Caracterização da enzima celobiase com modelagem da cinética de hidrólise da celobiose

• Main Author: Calsavara, Luiza Pedrina Vilxenski
• Publication Date: 1998
• Format: Master thesis
• Language: por
• Source: Repositório Institucional da Universidade Estadual de Maringá (RI-UEM)
• dARK ID: ark:/35916/0013000005dtk
• Download full: http://repositorio.uem.br:8080/jspui/handle/1/3714
• topic: Enzima celobiase; Indústria química; Energia alternativa; Enzima celobiase; Enzimas; Propriedades; Brasil.; Engenharias; Engenharia Química

The enzyme cellobiase Novozym 188, which is used for improving hydrolysis of bagasse with cellulase, was characterized in its commercial available form and integrated kinetic models were applied to the hydrolysis of cellobiose. The specific activity of this enzyme was determined for pH values from 3 to 7, and temperatures from 40 to 75 °C, with cellobiose at 2 g/L. Thermal stability was measured at pH 4.8 and temperatures from 40 to 70°C. Substrate inhibition was studied at the same pH, at 50°C, and cellobiose concentrations from 0.4 to 20 g/L. Product inhibition was determined at 40 and 50 °C, pH4.8, cellobiose concentrations of 2 and 20 g/L, and initial glucose concentration nearly zero or 1.8 g/L. The reaction was monitored by means of conversion data of cellobiose into glucose until nearly 100%. The enzyme has shown the greatest specific activity, 17.8 mmol/min.mg protein, at pH 4.5 and 65°C. Thermal activation of the enzyme followed Arrhenius equation with the energy of activation being equal to 11 kcal/mol for pH values between 4 and 5. Thermal deactivation was adequately modeled by the exponential decay model with energy of deactivation giving 81.6 kcal/mol. Substrate inhibition was clearly observed above 10 mM (3.4 g/L) cellobiose. Kinetic parameters obtained by fitting the experimental points to the substrate uncompetitive inhibition model were Km = 2.42 mM, Vmax = 16.3 mmol/min.mg protein and Ks = 54.2 mM, while for the substrate non-competitive inhibition model they were Km = 2.54 mM, Vmax = 17.1 mmol/min.mg protein and Ks = 51.7 mM. The conversion versus time experimental points for the hydrolysis reaction were fitted to the integrated kinetics models for the uncompetitive and non-competitive substrate inhibition and for competitive, uncompetitive and non-competitive product inhibition. Nevertheless, the kinetic parameters Ki and kcat calculated by these models were inconsistent, indicating that the hypotheses that were assumed were not able to describe the total extension of the reaction kinetics from zero to 100%