Enzyme immobilization to modified and patterned surfaces for biocatalytically active surface preparation

Abstract

Slate was cultivated at pH condition of 5, thus, the goal of this work was to evaluate their catalytic capacity of whole-cell by assay of para-nitrophenyl palmitate (pNPP). Bacterial cells were cultivated during 48 hours. Samples of different times were taken and cells were separated by centrifugation, washed three times and air dried at 40 • C. The reaction mixture consisted in 20 mg of cells, 0.5 ml of stock solution of pNPP (10 mM in n-heptane). To start the reaction 30 l of methanol or ethanol were added to the mixture an incubated at 40 • C and 160 rpm from 0.5 to 120 min. Then, 25 l of mixture were taken and mixed with 1 ml of 0.1 M of NaOH. Additionally, the reaction mixtures were incubated with no alcohol. The pNPP liberated was extracted by aqueous alkali phase and detected at 405 nm. The kinetics curves of samples demonstrated that the whole-cells cultivated at 12 hours and 24 hours presented the highest production of pNP with methanol during the first 10 min of reaction. After the lipase activity decreased and remains practically constant until the end of the experiment. The reaction with ethanol shows a maximal activity in samples of 24 hours of culture in 10 min of reaction. The lipase activity observed with no alcohol in the reaction was at a value of one third of that obtained in cells with methanol. The results presented here, using a whole-cell biocatalyst is considered promising for biodiesel production in industrial applications.Surface modification and enzyme immobilization studies were conducted for catalytically active surface preparation. Bare glass substrates were modified with metal oxide thin films by dip coating and gold nanoparticles (AuNPs) were introduced by physical entrapment into porous sol-gel matrix. According to surface morphology analysis conducted with atomic force microscopy (AFM) and scanning force microscopy (SEM), it was observed that homogeneous and smooth thin layers were obtained and presence of AuNPs were verified by UV spectroscopy and EDX analysis. Model enzyme invertase was immobilized covalently to the modified surfaces by thiolamine linker, cysteamine. The degree of enzyme immobilization was determined by following enzyme reactions spectrophotometrically. Further, by photolithographic methods, surface patterns were obtained to achieve different catalytically active site arrays which can be suitable for applications as bienzyme electrodes for sensors or as microreactors for biocatalytic synthesis and biotransformations.In the development of biosensors, studies have been focused on immobilization of enzymes onto electrode surfaces and the use of nanomaterials. Immobilization of enzymes onto thin films offers construction of smaller and fast response biosensors taking advantage of improved nanoscale properties. Gold nanoparticles are mostly used in biosensors as their biocompatibility and used as small conductive centers. In this study, conducting ITO glass substrates were coated by metal oxide thin films with gold nanoparticles, which were synthesized by so...