Flow injection analysis system for on-line cutinase activity assay

A FIA system based on a micellar system for a substrate (p-nitrophenylbutyrate) with low stability in aqueous phase was built to monitor cutinase activity in bioprocesses. All samples were previously diluted with 50 mM phosphate buffer pH 7.0 containing 11.6 mM sodium cholate. The cutinase activity in this diluted solution enhances about 40% in relation to phosphate buffer. Furthermore, the enzyme adsorption and consequent blocking/fouling of injector and tubes of the FIA system was eliminated due to excellent properties of sodium cholate as surface active agent. The cutinase activity is based in following the hydrolysis of pnitrophenylbutyrate to p-nitrophenol in the reaction stream through the formation of an absorbance peak at 400 nm proportional to enzyme activity. The compositions of reaction stream as well as its stability were studied in order to minimize non-enzymatic hydrolysis of p-nitrophenylbutyrate and maximize cutinase activity assay reproducibility. An excellent correlation was obtained between the FIA system and off-line method for determination of cutinase activity in the culture media, and during separation of Saccharomyces cerevisiae cells and cutinase concentration by micro and ultrafiltration, respectively.