title: 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.
authors.affiliation: Centre for Advanced studies in Botany, University of Madras, Guindy Campus, Chennai, India.
authors.forename: Renganathan
authors.initials: R
authors.lastname: Rajkumar
authors.forename: Kothilmozhian Ranishree
authors.initials: KR
authors.lastname: Jayappriyan
authors.forename: Ramasamy
authors.initials: R
authors.lastname: Rengasamy
mesh_terms: D001410:Bacillus megaterium / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification; D015373:Bacterial Typing Techniques; D001709:Biotechnology / Q000379:methods*; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D003902:Detergents / Q000378:metabolism*; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004795:Enzyme Stability; D006197:Hair / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D000461:Rhodophyta / Q000382:microbiology; D017422:Sequence Analysis, DNA; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D003902:Detergents; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; D012336:RNA, Ribosomal, 16S; D057057:Serine Proteases