Purification and characterization of an iron-activated alkaline phosphatase produced by rhizopus microsporus var. Microsporus under submerged fermentation using rye flour

Abstract

Current researches have been carried out to find microorganisms that can produce enzymes for different biotechnological purposes. Among the enzymes, the microbial phosphatases, responsible for hydrolyzing phosphoric acid anhydrides and esters, have been often employed in different sectors such as molecular biology experiments and clinical diagnosis. This work aims to purify and characterize the alkaline phosphatase produced by Rhizopus microsporus var. microsporus under submerged fermentation. This enzyme was purified 9.9-fold with 13% recovery. The molecular mass for the glycoprotein was 123 kDa estimated with gel filtration and 128 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that it is a monomeric enzyme. Optimal temperature and pH for the alkaline phosphatase was 45°C and 8.5, respectively, with halflife (t50) of 40 minutes at 50°C. Under alkaline pH, the phosphatase activity was above 50% for 24 hours. FeCl3 increased the phosphatase activity. Alkaline phosphatase hydrolyzed different substrates, especially p-nitrophenylphosphate, with Km of 0.45 and 0.38 mmol l−1, in presence and absence of FeCl3, respectively. Thus, alkaline phosphatase from R. microsporus var. microsporus was characterized, highlighting important characteristics and, thereby, making possible a future application.