Fig. 5.

Degradation products seen during reaction of partially purified chitinase (10.5 kD) with chito-oligosaccharides. 200ul of substrate (0.2 mM working conc.) and 200ul of enzyme solution (2 Units/mg protein) was mixed. The enzyme substrate mixture was incubated at 37 °C for 0, 5 s, 15 s, 30 s, 1 min, 5 min, 10 min, 15 min, 30 min, 1 h separately. Enzyme mixture with and without substrate served as negative control. The reaction was stopped by boiling the mixture at 100 °C and then concentrated using a vacuum evaporator (Eyela, Japan). The analysis was done on TLC plates (Silica gel 60, F ₂₅₄ (20 x 20 cm) with butanol:methanol:ammonia:water (5:4:2:1) as mobile phase and aniline diphenylamine reagent as spraying reagent. The plate was developed by heating at 120 °C for 10–15 minutes. Dark brown-greenish spots were identified and Rf value was compared to that of standards.

Lane 1, mixture of all substrates; lane 2, individual substrate; lane 3–10, 15 s, 30 s, 1 min, 5 min, 10 min, 15 min, 30 min, 1 h; lane 11–12, negative control (enzyme with/without substrate); lane 13, mixture of all substrates.