Purification and properties of the alkaline phosphatase ofSerratia marcescens

Abstract

Abstract. A mutant strain of Serratia marcescens produces alkaline phosphatase in high yield. High concentrations of inorganic phosphate do not suppress formation of the enzyme and inhibition of its activity by inorganic phosphate is relatively low. The enzyme has been purified to homogeneity as determined by analytical ultracentrifugation and gel electrophoresis; it has a molecular weight of 100000. Guanidinium chloride reduces the apparent molecular weight about 50~ suggesting that the native enzyme is a dimer. Only one isoenzyme species is observed after electrophoresis. Specific activity is high (3553 units/mg protein); catalytic and spectroscopic properties are similar to those of alkaline phosphatase from other species. It is present to the extent of about 2 ~ of the cellular protein.

Evidence on the presence of two distinct alkaline phosphatases inSerratia marcescens

“…The bacterial suspension was harvested and APase activity was isolated by the butanol extraction procedure [5] and dialyzed against Tris‐Mg 2+ buffer (Tris 10 mM, MgCl 2 5 mM, pH 8.0) for 48 h at 4°C. All the APase activity was sedimented with the bacterial cells after centrifugation, no cell‐free APase activity was detected in the cell‐free medium.…”

“…All the APase activity was sedimented with the bacterial cells after centrifugation, no cell‐free APase activity was detected in the cell‐free medium. Therefore, unlike Pseudomonas aeruginosa [9], Bacillus subtilis [10] and Haloarcula marismortui [11], APases of S. marcescens are not secreted into the growth medium during growth and are cell‐bound [5]. All other purification steps were carried out at 4°C.…”

“…Enzyme activity, protein concentrations and specific activity, starch gel electrophoresis and staining for APase activity were carried out as described earlier [5]. Thermal stability of the APases was performed according to the method described [6] at 95°C.…”

“…The effect of pH on the two types of APases was determined according to the method of Wilson et al [8]. Immunodiffusion was carried out as described [5].…”

Alkaline Phosphatases from Mycobacterium smegmatis

“…In some, the alkaline phosphomonoesterase is synthesized only when the concentration of orthophosphate in the medium is limiting (Bhatti, 1974;Garen & Echols, 1962;Horiuchi, Horiuchi & Mizuno, 1959;Stewart & Stewart, 1971 ;Taniguchi & Tsugita, 1966;Torriani, I 960). In Staphylococcus aureus and in Bacteroides ruminicola, the enzyme is constitutive (Cheng & Costerton, 1973;Kuo & Blumenthal, 1961).…”