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Exhaustive Study of the Novel Hyper Alkalophil, Thermostable, and Chelator Resistant Metalloprotease

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Abstract

Our newly discovered metalloprotease, designated as ALP NS12 was selected using gelatin agar plates with incubation at 100 °C. Subcloning of the fragments in to pUC118 to make E. coli HB101 (pPEMP01NS) with following two-step chromatography using diethylaminoethyl sepharose (DEAE-sepharose) and Sephadex G-100 columns to purify 97-kDa expressed enzyme was performed. Although activity of immobilized ALP NS12 on glass surface was established at temperatures between 70 and 120 °C and pH ranges 4.0–13.0, the optimum temperature and pH were achieved at 100 °C and 11.0, respectively. Enhancement of enzyme activity was obtained in the presence of 5 mM MnCl2 (91 %), CaCl2 (357 %), FeCl2 (175 %), MgCl2 (94 %), ZnCl2 (412 %), NiCl (86 %), NaCl (239 %), and Na-sulfate (81 %) while inhibition was observed with EDTA (5 mM), PMSF (3 mM), urea (8 M), and SDS (1 %) at 65, 37, 33, and 42 %, respectively. Consequently, the enzyme was well analyzed using crystallography and protein modeling. ALP NS12 can be applied in industrial processes at extreme temperatures and under highly basic conditions, chelators, and detergents.

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Acknowledgments

With special thanks to Nooshin Mohebali, Alex Thompson and Zahra Ghasemzadeh who helped us a lot in current research.

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Authors and Affiliations

  1. Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603, Malaysia

    Nima Samie, Batoulsadat Haerian & Masoumeh Ashouri

  2. Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI, 48109-5632, USA

    Nima Samie & David Green

  3. Drug and Research Unit, Department of Pathology, Hospital Kuala Lumpur (HKL), Kuala Lumpur, 50586, Malaysia

    Nima Samie

  4. Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603, Malaysia

    Sekaran Muniandy

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  1. Nima Samie
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Correspondence to Nima Samie.

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Samie, N., Haerian, B., Muniandy, S. et al. Exhaustive Study of the Novel Hyper Alkalophil, Thermostable, and Chelator Resistant Metalloprotease. Appl Biochem Biotechnol 175, 3397–3417 (2015). https://doi.org/10.1007/s12010-015-1513-6

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