Substrate specificity of chitinases from two species of fish, greenling, Hexagrammos otakii, and common mackerel, Scomber japonicus, and the insect, tobacco hornworm, Manduca sexta.
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 parameters, 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 alpha-chitin > beta-chitin >> silkworm cuticle alpha-chitin. On the other hand, the relative activities of HoChiB and HoChiC were beta-chitin >> silkworm alpha-chitin > shrimp and crab alpha-chitin. MsChi535 preferred silkworm alpha-chitin to shrimp and crab alpha-chitins, and no activity was observed toward beta-chitin. MsChi386, which lacked the C-terminal linker region and the chitin-binding domain, did not hydrolyze silkworm alpha-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.
Matsumiya
Masahiro
M
Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa, Japan. matsumiya@brs.nihon-u.ac.jp
Arakane
Yasuyuki
Y
Haga
Atsunobu
A
Muthukrishnan
Subaratnam
S
Kramer
Karl J
KJ
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D048271:Chitosan / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence; D005399:Fishes / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D018402:Manduca / Q000737:chemistry / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D012995:Solubility; D013270:Stomach / Q000737:chemistry / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature
D048271:Chitosan; D002688:Chitinases