Purification and characterization of a protease produced by Bacillus megaterium RRM2: application in detergent and dehairing industries

Abstract

An alkaline serine protease produced by Bacillus megaterium RRM2 isolated from the red alga, Kappaphycus alvarezii (Doty) Doty ex Silva was studied for the first time and the same analyzed for the production of protease in the present study. Identification of the bacterium was done on the basis of both biochemical analysis and by 16S rDNA sequence analysis. The extracellular protease obtained from B. megaterium RRM2 was purified by a three-step process involving ammonium sulphate precipitation, gel filtration (Sephadex G100) and Q-Sepharose column chromatography. The purity was found to be 30.6-fold with a specific activity of 3591.5 U/mg protein with a molecular weight of 27 kDa. The metal ions Ca(2+), Mg(2+), K(+) and Na(+) marginally enhanced the activity of the purified enzyme while Hg(2+), Cu(2+), Fe(2+), CO(2+) and Zn(2+), had reduced the activity. The enzyme was found to be active in the pH range of 9.0-10.0 and remained active up to 60 °C. Phenyl Methyl Sulfonyl Fluoride (PMSF) inhibited the enzyme activity, thus, confirming that this enzyme is an alkaline serine protease. Likewise, DTT also inhibited the enzyme thus confirming the disulfide nature of the enzyme. The enzyme exhibited a high degree of tolerance to Sodium Dodecyl Sulphate (SDS). The partially purified protease when used as an additive in the commercial detergents was found to be a suitable source for washing clothes especially those stained with blood. Further, it showed good dehairing activity within a short duration in goat skin without affecting its collagen component.

Keratinolytic Protease Production by Bacillus Cereus Strain Ps03 under Submerged Fermentation: Optimization and Characterization

“…The enzyme was found to be stable upto pH 9 for 1 h. Results of similar trend were observed by other researchers when characterizing Bacillus sp. producing proteolytic and keratinolytic enzymes (Rajkumar et al, 2011;El-Hadj-Ali et al, 2007;Haile and Gessesse, 2012). Rao et al (1998) have reported that the commercial bacterial keratinolytic enzymes have pH optima in the range from 8 to 12 and the results of the present study also found in line with his findings.…”

Isolation and Screening of Protease Producing Bacteria from Local Environment for Detergent Additive

“…Also it was compatible with all surfactants found in commercial detergent (Ariel), which indicates its potential candidate in detergent formulation. This result is well established by many authors [25,28,30]. Such practice may find application in removing blood stains from materials of medical importance.…”

“…Maximum loss of enzymatic activity was observed in Fe +2 . The presence of Ca 2+ did not affect the enzyme activity [28]. However, in another report, metal ions like Mn 2+ and Ca 2+ were found to be potent enhancers [29].…”

“…However, currently proteases constitute the largest product segment in the global industrial enzymes market. The detergent enzyme market has grown nearly 10-fold during the past 20 years [8].…”

Microorganisms living on macroalgae: diversity, interactions, and biotechnological applications

“…In other bacterial strains also found on Sargassum sp., amylase, carboxymethylcellulase, and protease activities were found (Mohapatra et al 2003). An alkaline serine protease with potential use in the laundry industry was found in Bacillus megaterium RRM2, isolated from a red alga species (Rajkumar et al 2011). Furthermore, bacterial enzymes such as lipases and esterases (Rajkumar et al 2011), cellulases (Dong et al 2010;Fu et al 2010;Gibbs et al 1992), proteases (Cristóbal et al 2011;Yang et al 2013), amylases (Both et al 1993;Liu et al 2012), laccases (Fang et al 2012;Ge et al 2011), and beta-glucosidases (Cristóbal et al 2009;Mai et al 2013), distantly related to terrestrial ones and displaying original biochemistry, are increasingly being isolated from the marine environment.…”