title: Domain C of thermostable α-amylase of Geobacillus thermoleovorans mediates raw starch adsorption.
abstract: The gene (1,542 bp) encoding thermostable Ca(2+)-independent and raw starch hydrolyzing α-amylase of the extremely thermophilic bacterium Geobacillus thermoleovorans encodes for a protein of 50 kDa (Gt-amyII) with 488 amino acids. The enzyme is optimally active at pH 7.0 and 60 °C with a t 1/2 of 19.4 h at 60 and 4 h at 70 °C. Gt-amyII hydrolyses corn and tapioca raw starches efficiently and therefore finds application in starch saccharification at industrial sub-gelatinisation temperatures. The starch hydrolysis is facilitated following adsorption of the enzyme to starch at the C-terminal domain, as confirmed by the truncation analysis. The adsorption rate constant of Gt-amyII to raw corn starch is 37.6-fold greater than that for the C-terminus truncated enzyme (Gt-amyII-T). Langmuir-Hinshelwood kinetic analysis in terms of equilibrium parameter (K R) suggested that the adsorption of Gt-amyII to corn starch is more favourable than that of Gt-amyII-T. Thermodynamics of temperature inactivation indicated a decrease in thermostabilisation of Gt-amyII upon truncation of its C-terminus. The addition of raw corn starch increased t 1/2 of Gt-amyII, but it has no such effect on Gt-amyII-T. It can, therefore, be stated that Gt-amyII binds to raw corn starch via C-terminal region that contributes to its thermostability. Phylogenetic analysis confirmed that starch binding region of Gt-amyII is, in fact, the non-catalytic domain C, and not the typical SBD of CBM families. The role of domain C in raw starch binding throws light on the evolutionary path of the known SBDs.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.
authors.forename: Deepika
authors.initials: D
authors.lastname: Mehta
authors.forename: T
authors.initials: T
authors.lastname: Satyanarayana
mesh_terms: D000327:Adsorption; D004795:Enzyme Stability; D056506:Geobacillus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D002365:Manihot / Q000737:chemistry; D008970:Molecular Weight; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D003313:Zea mays / Q000737:chemistry; D000516:alpha-Amylases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D013213:Starch; D000516:alpha-Amylases