Functional analyses of chitinolytic enzymes in the formation of calcite prisms in Pinctada fucata.

Abstract Background The mollusk shells present distinctive microstructures that are formed by small amounts of organic matrices controlling the crystal growth of calcium carbonate. These microstructures show superior mechanical properties such as strength or flexibility. The shell of Pinctada fucata has the prismatic layer consisting of prisms of single calcite crystals. These crystals contain small angle grain boundaries caused by a dense intracrystalline organic matrix network to improve mechanical strength. Previously, we identified chitin and chitinolytic enzymes as components of this intracrystalline organic matrix. In this study, we analyzed the function of those organic matrices in calcium carbonate crystallization by in vitro and in vivo experiments. Results We analyzed calcites synthesized in chitin gel with or without chitinolytic enzymes by using transmission electron microscope (TEM) and atom probe tomography (APT). TEM observations showed that grain boundary was more induced as concentration of chitinolytic enzymes increased and thus, chitin became thinner. In an optimal concentration of chitinolytic enzymes, small angle grain boundaries were observed. APT analysis showed that ion clusters derived from chitin were detected. In order to clarify the importance of chitinolytic enzymes on the formation of the prismatic layer in vivo, we performed the experiment in which chitinase inhibitor was injected into a living Pinctada fucata and then analyzed the change of mechanical properties of the prismatic layer. The hardness and elastic modulus increased after injection of chitinase inhibitor. Electron back scattered diffraction (EBSD) mapping data showed that the spread of crystal orientations in whole single crystal also increased by the effect of inhibitor injections. Conclusion Our results suggested that chitinolytic enzymes may function cooperatively with chitin to regulate the crystal growth and mechanical properties of the prismatic layer, and chitinolytic enzymes are essential for the formation of the normal prismatic layer of P. fucata.