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The role of N1 domain on the activity, stability, substrate specificity and raw starch binding of amylopullulanase of the extreme thermophile Geobacillus thermoleovorans

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Abstract

In order to understand the role of N1 domain (1–257 aa) in the amylopullulanase (gt-apu) of the extremely thermophilic bacterium Geobacillus thermoleovorans NP33, N1 deletion construct (gt-apuΔN) has been generated and expressed in Escherichia coli. The truncated amylopullulanase (gt-apuΔN) exhibits similar pH and temperature optima like gt-apu, but enhanced thermostability. The gt-apuΔN has greater hydrolytic action and specific activity on pullulan than gt-apu. The k cat (starch and pullulan) and K m (starch) values of gt-apuΔN increased, while K m (pullulan) decreased. The enzyme upon N1 deletion hydrolyzed maltotetraose as the smallest substrate in contrast to maltopentaose of gt-apu. The role of N1 domain of gt-apu in raw starch binding has been confirmed, for the first time, based on deletion and Langmuir–Hinshelwood kinetics. Furthermore, N1 domain appears to exert a negative influence on the thermostability of gt-apu because N1 truncation significantly improves thermostability.

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Acknowledgments

The authors gratefully acknowledge financial assistance from the Department of Science and Technology, Govt. of India, New Delhi, as the PURSE grant. Thanks are also due to Council of Scientific and Industrial Research (CSIR) and Indian Council of Medical Research (ICMR), Govt. of India, for awarding fellowship to NM.

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  1. Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110 021, India

    M. Nisha & T. Satyanarayana

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  1. M. Nisha
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Correspondence to T. Satyanarayana.

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Nisha, M., Satyanarayana, T. The role of N1 domain on the activity, stability, substrate specificity and raw starch binding of amylopullulanase of the extreme thermophile Geobacillus thermoleovorans . Appl Microbiol Biotechnol 99, 5461–5474 (2015). https://doi.org/10.1007/s00253-014-6345-8

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