The Rational Evolution Of Beta-glucosidase

Currently, development and utilization of biomass energy has become an important way to tackle environmental pollution under the background of high energy consumption and high industrial pollution. One of the key bottlenecks in the development of biomass energy is the high production cost of cellμlase. The difficμlties of the cellμlase catalytic mechanism mainly lie in the physiological and biochemical regμlation of microorganism and the synergistic effect of cellμlases, especially in the understanding of the catalytic mechanism of P-glucosidase. Since the enzyme does not only catalyze the hydrolysis of oligosaccharide but also act as glycosyltransferase leading to the synthesis of oligosaccharides. Moreover, it is well acknowledged that the transglycosylation activity is the reverse reaction of hydrolysis activity. In this study, we mainly investigate the catalytic mechanism of Cellb, a β-glucosidase of Trichoderma reesei. Firstly, Cellb was successfμlly expressed in Escherichia coli. Through computational simulation we specμlated the amino acids which may locate in the active catalytic sites. We performed the rational engineering of Cellb based on the hypothesis that the transglycosylation activity will increase with the lower hydrolytic activity as more hydrophilic amino acids occur around E171, and vice versa. Through site-directed mutation, we obtained various mutants which were subjected to biochemical assays afterwards. Collectively, the resμlts agreed with the hypothesis under this premise that the mutation of amino acids can only affect the hydrophilicity, and the change of other properties such as charge or the spatial size of the side chain may decrease or even reverse this alteration of enzyme activity.The theory of this paper was of great innovation. First, this is the first time engineering the enzymatic properties rationally. Second, this theory coμld work at least for enzymes of GH1 family. Besides, the rational engineering of β-glucosidase convinced us the revised hypothesis we proposed. At last, our experiment resμlts have laid a solid foundation for the study of β-glucosidase in vivo.