Carbonic anhydrase (CA) is a native enzyme that facilitates the hydration of carbon dioxide into bicarbonate ions. This study reports the fabrication of thin films of active CA enzyme onto a porous membrane substrate using layer-by-layer (LbL) assembly. Deposition of multilayer films consisting of polyelectrolytes and CA was monitored by quartz crystal microgravimetry, while the enzymatic activity was assayed according to the rates of p-nitrophenylacetate (p-NPA) hydrolysis and CO2 hydration. The fabrication of the films onto a nonporous glass substrate showed CO2 hydration rates of 0.52 +/- 0.09 mu mol cm(-2) min(-1) per layer of bovine CA and 2.6 +/- 0.7 mu mol cm(-2) min(-1) per layer of a thermostable microbial CA. The fabrication of a multilayer film containing the microbial CA on a porous polypropylene membrane increased the hydration rate to 5.3 +/- 0.8 mu mol cm(-2) min(-1) per layer of microbial CA. The addition of mesoporous silica nanopartides as a film layer prior to enzyme adsorption was found to increase the activity on the polypropylene membranes even further to a rate of 19 +/- 4 mu mol cm(-2) min(-1) per layer of microbial CA. The LbL treatment of these membranes increased the mass transfer resistance of the membrane but decreased the likelihood of membrane pore wetting. These results have potential application in the absorption of carbon dioxide from combustion flue gases into aqueous solvents using gas-liquid membrane contactors.