Purification and partial characterization of serine alkaline metalloprotease from Bacillus brevis MWB-01

Abstract

Background: Proteases from bacteria are among the most important hydrolytic enzymes that have been studied due to their extracellular nature and high yield of production. Of these, alkaline proteases have potential for application in detergent, leather, food, and pharmaceutical industries. However, their usefulness in industry is limited by low activity and stability at high temperatures, extreme pH, presence of organic solvents and detergent ingredients. It is therefore very crucial to search for new alkaline proteases with novel properties from a variety of microbial sources. Results: In the present study, 21 Bacillus species isolated from organic waste sites were screened for proteolytic activity on casein agar. Bacillus brevis MWB-01 exhibited highest proteolytic activity with a clear zone diameter of 35 mm. Production of protease from B. brevis MWB-01 was investigated in optimized media after 48 h of cultivation with shaking (180 rpm) at 37°C. The protease was partially purified in a two-step procedure using ammonium sulphate precipitation and gel filtration chromatography on Sephadex G-200 column. The enzyme was purified 2.1-fold with yield of 4.6%. The purified protease had optimum temperature of 40°C with relative activity of about 50% at 50°C and was uniquely stable up to 60°C after 30 min of incubation exhibiting 63% residual activity. The enzyme had optimum pH of 8.0 and remarkably showed relative activity above 70% at pH 9.0 to 11.0 and 53% at pH 12.0, respectively and was very stable over a wide pH range (6.0 to 12.0). Ca 2+ and Mn 2+ increased protease activity with 9.8% and 3.5%, respectively; Hg 2+ and Zn 2+ strongly inhibited protease activity by 89% and 86%. The almost complete inhibition of the enzyme by phenylmethylsulphonyl fluoride (PMSF) and ethylene diamine tetra acetic acid (EDTA) confirmed the enzyme as a serine metalloprotease. The enzyme had highest compatibility with Sunlight, a commercial laundry detergent.

Production and partial characterization of dehairing alkaline protease from Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 by using organic municipal solid wastes

“…It is reported that the activity of alkaline protease from Bacillus sp. and Bacillus megaterium RRM2 is increased by Ca 2+ ( Gupta et al., 2005 ; Rajkumar et al., 2011 ) and decreased by Hg 2+ , Zn 2+ and Co 2+ ( Venugopal and Saramma, 2007 ; Olajuyigbe and Falade, 2014 ).…”

Characterization and overexpression of a novel keratinase from Bacillus polyfermenticus B4 in recombinant Bacillus subtilis

“…Cross-linking in keratin fibers occurs through disulfide bonds, hydrogen bonds, and hydrophobic interactions. General proteases such as trypsin and pepsin cannot degrade keratin (Gupta and Ramnani 2006;Kreplak et al 2004;Olajuyigbe and Falade 2014).…”

Optimization of thermotolerant alkaline protease production from Brevibacillus brevis strain BT2 using surface response methodology

“…PLI-1 from Indonesia's hot springs. In contrast, thermotolerant alkaline protease producing strain, Bacillus brevis MWB-01 was isolated from sub-soil from organic waste sites (Olajuyigbe and Falade, 2014). The members of the genus Bacillus are spore formers and also produce a number of biocidal metabolites/enzymes which makes them a common inhabitant of diverse extreme habitats (Nicholson et al, 2000).…”

“…With the increasing emphasis on cost effectiveness for enzyme production, the use of low-cost fermentation medium and process optimization is of paramount importance for commercial gain. Recently, only a few studies investigated the protease producing potential of the Brevibacillus brevis ( Jaouadi et al, 2013 ;Olajuyigbe et al, 2014) but there is no previous report on process optimization using the design of experiment (DOE). In view of this, a statistical experimental design was employed to optimize culture conditions for higher alkaline protease using Brevibacillus brevis BT2 strain under submerged fermentation.…”