Purification and characterization of beta-N-acetylhexosaminidase from maize seedlings.
Enzymatic activity of beta-N-acetyhexosaminidase (EC 3.2.1.52) was analysed in seeds and young seedings of maize (Zea mays) using di-N-acetylchitobiose as a substrate. Substantial activity was detected in dry seeds. Activity increased before germination (48 h) but exclusively in the embryo. In seedlings, most of the activity was found in the scutellum, and lower levels in shoots and roots immediately after germination. An isoform of the enzyme was purified from scutellum (72 h after the start of imbibition) by heat treatment of crude extract and four steps of chromatography. Purified beta-N-acetyl-hexosaminidase showed a single band on SDS-PAGE of around 70 kDa. This was almost the same as the molecular weight estimated by size exclusion chromatography, indicating a monomeric form of the active enzyme. The relative activity of the enzyme for di-N-acetylchitobiose was about 15 times greater than that for p-nitrophenyl-N-acetylglucosaminide or p-nitrophenyl-N-acetylgalactosaminide. Analysis of the reaction with oligo-N-acetylchitooliogsaccharides [(GlcNAc)n] revealed an exotype enzyme producing predominantly (GlcNAc)n-1 and N-acetylglucosamine. The optimum pH, temperature, and isoelectric point (pl) were 4.5, 55 degrees C, and 6.75, respectively. The activity was almost completely inhibited in the presence of 5 mmol/L Ag+, Hg2+, or Fe3+.
Division of Applied Life Sciences, Graduated School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. oikawa@kais.kyoto-u.ac.jp
Akira
A
Oikawa
Eriko
E
Itoh
Atsushi
A
Ishihara
Hajime
H
Iwamura
D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D036226:Seedlings / Q000201:enzymology; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D003313:Zea mays / Q000201:enzymology* / Q000254:growth & development; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D009844:Oligosaccharides; D001619:beta-N-Acetylhexosaminidases