Endochitinase has an important application in the biological treatment of chitin, the second most abundant and renewable resource in nature. In order to enhance its activity, a double mutant strain MECH-Y185/S226 was obtained by the directed evolution using the error-prone PCR with the mature endochitinase cDNA from Trichoderma viride as the template. Compared to those of the primitive strain MEd-I, endochitinase activities of the mutant one were 1.8-fold higher towards 4-nitrophenyl-N-acetyl-beta-D-glucosaminide and 3.5-fold higher towards the colloidal chitin. Sequence alignments indicated that 9 nucleotides and 2 amino acids (Y185F and S226P) were mutant. The SDS-PAGE analysis showed that a single band with an estimated molecular weight of 46 kDa was obtained when the Ech42-Y185/S226 was purified sequentially by ammonium sulfate precipitation, DE52 anion-exchanging column chromatography and Sephadex G-100 column chromatography. Kinetic parameters K-m and V-max of the Ech42-Y185/S226 were 0.25 +/- 1 0.02 mmol/land 4.59 +/- 0.32 timol/Imin, respectively. The analysis of enzymatic properties showed that the Ech42-Y185/S226 had a higher thermal stability at higher temperatures and a higher pH stability within a wider pH range than the Ech42. Observed activities of the Ech42-Y185/S226 are the highest in the presence of Mg2+ and the lowest in the presence of Zn2+. (c) 2012 Elsevier Ltd. All rights reserved.