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°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°C, the difference in chitinase activities at 10°C and 37°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 Km was 0.03 mm and Vmax was 0.095, using p-nitrophenyl N-acetyl-β-d-glucosaminide as a substrate. Among the metal ions tested for inhibitory activity, Ag+, Hg2+, and Pb2+ strongly inhibited enzyme activity, whereas Mg2+ and Fe2+ 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".