A Viscometric Determination of -Amylase Activity on Cassava Starch: Technique Optimization and Validation for Endomycopsis fibuligera -Amylase (poster)

C.F. GONZÁLEZ; J.I. FARIÑA; N.I. PEROTTI; L.I.C. FIGUEROA

Chicago

Congreso; 99th GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY; 1999

ASM

The colorimetric methods for endoglucanase determination are based on the release of reducing groups from soluble substrates. This assays usually are time-efficient but the main drawbacks are the sensitivity and the interference of reducing sugars in the enzyme sample or generated by exo-acting enzymes. Likewise, the feasibility of alkaline hydrolysis during the reducing sugars determination could determine an inespecific increase not due to the enzyme activity. In this work a viscometric method to measure alpha-amylase activity with high viscosity cassava starch aqueous solutions was developed, considering the advantage that this technique is not interfered by reducing sugars. It was observed an exponential relationship between the apparent viscosity and the concentration of starch, showing a non-newtonian, pseudoplastic and tixotropic behavior in association with a high consistency coefficient and a low flow behavior index. No significant differences were found among the diverse starch preparations. It was selected the optimal starch concentration (5% w/v) and shear rate (0.168 1/s) for working over a reliable viscosity measurement range. Measurements were carried out in optimal conditions for enzyme activity. The determination of the slope corresponding to the decrease in viscosity of the starch solution was expresed as arbitrary viscometric units and correlated to the commercial enzyme activity (IU). It was found a linear relationship and a very good correlation factor until 0.1 IU. Taking into account the final reaction volume, this value corresponded to 0.01 IU/ml, denoting a high sensitivity of the method. We further proceeded to determine the activity of the Endomycopsis fibuligera alpha-amylase and similar results were found. Likewise, the correlation with conventional methods such as the determination of reducing sugars and the colorimetric assay with iodine was also evaluated.