Detection of Neuraminidase Activity in Pseudomonas aeruginosa PAO۱

Objective(s) Some properties of neuraminidase produced by Pseudomonas aeruginosa PAO۱ growth in a defined medium (BHI) were examined and evaluated for its features. Materials and Methods The obtained supernatant enzyme of P. aeruginosa PAO۱ cultures was used in a sensitive fluorometric assay by using ۲'-(۴-methylumbelliferyl) a-D-N acetylneuraminic acid as substrate. As hydrolyzing MUN with neuraminidase; free N-acetylneuraminic acid and ۴-methylumbelliferone were formed with a shift in the fluorescence spectra from ۳۱۵/۳۷۴ nm (substrate) to ۳۶۵/۴۵۰ nm (product). Enzyme activity was then measured by the fluorescence of ۴-methylumbelliferone at ۴۵۰ nm. Results Among the culture media to determine the enzyme production, the highest production of P. aeruginosa PAO ۱ neuraminidase was found in BHI culture media. Neuraminidase production in P. aeruginosa PAO۱ paralleled bacterial growth in defined medium (BHI) and was maximal in the late logarithmic phase of growth but decreased during the stationary phase, probably due to protease production or thermal instability. The neuraminidase of P. aeruginosa PAO۱ possessed an optimum temperature of ۵۶ °C and the activity was pH-dependent with maximal activity at pH ۵. Heating the enzyme at ۵۶ °C for ۴۵ min in the presence of bovine serum albumin destroyed ۳۳.۱% of the activity while the addition of Ca+۲, EDTA and N-acetyl neuraminic acid (NANA) decreased activity markedly. Conclusion Overall, the results indicated that neuraminidase of P. aeruginosa PAO۱ is more an extracellular enzyme than K. pneumonia neuraminidase is.

Keywords:

Fluorescence spectrometry , Klebsiella pneumoniae , Neuraminidase , Pseudomonas aeruginosa PAO۱ , Specific activity