Substrate inhibition hinders chitinolytic beta-N-acetyl-D-hexosaminidases in producing N-acetyl-D-glucosamine (GlcNAc), the valuable chemical widely applied in medical and food industries. Here we focused on a promising chitinolytic enzyme, OfHex1 from the insect, Ostrinia furnacalis. By structural analysis of OfHex1, five residues nearby the active pocket including V327, E328, Y471, V484 and W490 were chosen and nine mutants including V327G, E328Q, E328A, Y471V, V484R, W490A, W490H, V327G/V484R/W490A and V327G/Y471V/W490H were constructed and recombinantly expressed in Pichia pastoris. The best-performing mutant, W490A, obtained by a higher yield of 5 mg/L, did not show substrate inhibition even when 5 mM of the substrates, (GlcNAc)(2-4), were applied. The k(cat)/K-m values for (GlcNAc)(2-4) are 239.8, 111.3 and 79.8 s(-1) mM(-1), respectively. Besides, the pH stability of the mutant ranges from pH 4 to 11 and the thermal stability is up to 50 degrees C. This work suggests the W490A mutant might be an ideal biocatalyst for GlcNAc production from chitin. (c) 2012 Elsevier Ltd. All rights reserved.