Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3

• Published in: The FEBS journal 2011-04, Vol.278 (8), p.1345-1357
• Main Authors: Wu, Hui-Jun, Yang, Yang, Wang, Shuai, Qiao, Jun-Qing, Xia, Yan-Fei, Wang, Yu, Wang, Wei-Duo, Gao, Sheng-Feng, Liu, Jun, Xue, Peng-Qi, Gao, Xue-Wen
• Format: Article
• Language: English
• Subjects: alpha-ketoglutaric acid; Amino Acid Sequence; amino acid sequences; aspartate aminotransferase; Aspartate Aminotransferases - genetics; Aspartate Aminotransferases - isolation & purification; Aspartate Aminotransferases - metabolism; aspartate transaminase; aspartic acid; Bacillus circulans; Bacillus subtilis; Bacillus subtilis - enzymology; Base Sequence; Cloning, Molecular; conserved active residues; Escherichia coli - metabolism; genes; genetic databases; glutamic acid; industrial applications; kinetic parameters; Kinetics; Molecular Sequence Data; molecular weight; nucleotide sequences; Phylogeny; protein sequence analysis; Recombinant Proteins - metabolism; Sequence Alignment; Substrate Specificity; thermal stability; transamination
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2011.08054.x

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 Km 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.