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.

β-d-Glucan nanoparticle pre-treatment induce resistance against Pythium aphanidermatum infection in turmeric

“…3b). There are reports that during plant-pathogen interaction, plants produce PIs to combat microbial proteases [26][27][28]. The interaction of plant cell with NP results in modification of plant gene expression and associated biological pathways were reported [29].…”

Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus,Alternaria solani

“…MALDI/TOF/MS data also confirmed AsP is homologous to the alkaline protease of A. fumigatus . The PMF analysis of alkaline protease showed fragmentation of nearly 22 peptides of them only one peptide 1485 m / z yielded good fragmentation (Supporting Information Table S1). The precursor ion at 1485 m / z was selected by the first TOF and allowed to react with the floating cell with inert gas; the MS/MS reported spectrum was obtained after ionization by second TOF operated in the reflectron mode.…”

“…The template protein sequence of AsP from A. solani was obtained from Chandrasekaran and Sathiyabama . It was ascertained that the 3D structure of the protein was not available in Protein Data Bank (PDB) (http://www.rcsb.org/pdb).…”

“…A. solani uses an infection strategy based on the secretion of numerous cell‐wall degrading enzymes . Remarkable progress has been made over the past decade on the contribution of proteolytic enzymes in elucidating plant–pathogen interaction mechanism . Proteases of various phytopathogenic fungi have been detected in infected host plant tissues, but their precise function has not been elucidated yet.…”

Lasiodiplodia theobromae as a Producer of Biotechnologically Relevant Enzymes

“…Proteases are known to have distinct functions in fungal biology, such as in nutrition and in virulence mechanisms. In industry, these proteases also have a high number of applications, such as in detergents, food industry and pharmaceutical industry or in bioremediation processes [ 35 , 36 ]. The higher levels of proteolytic activity found for some of the strains under study, especially when grown at 30 °C, as well as their thermostability up to 80 °C ( Figure 6 ), and the different profiles found by SDS-Zymography ( Figure 4 ), demonstrates the biotechnological potential of this species.…”