Bioinformatics Research On β-Agarase B And B And Amino Acid Mutation

Research on Agarase is one of the most important projects in the marine molecular biology. We successfully cloned and expressed two agarase gene from the marine microbe and promoted the research on agarase. With the development of the research on β -agarase A and B which were discovered in our lab, we have known more about the enzyme characteristic. In this paper, bioinformatics softwares were used to analyze the 3D characteristic of 3 -agarase A and B. Sequence search, profile search, homology modeling, family search and analysis and threading methods were used to decipher the unique enzyme characteristics of 3 -agarase A and B. Information given by the model and docking work helped me to design the moleculear biology experiment. Amino acid mutation experiment was carried out based on the result of the bioinformatics research.The brief introduction of my work:Bioinformatics software were used to analyze the β -agarase A. The sequence, motif, prosite information were collected and 3D structures were modeled with multi methods. The structure healthy check softwares were used to validate the model and the final complete structure was joint with the best model structure of the two domains. Visual analyze tools helped finding the key loop in the binding site. A hypothesis was developed that the loop comprising TRP244 may block the linear agarose going through the binding site which can explain the enzyme characteristic. A full flexible docking method was used to evaluate the energy changes of two different binding modes. The result also proveed the hypothesis.Sequence similarity search, motif search, distance homology recognition and threading method were used to find the secret of β -Agarase B. With low similarity with other known enzymes, the structure of β -agarase B could not be built by traditional homology method. Distance homology recognition and threading methodhelped building 5 model structures. Based on the alignment between the models and template enzymes and the knowledge of families information of the templates, 5 residues were proposed to be the potential catalytic residues which are D217, D221, D236, D245, D288. Based on the 5 models built with the distance homology recognition and threading method, Autodock software was used to dock disaccharides of agarose to the binding site automatically. The acid residues which are in 10 A distance to the disaccharides were collected. The D176, D221, D236, D245 may play the catalytic role in the binding site which will be researched in the molecular biology research.Mega primer PCR method was used to mutate amino acid in P -agarase A. Two mutated enzymes E124A and El29A were generated. The experiments have proven the mutation induced the lost of enzymatic capability. The hypothesis made with the bioinformatics was proved to be right and the molecule modeling method and protein engineering will help to identify the characters of other enzymes.Bioinformatics methods were used to analyze the two agarases and built high quality models, and the docking methods were also used to get the detail information to guide the molecular biology experiments. This paper proves that it is very helpful to use the bioinformatics method in the biology experiment, especially the protein structure-activity analysis.