title: Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3.
abstract: In the present study, we report the identification of a new gene from the Bacillus subtilis B3 strain (aatB3), which comprises 1308 bp encoding a 436 amino acid protein with a monomer molecular weight of 49.1 kDa. Phylogenetic analyses suggested that this enzyme is a member of the Ib subgroup of aspartate aminotransferases (AATs; EC 2.6.1.1), although it also has conserved active residues and thermostability characteristic of Ia-type AATs. The Asp232, Lys270 and Arg403 residues of AATB3 play a key role in transamination. The enzyme showed maximal activity at pH 8.0 and 45 °C, had relatively high activity over an alkaline pH range (pH 7.0-9.0) and was stable up to 50 °C. AATB3 catalyzed the transamination of five amino acids, with L-aspartate being the optimal substrate. The K(m) values were determined to be 6.7 mM for L-aspartate, 0.3 mM for α-ketoglutarate, 8.0 mM for L-glutamate and 0.6 mM for oxaloacetate. A 32-residue N-terminal amino acid sequence of this enzyme has 53% identity with that of Bacillus circulans AAT, although it is absent in all other AATs from different organisms. Further studies on AATB3 may confirm that it is potentially beneficial in basic research as well as various industrial applications.
authors.affiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China.
authors.forename: Hui-Jun
authors.initials: HJ
authors.lastname: Wu
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.forename: Shuai
authors.initials: S
authors.lastname: Wang
authors.forename: Jun-Qing
authors.initials: JQ
authors.lastname: Qiao
authors.forename: Yan-Fei
authors.initials: YF
authors.lastname: Xia
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Wei-Duo
authors.initials: WD
authors.lastname: Wang
authors.forename: Sheng-Feng
authors.initials: SF
authors.lastname: Gao
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Peng-Qi
authors.initials: PQ
authors.lastname: Xue
authors.forename: Xue-Wen
authors.initials: XW
authors.lastname: Gao
mesh_terms: D000595:Amino Acid Sequence; D001219:Aspartate Aminotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D001219:Aspartate Aminotransferases