Expression, purification and characterization of endoglucanase I from Trichoderma harzianum aiming the development of enzymatic blends for production of lignocellulosic ethanol
Monday, April 28, 2014
Exhibit/Poster Hall, lower level (Hilton Clearwater Beach)
Vanessa de Oliveira Arnoldi Pellegrini and Igor Polikarpov, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil
Cellulases have attracted much interest in recent years due to its ability in the bioconversion material lignocellulose in glucose, which can then be converted to ethanol by fermentation. In view of their high cellulolytic activity, the fungus Trichoderma harzianum has a great potential application in biomass, but his cellulolytic complex was not thoroughly characterized. One of the complex enzymes is the endoglucanase I, a cellulase of interest for degradation of lignocellulose and was heterologously expressed in Aspergillus niger. Was realized cloning, expression and characterization of the catalytic domain of the endoglucanase from  T. harzianum. First of all, established efficient protocol of purification and the biochemical characterization has shown that the protein has a molecular mass of 45 kDa. A wide panel of pH revealed that the construction has optimal pH below 3 and preference for acidic pH values in different buffers tested. The optimum temperature is 50°C. The protein has specific activity in CMC, substrate most commonly used, around 11 U/mg when tested in citrate buffer pH 3 and temperature  50°C. When the substrate changes to xyloglucan, this activity rises to 30U/mg. The influence of pH and temperature on the secondary and tertiary structures and enzymatic activity were analyzed by CD spectroscopy, and these studies showed that regular protein tertiary structure loses from pH 5, but its secondary structure is disordered only extremely basic pHs, while above 8 at 25 ° C.