Research On Modification Of Catalytic Character Of Enzymes Base On Bioinformatics

As the rapid progress of information technology in the aspect of hardware,software, and machine's deep learning, and the better understanding of the essence of life, bioinformatics, as a part of the general biology, has been rapidly developed. Bioinformatics has been employed to research on molecular biology, genomics, proteomics, and structural biology. The developing databases, theories and methods (e.g. molecular mechanics,molecular dynamics and quantum mechanics), and computing technology are informational foundation, technical foundation and efficient foundation of bioinformatics, respectively. Hence, the main work of this thesis focuses on how to combine experimental technology and bio-informational methods to modify the catalytic character of enzymes.Protein is the final form of genetic expression with the vitality of life.Bioinformatics not only could facilitate people to understand the relationship among origin, evolution, structure and function; but also could guide people to tailor protein in order to obtain the novel biomolecules. Enzyme is a kind of protein and is a technical core of biocatalysis. However, with the development of the bio-catalytic technology in industrial field, the catalytic characters of natural enzyme could not afford the requirements of bio-catalysis from the view of more stable catalytic processes, the wider catalytic substrates/products and the cheaper application cost.Hence, the main work of this thesis focuses on how to understand the scientific discipline among evolution, structure and catalytic character; and which strategies is suitable to modify the catalytic character of enzyme. Our work developed three computing models based on bioinformatics in order to solve three general problems in the field of directed evolution, rational design and surface modification, respectively; verified and optimized the computing model of bioinformatics in the relative enzymatic system in practice.The study includes the following three parts:1. In the process of natural evolution, a huge number of random mutation and the accumulation of large population and a long time must be faced. A general problem of directed evolution is how to refine the larger genetic library with the knowledge of natural evolution in order to develop the smart library. Our research started from the analysis of the energy of unfolding of protein (AG) predicted by FoldX, explored the relationship among sequence,structure, and function, developed a selection model for building DNA recombination smart library. Our research employed Bacillus subtilis lipase A(BSLA) to evolve recombinant mutants towards the ionic liquid ([BMIM][Cl])resistance, reduced the ratio of inactive mutant in the DNA recombination library, and developed a virtual directed evolution process towards the ionic liquid ([BMIM][C1]) resistance of lipase (BSLA) combining evolution-based approach and energy-based approach. The desired mutant(F17S-V54K-D64N-D91E) obtained from virtual directed evolution strategy was the same with the desired mutant, and its substituted sites on F17 and V54 amino acid position of lipase (BSLA) was the same with the desired mutant from experimental directed evolution.2. Exploring the relationship between enzyme and substrate is the effective method to understand the catalytic mechanism and catalytic specificity, and is the important basis of rational design towards the substrate specificity. Our research started from the analysis of the binding energy between receptor and ligand, developed the model of molecular mechanics to explore the interaction between polyphosphate kinase and substrate.According to the model of molecular mechanics base on molecular docking,our research employed Corynebacteriunm glutawmicum polyphosphate kinase(PPK2 (NCg12620) to reveal the dimer catalytic mechanism which could offer an alternative ADP binding pocket to overcome the inhibition of short polyphosphate (PolyP). Then, our work tailored Sinorhizobium meliloti polyphosphate kinase (PPK2(SMc02148)) to rebuild the ADP binding pocket(His102Lys-Ala106Glu-Val115Thr) of PPK2(SMc02148) base on the dimer catalytic mechanism of PPK2 (NCgl2620) , developed an in vitro ATP regeneration system with the mutant of PPK2(SMc02148) utilizing PolyP(4) as phosphate donor, and produced glutathione titer (38.79 mM) and glucose-6-phosphate titer (87.35 mM) in cascade reactions with ATP regeneration using the mutant of PPK (SMc02148).3. How to understand and predict the conformation-function relationship between enzyme and modification molecule is a general problem of high-efficient screening towards modification molecules. Our research developed the molecular dynamic simulation model to explore the interaction between enzyme and modification molecule in solvents. According to the above model, our research employed Yarrowia lipolytica Lipase 2 (YLLIP2)to investigate the mechanism of the thermo-induced inactivation and polar organic solvent-induced inactivation of YLLIP2, revealed the improvement mechanism of ?-cyclodextrin on the thermo-stability and methanol resistance of YLLIP2 through the interaction between the hydroxyl group of(3-cyclodextrin and the polar residues on the surface of YLLIP2, indicated the positive effect of glucose-enzyme super-molecular conformation on the methanol resistance of YLLIP2 through surface modification according to the modification molecular hydroxyl group clues, and proved that glucose as additive improved the enzymatic synthesis of biodiesel with YLLIP2.