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Domain C of thermostable α-amylase of Geobacillus thermoleovorans mediates raw starch adsorption

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Abstract

The gene (1,542 bp) encoding thermostable Ca2+-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.

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Acknowledgments

The authors gratefully acknowledge financial assistance from the Council of Scientific and Industrial Research, New Delhi, Government of India while carrying out the work presented in the manuscript.

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  1. Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India

    Deepika Mehta & T. Satyanarayana

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  1. Deepika Mehta
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Correspondence to T. Satyanarayana.

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Mehta, D., Satyanarayana, T. Domain C of thermostable α-amylase of Geobacillus thermoleovorans mediates raw starch adsorption. Appl Microbiol Biotechnol 98, 4503–4519 (2014). https://doi.org/10.1007/s00253-013-5459-8

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