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

Abstract The mollusk shells present distinctive microstructures that are formed by small amounts of organic matrices controlling the crystal growth of calcium carbonate. The shell of Pinctada fucata has the prismatic layer consisting of prisms of single calcite crystals and the nacreous layer consisting of aragonite tablets. The calcite crystal of prisms contains 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, to reveal the function of those organic matrices in calcium carbonate crystallization, calcites synthesized in chitin gel with or without chitinolytic enzymes were analyzed by using transmission electron microscope (TEM) and atom probe tomography (APT), showing ion clusters derived from chitin inside of a calcite and small angle grain boundaries at optimal chitinolytic concentration. Furthermore, we performed the experiment in which chitinase inhibitor was injected into a living P. fucata. Nanoindentation and electron back scattered diffraction (EBSD) show that mechanical properties and crystal orientation were changed. These results suggested that chitinolytic enzymes work cooperatively with chitin to regulate the crystal growth and mechanical properties of the prismatic layer.