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, 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, XA
L601, The enzyme also had moderate homology with amylopullulanases from the
rmophilic anaerobic bacteria. Low levels of homology were observed between
the ApuTs of B. stearothermophilus Ts-23 and amylopullulanases of Pyrococcu
s abyssi Orsay, P, furiosus and Bacillus sp, KSM1378, When the intact codin
g region of apuTS was expressed in Escherichia coli under the control of th
e lac promoter, the product was degenerate, as revealed by amylase activity
staining after SDS/PAGE, The largest active polypeptide had an M, of about
220 000, while the smallest one had an M, 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.