Characterisation of a β-N-acetylhexosaminidase from a commercial papaya latex preparation

A beta-N-acetylhexosaminidase (beta-NAHA) (EC 3.2.1.52) with molecular mass of 64.1 kDa and isoelectric point of 5.5 was purified from a commercial papaya latex preparation. The optimum pH for p-nitro-phenyl-N-acetyl-beta-D-glucosaminide (pNP-beta-GlcNAc) hydrolysis was five; the optimum temperature was 50 degrees C; the K-m was 0.18 mM, V-max was 37.6 mu mol min(-1) mg(-1) and activation energy (E-a) was 10.3 kcal/mol. The enzyme was thermally stable after holding at 30-45 degrees C for 40 min, but its activity decreased significantly when the temperature exceeded 50 degrees C. Heavy metal ions, Ag+ and Hg2+, at a concentration of 0.25 mM and Zn2+ and Cu2+, at a concentration of 0.5 mM, significantly inhibited enzyme activity. The beta-NAHA had only one active site for binding both pNP-beta-GlcNAc and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNP-beta-GaINAc). A prototropic group with pKa value of about five on the enzyme may be involved in substrate binding and transformation, as examined by Dixon-Webb plots. (C) 2010 Elsevier Ltd. All rights reserved.