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Biotechnology and Applied Biochemistry (2001) 33, (189–199) (Printed in Great Britain)

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

Jen-Tao Chen, Ming-Chu Chen, Li-Lin Chen and Wen-Shen Chu¹

Food Industry Research and Development Institute, P.O. Box 246, Hsinchu 30099, Taiwan, Republic of China

Abbreviations used: IPTG, isopropyl b-D-thiogalactoside; ORF, open reading frame.

¹ To whom correspondence should be addressed (e-mail cws@firdi.org.tw).

The nucleotide sequences of apuTS and orfX and the flanking regions reported here have been deposited in the GenBank Nucleotide Sequence database under the accession number AF272660.

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.

Received 8 January 2001/18 February 2001; accepted 26 February 2001

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