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Thermostable alkaline protease from Thermomyces lanuginosus: optimization, purification and characterization

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Abstract

A serine alkaline protease (EC.3.4.21) was isolated, purified and characterized from culture filtrate of the thermophilic fungus Thermomyces lanuginosus Tsiklinsky. Fructose (1.5 %) and gelatin (0.5 %) proved to be the best carbon and nitrogen sources, giving a maximum enzyme yield of 9.2 U/mL. Dates waste was utilized as a sole organic source to improve enzyme productivity, and the yield was calculated to be 11.56 U/mL. This yield was expressed also as 231.2 U/g of assimilated waste. The alkaline protease produced was precipitated by iso-propanol and further purified by gel filtration through Sephadex G-100 and ion exchange column chromatography on diethyl amino ethyl (DEAE)-cellulose with a yield of 30.12 % and 13.87-fold purification. The enzyme acted optimally at pH 9 and 60 °C and had good stability at alkaline pH and high temperatures. The enzyme possessed a high degree of thermostability and retained full activity even at the end of 1 h of incubation at 60 °C. Michaelis–Menten constant (K m), maximal reaction velocity (V max) and turnover number (K cat) of the purified enzyme on gelatin as a substrate were calculated to be 4.0 mg/mL, 18.5 U/mL and 1.8 s−1, respectively. The best enzyme activators were K+, Ca2+ and Mn2, respectively, while phenylmethylsulfonyl fluoride (PMSF) was the strongest inhibitory agent, thus suggesting that the enzyme is a serine type protease. The enzyme is a glycoprotein with molecular mass of 33 kDa as determined by SDS-PAGE. It retained full activity after 15 min incubation at 60 °C in the presence of the detergent Ariel, thus indicating its suitability for application in the detergent industry.

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Authors and Affiliations

  1. Biological & Geological Sciences Department, Faculty of Education, Ain Shams University, Heliopolis, Roxy, Cairo, 1143, Egypt

    Mohamed Ghareib & Eman M. Fawzi

  2. Ministry of Higher Education, Kingdom of Saudi Arabia, Riyadh, Saudi Arabia

    Nouf A. Aldossary

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  1. Mohamed Ghareib
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  2. Eman M. Fawzi
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  3. Nouf A. Aldossary
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Correspondence to Eman M. Fawzi.

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Ghareib, M., Fawzi, E.M. & Aldossary, N.A. Thermostable alkaline protease from Thermomyces lanuginosus: optimization, purification and characterization. Ann Microbiol 64, 859–867 (2014). https://doi.org/10.1007/s13213-013-0725-7

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  • DOI: https://doi.org/10.1007/s13213-013-0725-7

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