Production, Purification and Characterization of L-Asparaginase from Marine Endophytic Aspergillus sp. ALAA-2000 under Submerged and Solid State Fermentation

Abstract

Among all endophytic fungi recovered from the marine soft sponge Aplysina fistularis, 72.2% were able to produce L-asparaginase. Among all obtained isolates, Aspergillus sp. ALAA-2000, the hyperactive producer for the anticancer agent, L-asparaginase, under submerged fermentation (SMF) and solid state fermentation (SSF) of different agriculture wastes was selected for the optimization of extraction process; optimization of physicochemical parameters, which affecting the production of L-asparaginases in SSF and optimization of the purified L-asparaginases parameters. Maximum L-asparaginase activity 23.34 U/ml was recovered from soybean with hot water at 40 °C and 150 rpm for 30 min under SSF and 30.64 U/ml under submerged fermentation using glucose as carbon source and asparagine as nitrogen source. Two types of L-asparaginase (AYA-1 and AYA-2) were purified from the culture supernatant of Aspergillus sp. ALAA-2000 through ammonium sulfate precipitation and gel filtration chromatography (sephadex G-200). Molecular weights of the enzymes were 25 kDa (AYA-1) and 31 kDa (AYA-2). The parameters of purified L-asparaginase were optimized for AYA-1 (pH 6.0, stable at 30°C to 50°C for 60 min, reaction time 15 min, and substrate concentration 1.275 mg/ml) and AYA-2 enzyme (pH 10, stable at 30°C to 70°C for 60 min, reaction time 15 min, and substrate concentration 1.275 mg/ml). Whereas inhibitors of metaloproteases, chelating agents EDTA, had no effect on L-asparaginase. These finding suggest that L-asparaginase was not metaloproteases.