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Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens

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Abstract

Raw starch is the most abundant source of glucose in the world. Therefore, finding enzymes capable of digesting raw starch would find high industrial demand. The α-amylase gene of Bacillus amyloliquefaciens ATCC 23842 was amplified, cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme was purified to apparent homogeneity using ion exchange and gel filtration chromatography. The raw-starch digestibility of the purified enzyme was characterized by studying the hydrolysis and adsorption rate on a variety of raw starches (potato, cassava, corn, wheat and rice). The raw-starch digestion was further confirmed by scanning electron microscopy studies, which revealed an effective rate of hydrolysis. The kinetic studies revealed a relatively low K m of 2.76 mg/mL, exhibiting high affinity towards the soluble starch as the most preferred substrate and the inhibition kinetic studies revealed a high K i value (350 mM).

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Abbreviations

CBM:

carbohydrate-binding module

IPTG:

isopropyl β-D-thiogalactopyranoside

LB:

Luria Bertani

SBD:

starch-binding module

SEM:

scanning electron microscopy

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Authors and Affiliations

  1. Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR, Trivandrum, 695 019, Kerala, India

    Dhanya Gangadharan, Ashok Pandey & K. Madhavan Nampoothiri

  2. Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625 021, India

    Priya Ramachandran & Gunasekaran Paramasamy

Authors
  1. Dhanya Gangadharan
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  2. Priya Ramachandran
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  3. Gunasekaran Paramasamy
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  4. Ashok Pandey
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  5. K. Madhavan Nampoothiri
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Correspondence to K. Madhavan Nampoothiri.

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Gangadharan, D., Ramachandran, P., Paramasamy, G. et al. Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens . Biologia 65, 392–398 (2010). https://doi.org/10.2478/s11756-010-0042-6

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  • DOI: https://doi.org/10.2478/s11756-010-0042-6

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