Crystal structure studies of Thermotoga maritima cellulase TmCel5A and enzyme engineering of Escherichia coli phytase EcAppA
Date Issued
2014
Date
2014
Author(s)
Wu, Tzu-Hui
Abstract
Exogenous enzymes have been produced and applied to many industrial fields for their abilities of accelerating the rates of the chemical reactions in a substrate specific manner. However, for the industrial applications, the traits of a natural derived enzyme need to be amended, such as the optimal conditions and the stability. Protein structures can be visualized by X-ray diffraction crystallography at atomic level. Thus, the interactions between the residues within protein structures and between substrates can be revealed for the rational design enzyme engineering. In our laboratory, we are devoted to utilize the information from protein structures for further protein engineering. In the thesis, two enzymes were studied. First, the structures of apo form and complex form of a thermostable cellulase were solved to give the whole picture of how the diverse substrates can be accommodated to the active site. Second, by structure-based ration design, the specific activity and thermostability of a phytase were improved.
Lignocellulosic bioethanol is a highly potential alternative energy. Enzymes with thermostablility and multiple substrate specificities are preferred to cooperate synergistically, owing to the complexity of the lignocellulosic biomass composition and the production process. A thermostable cellulase Cel5A from Thermotoga maritima, characterized with activity toward both glucan- and mannan-based mainchain and simple and branched chains, is an excellent candidate for industrial application. Here, we report the crystal structures in apo-form and in complex with three ligands, cellotetraose, cellobiose and mannotriose, at 1.29 A to 2.40 A resolution. The open carbohydrate-binding cavity which can accommodate oligosaccharide substrates with extensively branched chains explained the dual specificity of the enzyme. Moreover, our results also suggest that the wide spectrum of TmCel5A substrates might be due to the lack of steric hindrance around the C2-hydroxyl group of glucose or mannose unit from the active-site residues.
Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6 to 9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 33.1% residual activity.
In conclusion, the structural information of TmCel5A in complex with its ligands was provided and open up a sight for future dual substrate activity engineering of enzymes in the large glycosyl hydrolase family 5 (GH5) for applications. On the other hand, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications.
Subjects
X-光晶體繞射法
酵素工程
理性設計法
纖維素酶
葡聚糖內切酶
植酸酶
醣基化
Type
thesis
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