Effect of Carbonic Anhydrase Immobilized on Eggshell Membranes on Calcium Carbonate Crystallization In Vitro
The eggshell membranes (ESM) serve as the first interface with the inorganic phase during eggshell formation. During mineral growth, crystals nucleate on the outer side of the ESM at specialized sites called mammillae, mainly consisting of mammillan, a keratan sulfate proteoglycan together with the activity of carbonic anhydrase (CA).
In order to get insight into the mechanisms of chicken eggshell mineralization, ESM was used as a biotemplate for immobilizing carbonic anhydrase (CA) and study in vitro calcite crystallization. Here, we showed that when the eggshell membrane supplemented with immobilized or dissolved carbonic anhydrase is located at the gas-liquid interface, calcite nucleation and growth are sequestered by the ESM scaffold from solution, thus affecting the morphology and size of the crystals formed.
KeywordsEggshell membrane Carbonic anhydrase Biomineralization
Biomineralization is a widespread phenomenon in nature leading to the formation of a variety of solid inorganic structures by living organisms, such as intracellular crystals in prokaryotes; exoskeletons in protozoa, algae, and invertebrates; spicules and lenses; bone, teeth, statoliths, and otoliths; eggshells; plant mineral structures; and also pathological biominerals such as gall stones, kidney stones, and oyster pearls (Lowenstam and Weiner 1989; Mann et al. 1989; Simkiss and Wilbur 1989; Heuer et al. 1992; Arias and Fernandez 2008). By this process living organisms precipitate inorganic minerals on organic matrices. The resulting biominerals are deposited in elaborated shapes and hierarchical structures by interaction at the organic-inorganic interface, where the rate of crystal formation is regulated by the control of the microenvironment in which such mineralization events take place. One of the main biomineralization resulting mineral minerals is calcium carbonate (CaCO3), especially the more stable form, calcite. The avian eggshell is a good example of multifunctional biomineral where an organic scaffold (or matrix), the eggshell membrane, plays a crucial role in regulating mineral nucleation and growth by incorporating inorganic precursors, such as ions, ion clusters, and amorphous phases (Rao et al. 2017); other organic matrices complete the formation of a calcified layer (i.e., palisade) composed of calcite columns (Panheleux et al. 1999). Structurally, the eggshell is a multilayered calcitic bioceramic. As the egg migrates through the oviduct, the biomineral matures under the influence of biomolecular additives and an extracellular matrix (Nys et al. 1999; Fernandez et al. 2001). The matrix, primarily composed of collagen fibers, constitutes the eggshell membranes (ESM) (Arias et al. 1991). Each fiber exhibits a core surrounded by a glyco-proteinous material termed as the mantle. This serves as the first interface with the inorganic phase. During mineral growth, crystals nucleate on the outer side of the ESM at specialized sites called mammillae, mainly consisting of mammillan, a keratan sulfate proteoglycan together with the activity of carbonic anhydrase (CA). This metalloenzyme catalyzes the reversible hydration of carbonic dioxide to bicarbonate and a proton. In fact, it has been possible to mimic eggshell formation in vitro by adding the main organic components including CA, where an increase in calcium carbonate crystals growth and fusion was observed (Fernandez et al. 2004).
However, the use of enzymes in vitro is difficult, because of instability and the complexity for maintaining the catalytic function in chemical reactions (Lu et al. 2013). For that reason enzymatic immobilization on a solid substrate appears as a solution for this problem (Wanjari et al. 2013).
For in vitro biomineralization, a specific confined environment is needed, that means an inert scaffold which generates an almost two dimensional interface where the crystal nucleation takes place. Currently, many kinds of surfaces and/or supports are used for enzyme immobilization, and the ESM meets all the requirements to be used as a natural support for CA immobilization and in vitro biomineralization experiments.
In order to get insight into the mechanisms of chicken eggshell mineralization, ESM was used as a biotemplate for immobilizing carbonic anhydrase (CA).
4.2 Material and Methods
4.2.1 Carbonic Anhydrase (EC 220.127.116.11) Immobilization on ESM
ESM were obtained after 30 min incubation of an empty egg in 1% acetic acid to detach the membrane from de shell, and then ESM was incubated for another 48 h in 1% acetic acid to eliminate any remaining calcium carbonate crystals and then washed in deionized water three times (Arias et al. 2008).
For enzyme immobilization 1 mg of carbonic anhydrase (2500 units/mg, Sigma, St. Louis, MO, USA) in 1 mL deionized water was used; membranes were incubated for 1 h with 100 μL of this solution; then 20 μL of 2.5% glutaraldehyde for 30 min was used as cross-linking agent (Tembe et al. 2008). Then, membranes were washed with TRIS buffer solution pH 9 at 4 °C.
4.2.2 Crystallization Experiments
4.3.1 ESM on Bottom of the Microbridge 24 H Incubation
Size of calcite crystals deposited on eggshell membrane (ESM) measured under different conditions
μm ± S.D
ESM at the bottom of the microbridge
21.33 ± 1.97
31.0 ± 2.08
CA in solution
20.0 ± 3.68
ESM at the top of the microbridge facing calcification solution
22.1 ± 1.91
35.83 ± 3.53
CA in solution
27.83 ± 2.47
4.3.2 ESM on Top of the Microbridge Facing the Calcification Medium After 24 H of Incubation
When in vitro CaCO3 crystallization experiments are done in a gas-liquid interface diffusion environment, the favorite place for crystal nucleation and growth must be considered. In fact, in this chamber-mediated experiment, CO2 comes from the gas phase, while carbonate ions are in aqueous solution. If the reaction is done without any additional scaffold, such as the eggshell membrane, it is expected that nucleation of CaCO3 crystals occurs close to the gas-liquid interface, and then, after a determined period of growth, crystals formed precipitate reaching the bottom of the reaction vessel (microbridge). However, here we showed that when an active heterogeneous nucleator scaffold, such as the eggshell membrane supplemented with immobilized or dissolved carbonic anhydrase, is located upside down at the gas-liquid interface for avoiding gravity effect, calcite nucleation and growth are sequestered by the scaffold from solution, thus affecting the morphology and size of the crystals formed. The morphology changes, including calcite aggregations occurs in a way that resembles the calcite column aggregation and fusion observed during natural eggshell formation.
Work supported by Fondecyt project 1150681 from CONICYT.
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