PURIFICATION AND CHARACTERIZATION OF AN ENDOCHITINASE FROM MYROTHECIUM-VERRUCARIA

The endochitinase from the culture filtrate of Myrothecium verrucaria was purified by ultrafiltration using an Amicon PM-10 membrane and preparative polyacrylamide gel electrophoresis at pH 8.9. The purified enzyme showed a single protein band in SDS gel electrophoresis and polyacrylamide gel electrophoresis run at pH 8.9, 4.3 and 2.9. Isoelectric focusing in the pH range of 3.5-10.0 in 7.5% polyacrylamide gel also revealed a single protein band. The enzyme had an average molecular weight of 30,000 as estimated from SDS gel electrophoresis and gel filtration studies. The optimum pH and temperature using ethylene glycol chitin as a substrate were 5.0 and 50-degrees-C, respectively. The enzyme showed maximum activity towards carboxymethyl chitin and ethylene glycol chitin as compared to colloidal chitin. The apparent K(m) (mg/ml) was 1.33 and 2.85 for ethylene glycol chitin and carboxymethyl chitin, respectively. A V(max) (mumol NAG equivalents/min/mumol of enzyme) of 2.904 x 10(3) and 7.67 x 10(3) was estimated for ethylene glycol chitin and carboxymethyl chitin, respectively. The viscometric studies using carboxymethyl chitin (0.2%) revealed endotype action for the purified enzyme.