Enzymatic Hydrolysis of Soluble Starch with an r-Amylase from Bacillus licheniformis V. Bravo Rodrı ´guez,* E. Jurado Alameda, J. F. Martı ´nez Gallegos, A. Reyes Requena, and A. I. Garcı ´a Lo ´ pez Departamento de Ingenierı ´a Quı ´mica, Universidad de Granada, Avd. Fuentenueva s/n, 18071 Granada, Spain The enzymatic hydrolysis of soluble starch with an R-amylase from Bacillus licheniformis (commercial enzyme Termamyl 300 L Type DX) have been experimentally studied at pH 7.5, within the temperature range of 37-75 °C, at initial substrate concentrations of between 0.25 and 2.00 g/L, and enzyme concentrations of between 0.575 × 10 -4 and 13.8 × 10 -4 g/L. To follow the reaction a procedure based on the iodometric method for measuring R-amylase activity was used. The kinetics of the enzymatic hydrolysis was fitted to the Michaelis-Menten equation using the integral method, taking into account that the thermal deactivation of the enzyme follows a second-order kinetic. These parameters were fitted to the Arrhenius equation obtaining activation energies of 24.4 and 41.7 kJ/mol and preexponential factors of 734.9 g/L and 1.74 × 10 8 min -1 for K M and k, respectively. 1. Introduction The amylases (R-amylases, -amylases, and glucoamylases) are one of the most important families of enzymes in the field of biotechnology. Among these the R-amylases have the widest range of industrial applications. Their inclusion in detergents, for example, affords numerous advantages such as the saving of energy because they require lower temperatures and the reduction or replacement of other components that may be more harmful to the environment. They are also biodegradable and act without risking aquatic life or having a negative effect upon wastewater treatment. R-Amylase (E.C. 3.2.1.1., 1,4-R-D-glucan glucanohydrolase) hydrolyzes starch by randomly cleaving the internal R-1,4 glucosidic bonds, thus giving rise to smaller, more soluble compounds and facilitating the elimination of starchy dirt. This process allows more moderate washing temperatures and pH conditions than those permitted by traditional detergent formulas. Numerous studies have been made into the kinetics of starch hydrolysis with R-amylases. The kinetic models used can be divided into two categories: deterministic and probabilistic (1), the former being the most favored. Within the deterministic approaches most authors have fitted their kinetic data to the Michaelis-Menten equation (2-12), whereas only a few have used empirical models (13-15). To calculate the parameters K M and k of the Michaelis-Menten equation it is usual to resort to the initial-reaction-rate method followed by Lineweaver- Burk linearization. This method facilitates the interpretation of the experimental results and avoids the influence on the reaction rate of factors such as loss of enzyme activity through changes in pH, thermal deactivation, and inhibition by the reaction products. Nevertheless, the kinetic model obtained in this way is only valid for the initial moments of the reaction and may not be so at later times. In the present work, to extend the validity of the model to experiments over longer periods of time, the integral method has been applied to the evaluation of K M and k. To do this it is necessary to know the thermal deactivation kinetic of the enzyme versus temperature, which was established in a previous publication (16). 2. Materials and Methods 2.1. Enzyme and Substrate. A commercial R-amylase (Termamyl 300 L Type DX) from Novozymes A/S was used. This enzyme is used particularly in laundry and dishwashing detergents. The enzyme was assayed at concentrations between 0.575 × 10 -4 and 13.8 × 10 -4 g/L at temperatures of be- tween 37 and 75 °C and with a constant pH value of 7.5 (0.1 M phosphate buffer). Soluble potato starch (Panreac Quı ´mica S.A., Barcelona, SP, 121096) was used as substrate at initial concentrations between 0.25 and 2 g/L, moist weight basis, although hereafter the concentrations referred to are dry weight basis. To measure the moisture in the starch, two 5 g samples were dried for 25 min at 100 °C on an infrared balance (model AD-4714A from AND). The average water content was 16%. 2.2. Experimental Procedure. To follow the enzyme reaction the procedure described in a previous publication (16) was used, based on Fuwa’s iodometric method for measuring amylase activity (17). The volume of reaction mixture was 5 mL. The enzyme was dissolved to the desired concentration with a solution of phosphate buffer (0.1 M, pH 7.5). As reaction substrate a stock solution of starch (0.5% w/v) in phosphate buffer (0.1 M, pH 7.5) was prepared. For the experiments the quantity of starch solution necessary for the final concentration desired was put into a test tube and made it up to 4.95 mL with phosphate buffer (0.1 M, pH 7.5). This solution was kept at the reaction temperature for 10 min before 0.05 mL of the enzyme solution was added and the reaction was left for the desired time. The reaction was stopped using the method described by Paolucci-Jeanjean et al. (18). Once the enzyme was deactivated the starch was stained with iodine according to Fuwa (17). After 10 min (19) the absorbance was measured at 575 nm in a spectrophotometer (model HEλIOS * To whom correspondence should be addressed. Ph: +34 958243310. Fax: +34 958248992. E-mail: vbravo@ugr.es. 718 Biotechnol. Prog. 2006, 22, 718-722 10.1021/bp060057a CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical Engineers Published on Web 03/31/2006