Purification and characterization of an alkaline protease Prot 1 from Botrytis cinerea : biodetergent catalyst assay.
Alkaline thiol protease named Prot 1 was isolated from a culture filtrate of Botrytis cinerea. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Thus, the enzyme was purified to homogeneity with specific activity of 30-fold higher than that of the crude broth. The purified alkaline protease has an apparent molecular mass of 43 kDa under denaturing conditions as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular mass (45 kDa), determined by gel filtration, indicated that the alkaline protease has a monomeric form. The purified protease was biochemically characterized. The enzyme is active at alkaline pH and has a suitable and high thermostability. The optimal pH and temperature for activity were 9.0-10.0 and 60 degrees C, respectively. This protease was stable between pH 5.0 and 12.0. The enzyme retained 85% of its activity by treatment at 50 degrees C over 120 min; it maintained 50% of activity after 60 min of heating at 60 degrees C. Furthermore, the protease retained almost complete activity after 4 wk storage at 25 degrees C. The activity was significantly affected by thiol protease inhibitors, suggesting that the enzyme belongs to the alkaline thiol protease family. With the aim on industrial applications, we focused on studying the stability of the protease in several conditions. Prot 1 activity was not affected by ionic strength and different detergent additives, and, thus, the protease shows remarkable properties as a biodetergent catalyst.
Abidi
Ferid
F
Biological Engineering Unit 99UR09-26, National Institute of Applied Sciences and Technology, Tunis, Tunisia.
Limam
Ferid
F
Marzouki
M Nejib
MN
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D020171:Botrytis / Q000378:metabolism*; D002384:Catalysis; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D004983:Ethanolamines / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
D001426:Bacterial Proteins; D003902:Detergents; D004983:Ethanolamines; D011506:Proteins; D012685:Sepharose; D010450:Endopeptidases; D010447:Peptide Hydrolases; C087822:alkaline protease; C007369:2-diethylaminoethanol