nat-b and a conserved region of N-acetyltransferases,digested total DNA of strain 10-L-2 showed one positive band.We cloned and sequenced the gene encoding Nat-b.The NH2-terminal amino acid sequence predicted from the open reading frame(768 base pairs)corresponded to that of purified Nat-b.The cloned Nat-b gene was expressed in Escherichia coli.The expressed enzyme(BcNAT)from the recombinant strain was partially purified and characterized.Nat-b from strain 10-L-2 and BcNAT from the recombinant strain were slightly different from each others in substrate specificity and thermostability.We examined the biotransformations of 2-aminophenols and phenylenediamines by the whole cells of the recombinant strain.The cells converted these compounds into their corresponding acetanilides.Only one amino group of phenylenediamines was acetylated.The cells utilized 4-nitroacetanilide as an acetyl donor instead of acetyl-CoA.4-Aminoacetanilide was produced and 4-nitroaniIine was released almost stoichiometrically.