Purification and characterisation of a novel alkalophilic alpha-D-mannosidase from Pseudomonas fluorescens

Abstract

An extracellular alkaline -D-mannosidase in the cell culture of a marine bacterium Pseudomonas fluorescens JK-02 was purified to homogeneity with a 30.7-fold by ammonium sulphate fractionation, anion-exchange chromatography and gel-filtration chromatography. The molecular weight of the purified enzyme was estimated to be 50.5 kDa based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature of the purified enzyme were 8.5 and 30 degrees C. The K-m and V-max values of the purified enzyme towards p-nitrophenyl--D-mannopyranoside were determined to be 77 mu M and 0.23 mu M min(-1)mg(-1) of protein, respectively. The -D-mannosidase showed higher substrate specificity to -1,3-mannobiose than other isomeric substrates such as -1,2- and -1,6-mannobiose. In addition, molecular characterisation of this enzyme reveals that it belongs to a class II -mannosidase from the glycosyl hydrolase family 38. To the best of our knowledge, this is the first report on the alkalophilic -1,3 D-mannosidase of Pseudomonas species, which has selective algal-lytic activity against Alexandrium tamarense, Akashiwo sanguine, Gymnodinium catenatum, Gymnodinium mikimotoi and Prorocentrum dentatum.