Cloning and sequencing of the Thermoanaerobacterium saccharolyticum B6A-RI apu gene and purification and characterization of the amylopullulanase from Escherichia coli

Abstract

The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an Mr of 142,000 + 2,000 and had the same catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase.

Recombinant bacterial amylopullulanases

“…66 The half-lives of the full-length and truncated G. thermoleovorans NP33 amylopullulanase were 30 min and 75 min, respectively for the α-amylase activity and 20 min and 30 min for the pullulanase activity at 90 °C. A 20% loss in the enzyme activity was attained in the amylopullulanase from T. saccharolyticum B6A-RA1 on incubation at 65 °C for 1 h. 11 The α-amylasepullulanase from C. thermohydrosulfuricum DSM 3783 retained 60% of the enzyme activity, even after 2 h at 85 °C. 21 The L. plantarum L137 amylopullulanase was found stable at pH 2.5-6.5 and was reported to be less stable above neutral pH in comparison with its C-terminal truncated variant.…”

“…KSM-1378 amylopullulanase, the putative catalytic triads were identified as Asp550-Glu579-Asp645 for the amylase activity and Asp1464-Glu1493-Asp1581 for the pullulanase activity. 16 The amylopullulanases of T. ethanolicus 39E 12 and T. saccharolyticum B6A-RI 11 have Asp597-Glu626-Asp703 and Asp594-Asp700-Glu623 as catalytic triads, respectively for amylase and pullulanase activities.…”

“…Site directed mutagenesis of the three catalytic amino acid residues to amides has led to complete loss of both α-amylase end of the barrel β-strands by using hydrophobic cluster analysis (HCA) in case of amylopullulanases from Bacillus sp KSM-1378, 16 T. ethanolicus 39E, 12 and T. saccharolyticum B6A-RI. 11 HCA is a 2-D illustration of the amino acid sequences of the protein and is used for comparing the shapes and relative location of hydrophobic clusters in proteins. The shape of hydrophobic clusters predicts the secondary structure of the protein and the similarity of the clusters suggests the similarity in the polypeptide folding of the proteins.…”

“…Amylopullulanases are classified into two subgroups based on the number of active sites within the protein. 9 The thermophilic anaerobes produce amylopullulanases possessing a single active site, [10][11][12] while amylopullulanases from aerobic microbes contain either one 13,14 or two active sites for hydrolyzing α-1,4 and α-1,6-glycosidic linkages. 8,16 Due to its bifunctionality, the enzyme is also referred to as α-amylase-pullulanase based on the presence of two active sites, each being specific for one bond type.…”

Purification and biochemical characterization of pullulanase type I from Thermus caldophilus GK-24

“…2). This sequence shows some similarity only to pullulanases from Thermo- anaerobacterium saccharolyticum B6A-RI [27], T. thermosulfurigens EM 1 [28], Thermoanaerobacter ethanolicus 39E [29], K. pneumoniae ATCC 1505 [30] and B. flavocaldarius [31], but not to the other enzymes mentioned above. Ala-Pro (positions l-2), Asn (51, and ILe-Gly (9-10) sequences coincide with sequences from the noted pullulanases.…”