PURIFICATION AND CHARACTERISATION OF ALKALINE PHOSPHOTASE ENZYME FROM THE PERIPLASMIC SPACE OF Escherichia coli C90 USING DIFFERENT METHODS

Alkaline phosphotase enzyme was purified from bacteria Escherichia coli C90 grown in phosphate-poor medium as stationary phase; using an ion exchange column packed with DEAE-cellulose as matrix and size exclusion chromatography using Sepharcryl S-300HR, equilibrated with Buffer A. The enzyme was extracted from the periplasmic space, external to the cell membrane. Previous studies with E. coli have shown increase in alkaline phosphatase activity upon phosphate deprivation with much of the enzyme released into the medium during osmotic shock, indicating that the enzyme is located either in the periplasmic space or is loosely bound to the cell wall. Initially, a DEAE column was used leading to 28% yield and 77 times fold purification, followed by Sephacryl gel filtration column giving 25% yield and 72 times fold purification; indicating loss of enzyme in the subsequent purification step. SDS PAGE analysis was carried out as a control and to compare the results with the spectroscopy. There was no clear decrease in the yield seen in the bands and the loss of enzyme was not observed with the gel analysis. It may, therefore, be assumed that the decrease in yield was due to some pipe tting errors in the spectroscopy measurements. The native gel results show clear distinct bands for the 3 alkalin e phosphotaseisoenzymes, confirming that, the purification procedure for the enzyme was a success.