Tropical Plant Research
An International Journal by Society for Tropical Plant Research
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2021, VOLUME 8 ISSUE 1Pages: 95-99
Extraction, purification and characterization of L-asparaginase from isolated Penicillium citrinum Thom. (Strain FMG 181)
Krishna Raju Patro and Nibha Gupta*
*Regional Plant Resource Centre, Bhubaneswar-751015 Odisha, India
Abstract:
Mass scale production of fungal culture was done by using a Glucose-asparagine medium of 4.5 pH under submerged shake culture conditions. The fungal strain opted 4.5 pH and 10 days for good growth and enzyme production. The purification of the enzyme from mass culture developed in growth media was done by following salt precipitation, sephadex G-100 filtration and ion-exchange chromatography by DEAE cellulose columns. We obtained the purified protein and enzyme preparation that was matched with standard (66 kDa) and exhibited approximately 37 k Dawith 2.5 × 10-3 M. M Km value. Partially purified enzymes prefer the alkaline pH (4–8), active at 45–50 ⁰C whereas DEAE separated enzyme was gradually decreasing its activity at higher temperature. It preferred aspartic acid, arginine and histidine besides L-asparagine for substrate requirement whereas DEAE purified enzyme exhibited good activity with L-arginine and L-aspartic acid. Sephadex purified enzyme was active at 45–50 ⁰C whereas DEAE separated enzyme was gradually decreasing its activity at the higher temperature. The present study was done considering Glucose-aspargine medium as basal medium. Further modifications of nutritional and cultural parameters may impact the enzyme production towards the higher side. Hence, more experimentations on C:N requirement and additional P, K and mineral sources is required before use this fungus for large scale production of the enzyme.
Mass scale production of fungal culture was done by using a Glucose-asparagine medium of 4.5 pH under submerged shake culture conditions. The fungal strain opted 4.5 pH and 10 days for good growth and enzyme production. The purification of the enzyme from mass culture developed in growth media was done by following salt precipitation, sephadex G-100 filtration and ion-exchange chromatography by DEAE cellulose columns. We obtained the purified protein and enzyme preparation that was matched with standard (66 kDa) and exhibited approximately 37 k Dawith 2.5 × 10-3 M. M Km value. Partially purified enzymes prefer the alkaline pH (4–8), active at 45–50 ⁰C whereas DEAE separated enzyme was gradually decreasing its activity at higher temperature. It preferred aspartic acid, arginine and histidine besides L-asparagine for substrate requirement whereas DEAE purified enzyme exhibited good activity with L-arginine and L-aspartic acid. Sephadex purified enzyme was active at 45–50 ⁰C whereas DEAE separated enzyme was gradually decreasing its activity at the higher temperature. The present study was done considering Glucose-aspargine medium as basal medium. Further modifications of nutritional and cultural parameters may impact the enzyme production towards the higher side. Hence, more experimentations on C:N requirement and additional P, K and mineral sources is required before use this fungus for large scale production of the enzyme.
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