The Study Of Enzyme’s Mechanism And Design Method Base On The Structure And Computation

With the development of biotechnology, people’s cognition of the life system is stronger and stronger, the same as wishing serve humanity by the control and transformation of the structure and function of living systems. One important object is the design and transformation of enzyme molecules. Unfortunately, the cognition of catalytic mechanism of the enzyme molecules and theoretical system of the functional design and transform is weak now. Research methods, especially computing theory and tools cannot meet the needs. Both of these restrain the development of relevant field. In this paper, taking lipase and glucosidase as the research object, we studyed a series of enzyme catalytic mechanisms and design transformation theory and methods, and has made some breakthroughs.In this paper, we took the lipase LipK107with adequate research base and excellent performance as the research object. First, we got its three-dimensional structure in the way of protein crystallization and X-ray diffraction method. Then, by means of bioinformatics and computational biology, based on the previous study of the lipase’s catalytic mechanism, we inferred the catalytic mechanism of this enzyme.In this paper, using the programming language C++, we have written a calculation and analysis software of protein thermal stability. This software, based on TK-SA protein model, analyzed amino groups which have a stronger contribution to the stability, as well as the overall stability of the protein by calculating electrostatic interaction between internal ionizable protein amino acid residues. At the same time, based on the calculation result of the software, we designed the mutagenesis experiments, and successfully improved the thermal stability of LipK107.As to the chiral selectivity of the lipase LipK107substrate, this paper innovatively proposed that conformation and energy are equally important and both are the key factors influencing the chiral selectivity. According to this theory, we designed some expected positive and negative mutant experiments based on a novel charge-biasing force strategy and in silico filter. The result was completely consistent with the expected results, thus supporting our hypothesis. And it also provides new ideas and methods for the transformation of enzyme substrates and the chiral selectivity.In consideration of fact that LipK107and glucosidase have the same substrate, but two different products, this paper proposed that the binding free energy of product and enzyme affects the product selectivity. Using MM/PBSA calculation method, we conducted the parallel control calculation for isomaltuloses of a seaweed ketose main production and a major producing glucosidase. The results showed that the binding free energy of the main products into enzyme is always less than the by-products, thus verifying our hypothesis which provides new resolution to the enzyme product option transformation.Using QM/MM calculation method, we calculated and analyzed the hydrolysis reaction path of lipase LipK107, identified the reaction path of the hydrolysis reaction and the potential barrier for the completion of this reaction path. At the same time, we achieved the establishment of a set of simple and efficient computational strategy, which provides valuable theoretical and practical reference for calculation and design of enzymes with high activity based on reducing the potential energy barrier.For the overall mechanism cognition of thermal stability, pH stability, substrate (chiral) selectivity, product selectivity of the catalytic reaction and catalytic reaction activity, we developed a number of useful computational tools and strategies, and put forward a number of unique theories and cognition of the mechanism. This provides a lot of reference materials for subsequent researchers.