Protein engineering of α2,3/2,6-sialyltransferase to improve the yield and productivity of in vitro sialyllactose synthesis
| Autor: | Il-Hyang Park, Dae-Hee Kim, Joonho Park, Kyoung-Soon Jang, Nahum Lee, Yun Hee Choi, Byung-Gee Kim, Jong Hoon Kim |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Pasteurella multocida
Sialyltransferase Mutant Lactose Protein Engineering Biochemistry Cloning Molecular beta-D-Galactoside alpha 2-6-Sialyltransferase biology Organisms Genetically Modified Chemistry business.industry Photobacterium Mutagenesis Rational design Protein engineering Sequence Analysis DNA Alanine scanning Sialyltransferases Biotechnology Photobacterium damselae biology.protein Mutagenesis Site-Directed Specific activity Mutant Proteins business |
| Zdroj: | Glycobiology. 24(2) |
| ISSN: | 1460-2423 |
| Popis: | In the large-quantity production of α2,3- and α2,6-sialyllactose (Neu5Ac(α2,3)Galβ1,4Glc (3'-SL) and Neu5Ac(α2,6)Galβ1,4Glc (6'-SL)) using sialyltransferases (STs), there are major hurdles to overcome for further improvement in yield and productivity of the enzyme reactions. Specifically, Pasteurella multocida α2,3-sialyltransferase (α2,3PST) forms a by-product to a certain extent, owing to its multifunctional activity at pH below 7.0, and Photobacterium damselae α2,6-sialyltransferase (α2,6PdST) shows relatively low ST activity. In this study, α2,3PST and α2,6PdST were successfully engineered using a hybrid approach that combines rational design with site-saturation mutagenesis. Narrowly focused on the substrate-binding pocket of the STs, putative functional residues were selected by multiple sequence alignment and alanine scanning, and subsequently subjected to site-saturation mutagenesis. In the case of α2,3PST, R313N single mutation improved its activity slightly (by a factor of 1.5), and further improvement was obtained by making the double mutants (R313N/T265S and R313H/T265S) resulting in an overall 2-fold improvement in its specific α2,3 ST activity, which is mainly caused by the increase in kcat. It was revealed that the R313 mutations to N, D, Y, H or T greatly reduced the α2,6 ST side-reaction activity of α2,3PST at below pH 7.0. In the case of α2,6PdST, single-mutation L433S/T and double-mutation I411T/L433T exhibited 3- and 5-fold enhancement of the α2,6 ST-specific activity compared with the wild-type, respectively, via increase in kcat values. Our results show a very good model system for enhancing ST activity and demonstrate that the generated mutants could be used efficiently for the mass production of 3'-SL and 6'-SL with enhanced productivity and yield. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|