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

Abstract

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.

Comparative Genomics of Two Closely Related Unicellular Thermo-Acidophilic Red Algae, Galdieria sulphuraria and Cyanidioschyzon merolae, Reveals the Molecular Basis of the Metabolic Flexibility of Galdieria sulphuraria and Significant Differences in Carbohydrate Metabolism of Both Algae

“…In addition, G. sulphuraria also possesses an amylopullulanase that is able to catalyze the hydrolysis of both a-(1,6)-and a-(1,4)-glycosidic linkages. This enzyme has not previously been reported for plants, but only for bacteria (Mathupala et al, 1993;Ara et al, 1995;Chen et al, 2001). Moreover, G. sulphuraria possesses an additional glucan-(1,4)-a-glucosidase that releases terminal b-Glc units from the nonreducing ends of the glucan.…”

Domain evolution in the GH13 pullulanase subfamily with focus on the carbohydrate-binding module family 48

“…KSM-1378 (Hatada et al 1996) and Bifidobacterium breve (Ryan et al 2006) and the amylopullulanases from Bacillus sp. XAL 601 (Lee et al 1994) and Geobacillus stearothermophilus (Chen et al 2001).…”

A High Molecular-Mass Anoxybacillus sp. SK3-4 Amylopullulanase: Characterization and Its Relationship in Carbohydrate Utilization

“…An identical stretch of 9 tandem repeats of PGSGTT was found in G. stearothermophilus TS-23 Apu [11]. The Apu of Bacillus sp.…”

“…XAL601 (224 kDa) [10], G. stearothermophilus TS-23 (220 kDa) [11], Bacillus sp. KSM-1378 (210 kDa) [7], and L. plantarum L137 (211 kDa) [19], Anoxybacillus ApuASK (225 kDa) is among the few Apus that exhibited high molecular-mass (Table 1).…”