title: Production, partial purification and characterization of protease from a phytopathogenic fungi Alternaria solani (Ell. and Mart.) Sorauer.
abstract: An alkaline serine protease producing strain Alternaria solani was optimized for its enzyme production under submerged conditions. The maximum production of protease by A. solani was achieved by using sodium nitrate at the optimum concentration of 0.2% w/v. A. solani produced higher quantities (3.75 [unit/mg of protein]) of an inducible extracellular proteases on day 9 after incubation in czapek's dox broth medium amended with 1% casein as an inducer at pH 8.5, temperature 27 °C and 3% sucrose as carbon source. Extracellular proteases were precipitated by ammonium sulphate saturation (80%) method and purified on Sephadex G-100 column chromatography. The molecular mass of SDS-PAGE and Sephadex G-100 Column Gel permeation chromatography purified protease was estimated to 42 kDa. In addition, trypsin digestion of 42 kDa protein band was carried out and analyzed by MALDI-TOF for the identification of protease. The sequence IKELATNGVVTNVK (378-391) segment of the alkaline serine protease was found by using MS/MS spectrum at 1485 m/z from the purified fraction. It showed optimal activity at 50 °C and pH 9-10 and broad pH stability between pH 6-12. The protease activity was inhibited by phenyl methyl sulfonyl fluoride (PMSF), all the results indicated that the presence of a serine residue in the active site and is thus most likely a member of the serine protease family. This may function as a virulence protein during pathogenesis by A. solani. The results suggested that the presence of appreciable extracellular proteolytic activity in filamentous fungi may serve as a marker of their phytopathogenicity.
authors.affiliation: Department of Plant Science, Bharathidasan University, Tiruchirappalli, 620 024, Tamilnadu, India.
authors.forename: Murugesan
authors.initials: M
authors.lastname: Chandrasekaran
authors.forename: Muthukrishnan
authors.initials: M
authors.lastname: Sathiyabama
mesh_terms: D000528:Alternaria / Q000201:enzymology* / Q000378:metabolism / Q000472:pathogenicity*; D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology; D020539:Sequence Analysis, Protein; D057057:Serine Proteases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013441:Sulfinic Acids / Q000737:chemistry
chemical_list: D015842:Serine Proteinase Inhibitors; D013441:Sulfinic Acids; C048899:sulfuryl fluoride; D057057:Serine Proteases
keywords: Alternaria solani; MALDI-TOF-MS; Phytopathogenic Fungi; Protease; Serine Protease