Purification and characterisation of alkaline phosphatase from fruticose lichen usnea
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Purification and Characterisation of Alkaline Phosphatase from Fruticose Lichen
Usnea.sp
Antik K. Bosea1
a1 Human Biology Division, Fred Hurtchinson Cancer Research Center, 1100 Fairview Avenue
N, Seattle, Washington -98109, USA
Abstract:
Biochemical properties of alkaline phosphatase obtained from fruticose lichen Usnea.sp have
been described. The enzyme was purified 38439.26 fold with 7.32% recovery by DEAE-cellulose
and Sephadex G-200 Chromatography. The molar mass was estimated by Sephadex G-200 was
132.7KD and by 6% SDS-PAGE, a single band of 66 KD was obtained indicating a homodimer.
Temperature and pH optima were 35 C and pH 8.5 respectively. The enzyme was moderately
glycosylated (42% saccharide content). The activity was enhanced by Mg2+
(6.07-128.3%), Mn2+
(18.66-85.1%), Na+ (6.07%-64.47%), Al
3+ ( 4.5-52.58% ), Ba
2+ (2.95-25.2%). 4-
nitrophenylphosphate was hydrolysed with KM and Vmax of 6.67mM-1 and 22.22µM of PNPP
consumed/min respectively at pH 8.5. Amino acids at the active site have been identified using
Diisopropylphosphofloridate ( DIPF) , Diethylpyrocarbonate ( DEPC ), Diazomethane and 1-
ethyl-3( 3-dimethyl) aminopropylcarbodiimide ( EDAC) to be serine, histidine and aspartic acid
respectively.
