Gene cloning and characterization of arylamine N-acetyltransferase from Bacillus cereus strain 10-L-2 Shinji Takenaka, 1 Minyi Cheng, 2 Mulyono, 2 Atsushi Koshiya, 1 Shuichiro Murakami, 1 and Kenji Aoki 1, ⁎ Department of Applied Biological Chemistry, Graduate School of Agricultural Science, Kobe University,1-1 Rokkodai, Nada-ku, Kobe, 657-8501, Japan 1 and Division of Biosystems Chemistry, Graduate School of Science and Technology, Kobe University,1-1 Rokkodai, Nada-ku, Kobe, 657-8501, Japan 2 Received 22 April 2008; accepted 1 September 2008 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 NH 2 -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 NH 2 -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 thermo-stability. 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-nitroaniline was released almost stoichiometrically. © 2008, The Society for Biotechnology, Japan. All rights reserved. [Key words: Bacillus cereus; Aniline; Aminophenols; Phenylenediamines; Acetanilides; Arylamine N-acetyltransferase; Acetylation; 4-nitroacetanilide] Known arylamine N-acetyltransferases (EC 2.3.1.5, NAT) catalyze the transfer of an acetyl group from acetyl-CoA to the amino group of hydrazine, arylamine drugs, and carcinogens. NATs of humans, mice, and intestinal microorganisms have been characterized (1, 2). Human NAT I possibly plays a role in the activation of various carcinogens (3) and in the metabolism of the folate catabolite p-aminobenzoylgluta- mate (4). In the environment, bacterial NAT has the potential of acetylating and thereby detoxifying various xenobiotic substances containing an amino group (5, 6). The acetyltransferases from Sal- monella typhimurium (7) and Pseudomonas aeruginosa (8) have been characterized in detail. PaNAT from P. aeruginosa has been crystallized and analyzed by X-ray diffraction. The enzyme showed broad substrate specificity toward anilines with a substituent group at the C4 position (9). Bacillus anthracis RTC50 synthesizes multiple NATs, and recently, three putative NAT genes and their products BanatA, BanatB, and BanatC were characterized (10). SgNAT from Streptomyces griseus with high activity toward aminophenols has also been characterized (11). We have comprehensively studied the metabolic fate of mono- or di-substituted anilines (12, 13) and have isolated two strains of Ba- cillus cereus, PDa-1 and 10-L-2, capable of metabolizing phenylene- diamine with NAT (14, 15). The acetyltransferases from both strains could play a role in detoxification. We have purified and characterized two arylamine N-acetyltransferases, Nat-a and Nat-b, from strain 10- L-2. The enzymes have similar pH- and thermo-stabilities, pH and temperature optima, and are affected similarly by several reagents. Both enzymes (i) convert a wide variety of anilines to their corresponding acetanilides, (ii) act on polycyclic amines, and (iii) acetylate only one amino group of phenylenediamine. In this study reported here, we cloned and expressed Nat-b in Escherichia coli. The expressed enzyme (BcNAT) was characterized, to compare with the properties of Nat-b from strain 10-L-2. We also examined the biotransformation of aminophenols and phenylenediamines in more detail and determined the acetyl donor by the whole cells of the recombinant strain. MATERIALS AND METHODS Bacterial strains, plasmids, and culture conditions B. cereus strain 10-L-2 was cultured in 4-phenylenediamine medium (15). Escherichia coli XL-1 Blue was used for plasmid maintenance and construction. The vector pBluescript KS(+) and pGEM-T Easy were used for cloning. E. coli was cultivated in Luria–Bertani (LB) medium supplemented, if necessary, with ampicillin (100 μg/ml), isopropyl-β-D-thiogalactopyranoside (IPTG, 0.2 mM), and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal, 0.04%) at 37 °C with shaking. The B. cereus Nat-b gene was expressed in E. coli XL-1 Blue as follows. The recombinant strain was grown in 70 ml LB medium containing ampicillin (200 μg/ml) at 30 °C with shaking at 140 rpm; when the OD 66 0 nm reached 0.5, IPTG (0.5 mM) was added, and the culture was further incubated for 12 h with shaking. Cells were harvested and used for enzyme assays, purification, or whole cell reactions. Journal of Bioscience and Bioengineering VOL. 107 No. 1, 27 – 32, 2009 www.elsevier.com/locate/jbiosc ⁎ Corresponding author. Tel.: +81 78 803 5891; fax: +81 78 882 0481. E-mail address: kaoki@kobe-u.ac.jp (K. Aoki). 1389-1723/$ - see front matter © 2008, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2008.09.012