Efficient Entrapment of Carbonic Anhydrase in Alginate Hydrogels Using Liposomes for Continuous-Flow Catalytic Reactions

Abstract

A versatile approach to entrap relatively small enzymes in hydrogels allows their diverse biotechnological applications. In the present work, bovine carbonic anhydrase (BCA) was efficiently entrapped in calcium alginate beads with the help of liposomes. A mixture of sodium alginate (3 wt %) and carbonic anhydrase–liposome conjugates (BCALs) was dripped into a Tris-HCl buffer solution (pH = 7.5) containing 0.4 M CaCl2 to induce the gelation and curing of the dispersed alginate-rich droplets. The entrapment efficiency of BCALs, which was defined as the amount of catalysts entrapped in alginate beads relative to that initially charged, was 98.7 ± 0.2% as determined through quantifying BCALs in the filtrate being separated from the beads. When free BCA was employed, on the other hand, a significantly lower entrapment efficiency of 27.2 ± 4.1% was obtained because free BCA could pass through alginate matrices. Because the volume of a cured alginate bead (10 μL) entrapped with BCALs was about 2.5 times smaller than that of an original droplet, BCALs were densely present in the beads to give the concentrations of lipids and BCA of 4.6–8.3 mM and 1.1–1.8 mg/mL, respectively. Alginate beads entrapped with BCALs were used to catalyze the hydrolysis of 1.0 mM p-nitrophenyl acetate (p-NA) at pH = 7.5 using the wells of a microplate or 10 mL glass beakers as batch reactors. Furthermore, the beads were confined in a column for continuous-flow hydrolysis of 1.0 mM p-NA for 1 h at a mean residence time of 8.5 or 4.3 min. The results obtained demonstrate that the conjugation of BCA to liposomes gave an opportunity to achieve efficient and stable entrapment of BCA in alginate hydrogels for applying to catalytic reactions in bioreactors.

Combining Active Carbonic Anhydrase with Nanogels: Enzyme Protection and Zinc Sensing

“…Many attempts have been made to immobilize CA in various supports, including hydrogels, for the biocatalysis of the reversible conversion of CO 2 to bicarbonate. [35][36][37][38][39][40][41] Studies by Fierke and Thompson have demonstrated another unique application of CA, namely as a zinc biosensor, with advantages well-discussed in the literature. [25][26][27][28][29][30][31] Here we report, for the first time, a proof-of-concept study of combining active CA with nanogels.…”

“… 35 Immobilizing CA on various supports has been used to improve its stability and reusability for CO 2 capture, among which a few attempts have successfully immobilized CA in a hydrogel network. For example, CA-liposome conjugates 36 and metal-organic hybrid nanocomposites 37 , 38 have been reported to be embedded into hydrogel beads and membranes during the polymerization process. CA was also encapsulated in silk protein hydrogel via a dual crosslinking strategy, which employed a photoinduced dityrosine chemical crosslinking followed by a dehydration-mediated physical crosslinking.…”

Enzyme immobilization in hydrogels: A perfect liaison for efficient and sustainable biocatalysis

“…Junshi Moriyama et al. entrapped bovine carbonic anhydrase (BCA) into calcium alginate hydrogel beads with the help of liposomes (BCALs) [ 116 ]. Using the BCALs, 98.7 ± 0.2% of the liposome‐enzyme‐adduct was entrapped compared to 27.2 ± 4.1% when the free BCA was used.…”

Polymers as Encapsulating Agents and Delivery Vehicles of Enzymes

“…They can easily undergo gelation with divalent cations under mild conditions suitable for the incorporation of biomacromolecules. Alginate-based microencapsulation is currently a favored approach for enzyme encapsulation [ 47 , 48 ]. A relatively simple and safe technique to entrap enzymes is the use of calcium alginate beads [ 49 , 50 , 51 ].…”