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 pipetting errors in
the spectroscopy measurements. The native gel results show clear distinct bands for the 3 alkaline phosphotase
isoenzymes, confirming that, the purification procedure for the enzyme was a success.