Malaysian Journal of Microbiology, Vol 8(1) 2012, pp. 1-5 http://dx.doi.org/10.21161/mjm.28310 1 ISSN (print): 1823-8262, ISSN (online): 2231-7538 Partial Purification and Characterization of a Thermostable Alkaline Protease from Lactobacillus brevis Titilayo Olufunke Femi-Ola * and Desmond Olayinka Oladokun Department of Microbiology, University of Ado-Ekiti, P. M. B. 5363, Ado-Ekiti, Nigeria. E-mail: titifemi2006@yahoo.com Received 30 November 2010; received in revised form 5 April 2011; accepted 13 August 2011 _______________________________________________________________________________________________ ABSTRACT Aims: The research was done to study the partial purification and characterization of thermostable alkaline protease from Lactobacillus brevis. Methodology and Results: The enzyme was purified in a two-step procedure involving ammonium sulphate precipitation and Sephadex G-150 gel permeation chromatography. The protease was purified 8.04 fold with a yield of approximately 30% after purification with Sephadex G-150 column. It has a relative molecular weight of 33.2 kDa and optimally active at a temperature of 60 o C and pH 9.0. The maximum velocity Vmax and Michaelis constant Km of the protease produced during the hydrolysis of casein were 66.66 U/mg protein and 3.33 mg/ml. It was strongly activated by Ca 2+ and ethylene diamine tetra acetic acid (EDTA), mildly inhibited by Na + , K + , Mg 2+ and Fe 2+ and strongly inhibited by Cu 2+ and Hg 2+ . The ability of the enzyme to improve the cleansing power of various detergents was also studied. Conclusion, significance and impact of study: The findings in this study suggest that the protease is a suitable candidate for detergent formulation and biotechnological applications. Keywords: protease, Lactobacillus, alkaline, inhibitors and detergent _______________________________________________________________________________________________ INTRODUCTION Proteases are one of the most important classes of enzymes and are expressed throughout the animal kingdom, plant and as well as microbes (Dubey et al., 2007). Among the various proteases, bacterial proteases are the most significant compared with those of animals and plants (Gupta et al., 2002). This enzyme accounts for nearly 60% of the total worldwide enzyme sales (Adinarayana et al., 2003; Beg et al., 2003). There are acidic, alkaline and neutral proteases, but of all these, alkaline proteases are employed primarily as cleansing additives (Ward, 1995; Nehra et al., 2004). Alkaline protease of microbial origin possess considerable industrial potential due to their biochemical diversity and wide applications in tannery and food industries, medicinal formulations, detergents and processes like waste treatment, silver recovery and resolution of amino acid mixtures (Rao et al.,1998; Agarwal et al., 2004; Devi et al., 2008). One of the major drawbacks affecting the stability at alkaline pH of enzymes recovered from thermophiles is that enzymes from alkalophile confer stability in a wide pH range but are usually thermolabile (Griffin et al., 1992). In this present study, the protease produced by Lactobacillus brevis was characterized. The results obtained showed that the protease may be applied as an effective detergent additive which is recommended for laundry/detergent producing industries. MATERIALS AND METHODS Organisms and culture conditions The test isolate, Lactobacillus brevis used for this research was isolated from the hindgut of kola nut weevil Balanogastris kolae Desbr. It was cultured on nutrient agar to obtain the pure culture of this organism. The organism was grown in a basal medium containing (g/L): K2HPO4, 1.5; KH2PO4, 0.5; MgSO4, 0.05; NaCl, 1.5; (NH4)2SO4, 1.0; CaCl2·2H2O, 0.02; FeSO4·7H2O 0.02; yeast extract, 0.5; sucrose, 0.5 and casein, 0.1. The inocula for the experiments were prepared by growing the organism in nutrient broth (NB, Oxoid) at 35 °C for 18 h on a rotary shaker (Gallenkamp). Sterilized medium (500 mL) in 1000 mL conical flasks was inoculated with 10mL of inocula (1.2 ×10 5 cells/mL). The flask was incubated at 35 °C on a rotary shaker (120 rpm) for 48 h and then centrifuged at 5000 rpm for 20 min at 4 °C to remove bacterial cells. The supernatant obtained was used as the crude extract for further studies. Protease assay The protease activity was determined a reaction mixture consisting of 1 mL of substrate solution (1% casein in Tris- HCl buffer, pH 8.0) and 1 mL of the enzyme solution. The reaction mixture was incubated for 60 min at 40 °C. The proteins were precipitated by adding 3 mL of 0.5% TCA and free amino acids released by protease from casein *Corresponding author