Isolation and characterization of extracellular thermostable alkaline phosphatase enzyme from bacillus spp.

ABSTRACT

Alkaline phosphatase (E C 3.1.3.1) belongs to the class of hydrolases and catalyzes the alkaline hydrolysis of a number of phosphoric acid esters, nucleotides etc. Alkaline phosphatase was produced from Bacillus spp, isolated from soil samples. The Bacillus spp. was identified by staining and standard biochemical tests after which screening was done using modified Pikovoskaya’s agar method. Production of alkaline phosphatase using different substrates like calcium phosphate along with casein, starch, glucose and glutamic acid was carried out. High activity was found in calcium phosphate along with the casein. The specific activity of the crude extract was found to be 0.825U and it was subjected to purification by DEAE-Cellulose ion exchange chromatography. Finally,36% recovery was obtained. The molar mass was estimated by using 10% SDS-PAGE and was found to be approximately 84 KD. The optimum activity was at pH 8.8 and temperature of 650C. Alkaline phosphatase activity was enhanced by Mg2++ upto 66% and 80% activity was inhibited by EDTA. Alkaline Phosphatase activity was also confirmed by zymography using malachite green staining method.