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.

Microbes Producing L-Asparaginase free of Glutaminase and Urease isolated from Extreme Locations of Antarctic Soil and Moss

“…Currently, L-Asparaginase is industrially produced mainly from prokaryotic microbes such as Escherichia coli and Erwinia chrysanthemi . However, the concomitant presence of the activities of glutaminase and urease means that additional purification steps are required to remove these two enzymes and maintain drug efficacy 9,10 . The presence of glutaminase destroys the necessary glutamine required for the growth of normal cells 11 .…”

Production of L-Asparaginase (L-ASN) from endophytic Lasiodiplodia theobromae hosted Teucrium polium in Egypt

“…Acute Lymphoblastic Leukemia (ALL) is widely distributed among children: 60% at the age of (0-14), 40% to other ages, several studies have been carried out to obtain a safe form of the L-Asparaginase out of fungal sources and they already succeed to get a safe forms of L-Asparaginase (Asha and Pallavi 2012;Abbas Ahmed 2015;Kumar and Sobha 2012). Our results revealed that L-Asparaginase activity were recorded in three native species namely: Fusarium oxysporum, Fusarium solani and Lasiodiplodia theobromae.…”

Bioactive Novel Natural Products from Marine Sponge: Associated Fungi

“…Therapeutic enzymes, it seems out, have a unique ability to facilitate high-affinity interactions with unrelated cancer-related proteins. Endophytic fungi recovered from the marine soft sponge Aplysina fistularis, produce L-asparaginase [57].…”