New trihydroxy-keto-carotenoids isolated from an astaxanthin-producing marine bacterium

Abstract

β-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from the liver of a prawn, Penaeus japonicus, by ammonium sulfate fractionation and chromatography with Sephadex G-100, hydroxylapatite, DEAE-Cellulofine, and Cellulofine GCL-2000-m. The purified enzyme showed a single band keeping the potential activity on both native PAGE and SDS–PAGE. The apparent molecular weight was 64,000 and 110,000 by SDS–PAGE and gel filtration, respectively. The pI was less than 3.2 by chromatofocusing. The aminoterminal amino acid sequence was NH2-Thr-Leu-Pro-Pro-Pro-Trp-Gly-Trp-Ala-?-Asp-Gln-Gly-VaI-?-Val-Lys-Gly-Glu-Pro-. The optimum pH and temperature were 5.0 to 5.5 and 50°C, respectively. The enzyme was stable from pH 4 to 11, and below 55°C. It was 39% inhibited by 10mM HgCl2. Steady-state kinetic analysis was done with the purified enzyme using N-acetylchitooligosaccharides (GlcNAcn, n = 2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-β-GlcNAcn, n= 1 to 3) as the substrates. The enzyme hydrolyzed all of these substrates to release monomeric GlcNAc from the non-reducing end of the substrate. The parameters of Km and kcat at 25°C and pH 5.5 were 0.137 mM and 598s–1 for pNp-β-GlcNAc, 0.117 mM and 298s–1 for GlcNAc2, 0.055 mM and 96.4s–1 for GlcNAc3, 0.044 mM and 30.1 s–1 for GlcNAc4 0.045 mM and 14.7 s–1 for GlcNAc5, and 0.047 mM and 8.3 s–1 for GlcNAc6, respectively. These results suggest that this β-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates. © 1996, Taylor & Francis Group, LLC. All rights reserved.