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Purification Studies on a Thermo-active Amidase of Geobacillus pallidus BTP-5x MTCC 9225 Isolated from Thermal Springs of Tatapani (Himachal Pradesh)

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Abstract

An intracellular aliphatic amide degrading inducible thermo-active amidase produced by Geobacillus pallidus BTP-5x MTCC 9225 was purified to apparent homogeneity using anion exchange and gel filtration chromatography, giving a yield of 6.7 % and a specific activity of 30.49 units mg−1. The purified protein migrated as a single band of estimated molecular mass of 158 kDa (homo-tetramer) in 8 % polyacrylamide gel electrophoresis and ∼38.5 kDa in 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Optima of pH and temperature varied widely in broad pH range (pH 6–9) and temperature range (45–70 °C). The purified amidase was stable up to 6 h at 50 °C, with a t 1/2 of 7 h at 55 °C. The multimeric nature of the holozyme (tetramer) contributed to protection of the enzyme against thermal denaturation. The enzyme showed resistance to metal chelating agents (EDTA, 8-hydroxyquinoline, and sodium azide), explaining its non-metallic nature, and is strongly inhibited by thiol reagents that means cysteine is involved in catalysis. The amidase of G. pallidus BTP-5x preferentially hydrolyzed only small aliphatic amides and has a narrow substrate spectrum. The K M value for acrylamide is 10.54 mM, V max 45.19 μmol−1 min−1 mg−1 protein, and k cat 4.29 min−1. The sequence of amino acids of the purified enzyme MRHGDISSSHDTVGI appears similar to thermophilic amidases. Sequence analysis of the amidase gene showed that the enzyme is 347 amino-acid-long with a molecular weight of 38.4 kDa (as observed in SDS-PAGE), theoretical pI 5.38, and show strong similarity to thermostable amidases, possessing unique restriction sites.

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Acknowledgments

We thank the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing financial assistance in the form of Senior Research Fellowship to Ms. Monica Sharma and Mr. Nitya Nand Sharma and N-terminal sequencing facility at the Institute of Microbial Technology (IMTECH), Sector 39A, Chandigarh (India).

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Authors and Affiliations

  1. Department of Biotechnology, Himachal Pradesh University, Summer Hill, 171005, Shimla, Himachal Pradesh, India

    Monica Sharma, Nitya Nand Sharma & Tek Chand Bhalla

  2. Department of Biotechnology, Delhi Technological University, Main Bawana Road, Shahbad Daulatpur, Delhi, 110042, India

    Monica Sharma

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  1. Monica Sharma
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  2. Nitya Nand Sharma
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  3. Tek Chand Bhalla
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Correspondence to Tek Chand Bhalla.

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Sharma, M., Sharma, N.N. & Bhalla, T.C. Purification Studies on a Thermo-active Amidase of Geobacillus pallidus BTP-5x MTCC 9225 Isolated from Thermal Springs of Tatapani (Himachal Pradesh). Appl Biochem Biotechnol 169, 1–14 (2013). https://doi.org/10.1007/s12010-012-9945-8

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