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 pre parative polyacrylamide gel electrophoresis at pH 8.9. The purified en zyme showed a single protein band in SDS gel electrophoresis and polya crylamide 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 r evealed a single protein band. The enzyme had an average molecular wei ght of 30,000 as estimated from SDS gel electrophoresis and gel filtra tion studies. The optimum pH and temperature using ethylene glycol chi tin as a substrate were 5.0 and 50-degrees-C, respectively. The enzyme showed maximum activity towards carboxymethyl chitin and ethylene gly col 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 chit in and carboxymethyl chitin, respectively. The viscometric studies usi ng carboxymethyl chitin (0.2%) revealed endotype action for the purifi ed enzyme.