Specificity Transformation Of Glucansucrase GTF-D Donor Substrate

Taking the cheap sucrose as its substrate, glucansucrases is able to be synthesized into various specific oligosaccharides or glycosylation products, widely used in food industry, forage production, medicine, cosmetology and many other aspects and it has gradually developed into an important kind of tool enzymes in biocatalysis and synthesis. The specificities of its products are relevant to that of enzymes and its reaction condition.The natural substrate of Glucosyltransferase-D from Streptococcus mutans GS5is sucrose, other disaccharides are not been hydrolyzed by GTF-D. In this experiment, the substrate specificity of GTF-D was transformed by directed evolution. The experimental results are as follows:1. This experiment focuses on saturate mutation of four amino acid sites(Arg463%His583、Asp584、Gln965) in GTF-D receptor binding domain deriving from streptococcus mutans and then transform colibacillus MC10061.Therefore, a High-level expression GTF-SM3will take into shape.2. At the same time, it also brings in the carrier of reporter gene which is sensitive to trans-glycosylating reaction by using the trans-glycosylating specificity of GTF-D. Then they can come into effects in the experiment of BW251131acZ"ebgA-.Then we can continue the high-throughput screening by designed GLACK plating.3. This experiment also discovers that EbgA in MCI0061can substitute lacZ in functions. Under the condition of this experiment, EbgA is highly likely conduct a mutation of]3-Galactosidase activity. When the ebgA is removed, false positive of mutants simultaneously disappear.4. After several selection, we finally pick up SMs-b which shows its obvious lactose hydrolysis activity in the host bacteria...