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Protein engineering of α2,3/2,6-sialyltransferase to improve the yield and productivity of in vitro sialyllactose synthesis

饱和突变 唾液酸转移酶 化学 突变体 酶动力学 突变 蛋白质工程 产量(工程) 立体化学 生物化学 突变 活动站点 基因 冶金 材料科学
作者
Yun‐Hee Choi,Jong‐Hoon Kim,Joon Ho Park,Nahum Lee,Dae‐Hee Kim,Kyoung‐Soon Jang,IL-Hyang Park,Byung‐Gee Kim
出处
期刊:Glycobiology [Oxford University Press]
卷期号:24 (2): 159-169 被引量:40
标识
DOI:10.1093/glycob/cwt092
摘要

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.
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