Insect beta-N-acetyl-D-hexosaminidases are of particular interest due to their multiple physiological roles in many life processes. Chitinolytic beta-N-acetyl-D-hexosaminidases, which function only in chitin degradation in insects, have long been regarded as species-specific target potentials in developing environmental friendly pesticides. Here the chitinolytic beta-N-acetyl-D-hexosaminidase from the insect Ostrinia furnacalis was cloned and expressed in the yeast strain, Pichia pastoris, to meet the demands of biochemical studies and drug development. Enzymatic assay as well as Western blot confirmed that the high-level expression could be achieved after the induction of methanol for 120 h. Through the sequential combination of ammonium sulfate precipitation, metal chelating chromatography as well as anion exchange chromatography, 7.7 mg of the recombinant OfHex1 with high purity was obtained from 1 liter of culture supernatant. The recombinant OfHex1, characterized as a homodimer with molecular weight of 130 kDa, exhibited the same enzymatic activities as its native form, which could efficiently degrade the chitooligosaccharide substrate (GlcNAc)(2) and release 4-methylumbelliferone (4MU) from substrates, 4MU-beta-GlcNAc and 4MU-beta-GalNAc. This work provides a low-costing and high-efficient purification procedure for the preparation of insect beta-N-acetyl-D-hexosaminidases. (C) 2009 Elsevier Inc. All rights reserved.