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

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 K_m was 0.03 mM and V_max was 0.095. using p-nitrophenyl N-acetyl-β-D-glucosa- minide 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".