Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/5/2005
Publication Date: 3/1/2006
Citation: Matsumiya, M., Arakane, Y., Haga, A., Muthukrishnan, S., Kramer, K.J., Beeman, R.W. 2006. Substrate specificity of chitinases from two species of fish, greenling, hexagrammos otakii, and common mackerel, scomber japonicus, and the insect, tobacco hornworm, manduca sexta. Insect Biochemistry and Molecular Biology 70: 971-979. Interpretive Summary: Chitin is one of the most abundant biomasses in the world, second only to cellulose, and is now regarded as a renewable resource. Enzymes such as chitinase hydrolyze the chitin polymer and are used to process chitin-containing waste materials for recycling purposes. Chitinases are widely distributed in organisms, and have been isolated and characterized from a variety of sources. In a collaborative project with scientists at Kansas State University, Nihon University, and the National Institute of Agrobiological Sciences in Japan, we have isolated three chitinases from two species of fish and one species of insect, and compared their catalytic properties using several different kinds of naturally occurring chitinous substrates. Our results demonstrate that fish and insect chitinases possess unique substrate specificities that are correlated with their physiological roles in the digestion of chitins found in foods and biological tissues. One or more of these enzymes could be used in the waste management industry to breakdown microbial and animal byproducts that contain chitin.
Technical Abstract: Three chitinase isozymes, HoChiA, HoChiB and HoChiC, were purified from the stomach of the greenling, Hexagrammos otakii, by ammonium sulfate fractionation, followed by column chromatography on Chitopearl Basic BL-03 and CM-Toyopearl 650S. The molecular masses and pIs of HoChiA, HoChiB and HoChiC are 62 kDa and pH 5.7, 51 kDa and pH 7.6, and 47 kDa and pH 8.8, respectively. Substrate specificities of these chitinases were compared with those of another fish stomach chitinase from the common mackerel, Scomber japonicus (SjChi), as well as two from the tobacco hornworm, Manduca sexta (MsChi535 and MsChi386). The efficiency parameter, kcat/Km, toward glycolchitin for HoChiA and SjChi were larger than those for HoChiB and HoChiC. The relative activities of HoChiA and SjChi toward various forms of chitin were as follows: shrimp shell or crab shell a-chitin > ß-chitin >> silkworm cuticle a-chitin. On the other hand, the relative activities of HoChiB and HoChiC were ß-chitin >> silkworm a-chitin > shrimp and crab a-chitin. MsChi535 preferred silkworm a-chitin to shrimp and crab a-chitins, and no activity was observed toward ß-chitin. MsChi386, which lacked the C-terminal linker region and the chitin-binding domain, did not hydrolyze silkworm a-chitin. These results demonstrate that fish and insect chitinases possess unique substrate specificities that are correlated with their physiological roles in the digestion of food or cuticle.