Identification of a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from marine Zoanthid Palythoa caribaeorum (Cnidaria, Anthozoa, Zoanthidea).

beta-N-Acetylhexosaminidases (EC 3.2.1.52) belong to an enzyme family that hydrolyzes terminal beta-d-N-glucosamine and beta-d-N-galactosamine residues from oligosaccharides. In this report, we purified a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from the marine zoanthid Palythoa caribaeorum by applying ammonium sulfate fractionation, affinity chromatography on a chitin column, followed by two rounds of size exclusion chromatography. SDS-PAGE analysis indicated a single band protein of apparent homogeneity with a molecular mass of 25kDa. The purified enzyme preferentially hydrolyzed p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylglucosamide (pNP-GlcNAc) and to a lesser extent p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylgalactosamide (pNP-GalNAc). Detailed kinetic analysis using pNP-GlcNAc resulted in a specific activity of 57.9 U/mg, a K(m) value of 0.53 mM and a V(max) value of 88.1 micromol/h/mg and k(cat) value of 0.61s(-1). Furthermore, purified Pcb-NAHA1 enzyme activity was decreased by Hg Cl(2) or maltose and stimulated in the presence of Na(2)SeO(4,) BaCl(2), MgCl(2,) chondroitin 6-sulfate, and phenylmethylsulfonylfluoride. The optimum activity of Pcb-NAHA1 was observed at pH 5.0 and elevated temperatures (45-60 degrees C). Direct sequencing of proteolytic fragments generated from Pcb-NAHA1 revealed remarkable similarities to plant chitinases, which belong to family 18, although no chitinase activity was detected with Pcb-NAHA1. We conclude that beta-N-acetylhexosaminidases, representing a type of exochitinolytic activity, and endo-chitinases share common functional domains and/or may have evolved from a common ancestor.

Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets.

The digestive system of Ceratitis capitata was characterized during its larval development and in the insect stage. Disaccharidases against maltose and sucrose were more evident in the 2nd and 3rd day of larval development and in the adult stage, respectively. Glycosil-hydrolyses with higher specific alpha-galactosidasic and beta-galactosidasic activities were detected in the 2nd and 3rd day of the larval stage, respectively. Specific proteolytic activities against azocasein showed an increase in the 4th and 5th day of the larval stage and in the adult stage. Specific hemoglobin activities were constant between 2nd and 6th day of the larval stage. The larvae used mainly serine proteinases, such as trypsin/chymotrypsin, and the adult insects only chymotrypsin-like enzymes in their digestive process. Two serine proteinases were separated from zymogram between the 4th and 5th day of larval development and in the adult stage. Effect of soybean trypsin inhibitor (SBTI, a serine proteinase inhibitor) on development of C. capitata was examined by bioassay. C. capitata was susceptible to SBTI which affected larval mass at ED50 3.01%.