A novel low temperature chitinase from the marine fungus Plectosphaerella sp strain MF-1

Abstract

The marine fungus Plectosphaerella sp. strain MF-1 was isolated from sea shells and found to produce a chitinase potentially active at low temperature. The fungal strain was characterized by morphological and molecular features. Chitinase production by Plectosphaerella MF-1 was detected by inoculating the fungus into M9 medium containing 0.5% colloidal chitin at 10 degrees C. Crude chitinase production in culture filtrates reached a maximum 14 days after inoculation. Crude chitinase was purified by ammonium sulfate fractionation, cellulose DEAE anion exchange, and sephadex gel filtration chromatography. Purified marine fungal chitinase had activity at 37 degrees C, the difference in chitinase activities at 10 degrees C and 37 degrees C was less than 0.01 U ml(-1) indicating chitinase was active at low temperature. The optimal pH for the low temperature active chitinase was 3-4. The K(m) was 0.03 mM and V(max) was 0.095, using p-nitrophenyl N-acetyl-beta-D-glucosaminide as a substrate. Among the metal ions tested for inhibitory activity, Ag(+), Hg(2+), and Pb(2+) strongly inhibited enzyme activity, whereas Mg(2+) and Fe(2+) had minimal inhibition. The molecular mass of purified chitinase was determined as 67 kDa by SDS-PAGE. The N-terminal amino acid sequence was determined to be "DNISQTGEHARYXPMVWFIKL''.