Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts.
An alkaline 5'-phosphodiesterase (5'-PDE) from barley (Hordeum distichum) malt sprouts was partially purified by thermal treatment and acetone precipitation to diminish phosphomonoesterase (PME) activity. 5'-PDE was purified 40-fold to a specific activity of 30 U mg(-1) protein with a final yield of about 32%. With synthetic substrate, the enzyme had an optimum pH of 8.9, maximum activity at 70 degrees C over 10 min, and a Km of 0.26 mM. The partially purified enzyme was activated by 10 mM Mg2+ up to 168% of the original activity, while Zn2+, Mn2+ and Cu2+ ions, chelating agent (EDTA) and NaN3 (1-10 mM), and 5'-ribonucleotides (1-5 mM) were inhibitory. Final enzyme preparation was stable over 8 d at 4 degrees C), at 70 degrees C for up to 120 min and without loss of activity over 90 d at -18 degrees C.
Beluhan
S
S
Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6/IV, 10000 Zagreb, Croatia. sunbel@pbf.hr
Karmelić
I
I
Novak
S
S
Marić
V
V
D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D004247:DNA / Q000737:chemistry; D004356:Drug Storage; D004789:Enzyme Activation; D004795:Enzyme Stability; D001467:Hordeum / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification*; D012313:RNA / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature
D008670:Metals; D012313:RNA; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5