Structure and expression of an amylopullulanase gene from Bacillus stearothermophilus TS-23

An amylopullulanase gene (apuTS) from Bacillus stearothermophilus TS-23 was cloned and characterized. apuTS consisted of an open reading frame of 6054 bp encoding a protein of 2018 amino acids with a calculated M_r of 223811. The deduced amino acid sequence revealed four highly conserved regions that are common among amylolytic enzymes. In the C-terminal region, a six-amino-acid sequence (Pro-Gly-Ser-Gly-Thr-Thr) is repeated nine times. It shared the highest degree of homology with the amylopullulanase of Bacillus sp. XAL601. The enzyme also had moderate homology with amylopullulanases from thermophilic anaerobic bacteria. Low levels of homology were observed between the ApuTS of B. stearothermophilus TS-23 and amylopullulanases of Pyrococcus abyssi Orsay, P. furiosus and Bacillus sp. KSM1378. When the intact coding region of apuTS was expressed in Escherichia coli under the control of the lac promoter, the product was degenerate, as revealed by amylase activity staining after SDS/PAGE. The largest active polypeptide had an M_r of about 220000, while the smallest one had an M_r of about 105000. Upstream of the apuTS gene, a gene orfX was fortuitously cloned. The putative OrfX protein was weakly related to the myosin heavy chain. It was predicted to contain a central, 179-residue-long, coiled-coil domain.