Study On Site-Directed Mutagenesis And Directed Evaluation Of Bacillus Amyloliquefaciens SacB1 Gene

Levansucrase is a kind of microbial enzyme which can catalyze sucrose to produce levan, accompanied by by-products of glucose, fructose and fructooligosaccharides. Levansucrase has very important applications in the fields of food, medicine, etc. BA-SacB1, the levansucrase from Bacillus amyloliquefaciens has a application potential because of its high catalytic activity and stability. However, until now no study about the catalytic mechanism of this enzyme has ever been reported. In this study, bioinformatics analysis about BA-SacBl was carried out, and some amino acids which may be related to catalytic activity were selected and performed site-directed mutagenesis, and the mutants were performed enzyme activity analysis to explore of the catalytic mechanism of BA-SacBl. In addition, DNA sequence encoding signal peptide was removed to investigate the effect of signal peptide on activity, stability and secretory expression properties of BA-SacBl. Additionally, BA-SacB1 was performed BLAST to find that it has a "redundant" section in its amino acid sequence when compared with that of levansucrase from Haloarcula japonica. Therefore, this "redundant" section was removed by overlap extension PCR, and the effect of this section on enzymatic characteristics of BA-SacB1 was studied, and the related information about molecular evolution was analyzed, all of which have never been reported.The activity of BA-SacBl, especially the levan-producing activity need to be further improved. Directed evolution is a very important and efficient technology for protein modification in recent years. This technology is equally efficient even when the catalytic mechanism of the enzyme is still not clear. Until now, no study about directed evolution of levansucrase has been reported, which may be due to lacking of efficient and high-throughput screening methods. To address this problem, in this study, we have established a new and high-throughput screening strategy based on the glucose assay kit. Then BA-SacBl random mutagenesis libraries were constructed by error-prone PCR, and directed evolution of this enzyme was performed. As a result, some interesting clones have been obtained, and further analysis is underway.We have ever tried to clone BA-SacBl using classical molecular cloning method, but has not been successful. To solve this problem, in this study we have established an improved cloning method, named "the modified digestion-ligation method", which can significantly improve the efficiency of cloning. As a result, cloning and mutagenesis of BA-SacBl gene were successfully realized. On this basis, the method was further optimized, and was proved to be similarly efficient even for some difficult cloning experiments, such as blunt-end DNA cloning and fusion gene construction. Therefore, in addition to providing a solution for molecular cloning in this study, this cloning method can also serve as a complement to classical molecular cloning techniques, and provide a valuable tool for similar studies.