Purification and enzymatic characterization of tobacco leaf β-N-acetylhexosaminidase

The kinetic properties of beta-N-acetylhexosaminidase purified from tobacco (Nicotiana tabacum L) leaves have been investigated. In addition to chromogenic pNP derivates, N,N'-diacetylchitobiose and N,N',N-triacetylchitotriose were also used as substrates of beta-N-acetylhexosaminidase. The highest reaction rate and the affinity for the substrate were observed for pNP-GlcNAc; however, an excess of this substrate inhibits the reaction. The reaction rate with pNP-GalNAc as the substrate was found to be about 85% of that obtained with pNP-GlcNAc. The hydrolysis of acetylated chitooligomers by beta-N-acetylhexosaminidase followed by separation and quantification using capillary electrophoresis was slower compared to pNP-GlcNAc. The pH optimum of beta-N-acetylhexosaminidase for individual substrates was found at 4.3-5.0 and the temperature optimum was 50-55 degrees C. Gel permeation chromatography and red native electrophoresis determined the relative molecular weight as 280 000 and the isoelectric point as 5.3. The inhibition of beta-N-acetylhexosaminidase by monosaccharides GlcN, GalN, GlcNAc, GalNAc in combination with substrates pNP-GlcNAc and pNP-GalNAc was studied and the type of inhibition and the inhibition constants were determined. (C) 2014 Elsevier B.V. and Societe francaise de biochimie et biologie Moleculaire (SFBBM). All rights reserved.