title: Purification and properties of detergent-compatible extracellular alkaline protease from Scopulariopsis spp.
abstract: A fungal alkaline protease of Scopulariopsis spp. was purified to homogeneity with a recovery of 32.2% and 138.1 U/mg specific activity on lectin-agarose column. The apparent molecular mass was 15 ± 1 kD by sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). It was a homogenous monomeric glycoprotein as shown by a single band and confirmed by native PAGE and gelatin zymography. The enzyme was active and stable over pH range 8.0-12.0 with optimum activity at pH 9.0. The maximum activity was recorded at 50°C and remained unaltered at 50°C for 24 hr. The enzyme was stimulated by Co(2+) and Mn(2+) at 10 mM but was unaffected by Ba(2+), Mg(2+), Cu(2+), Na(+), K(+), and Fe(2+). Ca(2+) and Fe(3+) moderately reduced the activity (∼18%); however, a reduction of about 40% was seen for Zn(2+) and Hg(2+). The enzyme activity was completely inhibited by 5 mM phenylmethylsulfonyl fluoride (PMSF) and partially by N-bromosuccinimide (NBS) and tocylchloride methylketone (TLCK). The serine, tryptophan, and histidine may therefore be at or near the active site of the enzyme. The protease was more active against gelatin compared to casein, fibrinogen, egg albumin, and bovine serum albumin (BSA). With casein as substrate, Km and Vmax were 4.3 mg/mL and 15.9 U/mL, respectively. An activation was observed with sodium dodecyl sulfate (SDS), Tween-80, and Triton X-100 at 2% (v/v); however, H2O2 and NaClO did not affect the protease activity. Storage stability was better for all the temperatures tested (-20, 4, and 28 ± 2°C) with a retention of more than 85% of initial activity after 40 days. The protease retained more than 50% activity after 24 hr of incubation at 28, 60, and 90°C in the presence (0.7%, w/v) of commercial enzymatic and nonenzymatic detergents. The Super Wheel-enzyme solution was able to completely remove blood staining, differing from the detergent solution alone. The stability at alkaline pH and high temperatures, broad substrate specificity, stability in the presence of surfactants and oxidizing and bleaching agents, and excellent compatibility with detergents clearly suggested the use of the enzyme in detergent formulations.
authors.affiliation: a Department of Biochemistry , Center for Post Graduate Studies, Jain University , Bangalore , India.
authors.forename: Francois Niyongabo
authors.initials: FN
authors.lastname: Niyonzima
authors.forename: Sunil
authors.initials: S
authors.lastname: More
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D003902:Detergents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D060645:Scopulariopsis / Q000201:enzymology*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D002614:Chelating Agents; D003902:Detergents; D005656:Fungal Proteins; D008670:Metals; D011480:Protease Inhibitors; D012710:Serum Albumin, Bovine; D010450:Endopeptidases; C087822:alkaline protease
keywords: Scopulariopsis spp.; alkaline protease; characterization; detergent additive; gelatin zymography; purification