Purification and Characterization of a Noble Thermostable Alpha-amylase from Anoxybacillus tengchongensis RA1-2-1 Isolated from Geothermal Spring of Nepal

Authors

  • Parash Mani Timilsina Kathmandu University, Dhulikhel, 6250, Nepal
  • Gyanu Raj Pandey 2Biotechnological Research and Developmental Centre Pvt. Ltd., Bharatpur, Chitwan, 44200, Nepal
  • Asmita Shrestha Biotechnological Research and Developmental Centre, Bharatpur, Chitwan, 44200, Nepal
  • Manish Ojha Kathmandu University, Dhulikhel, 6250, Nepal
  • Garima Baral Kathmandu University, Dhulikhel, 6250, Nepal
  • Tika Bahadur Karki Kathmandu University, Dhulikhel, 6250, Nepal

DOI:

https://doi.org/10.3126/jfstn.v12i12.31038

Keywords:

Alpha-amylase, Enzyme Kinetics, Thermostability, Liquefaction, Starch

Abstract

A thermophilic amylolytic strain, Anoxybacillus tengchongensis RA1-2-1 was isolated from geothermal spring of Rasuwagadi district of Nepal. The BLAST alignment of the 16s rRNA sequence revealed 99.3% similarity with the type strain Anoxybacillus tengchongensis T-11. The morphological, physiological and biochemical properties were similar to the type strain. The enzyme from the strain was purified to 40-fold purification by DEAE-cellulose ion exchange chromatography. The Km value of the enzyme was 0.68±0.05 mg/ml. The optimum pH and temperature were 7.0 and 70 °C. SDS-PAGE analysis showed a single band at 69 kDa. The half-life of the enzyme at 70°C and 80°C were 85.01min and 51.96 min respectively. TLC analysis of the hydrolysis product showed that the enzyme is maltogenic amylase. The calcium independent enzyme was completely inhibited by Hg2+ but showed inhibitory effect in the range of 100 %-30 % in the presence of other salts at 1-10 mM concentrations.

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Author Biographies

Parash Mani Timilsina, Kathmandu University, Dhulikhel, 6250, Nepal

Assistant Professor, Department of Biotechnology

Gyanu Raj Pandey, 2Biotechnological Research and Developmental Centre Pvt. Ltd., Bharatpur, Chitwan, 44200, Nepal

and Department of Biotechnology, Kathmandu University, Dhulikhel, 6250, Nepal

Asmita Shrestha, Biotechnological Research and Developmental Centre, Bharatpur, Chitwan, 44200, Nepal

Researcher

Manish Ojha, Kathmandu University, Dhulikhel, 6250, Nepal

Researcher, Department of Biotechnology

Garima Baral, Kathmandu University, Dhulikhel, 6250, Nepal

Researcher, Department of Biotechnology

Tika Bahadur Karki, Kathmandu University, Dhulikhel, 6250, Nepal

Professor, Department of Biotechnology

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Published

2020-12-19

How to Cite

Timilsina, P. M., Pandey, G. R., Shrestha, A., Ojha, M., Baral, G., & Karki, T. B. (2020). Purification and Characterization of a Noble Thermostable Alpha-amylase from Anoxybacillus tengchongensis RA1-2-1 Isolated from Geothermal Spring of Nepal. Journal of Food Science and Technology Nepal, 12(12), 49–58. https://doi.org/10.3126/jfstn.v12i12.31038

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Section

Research Papers