1144040-14-1Relevant articles and documents
Enhanced enzymatic reactions in a microchannel reactor
Kanno, Kenichi,Kawazumi, Hirofumi,Miyazaki, Masaya,Maeda, Hideaki,Fujii, Masayuki
, p. 687 - 690 (2002)
Organic and enzymatic reactions in microchannel reactors were discussed. Hydrolytic activity of a microchannel pre-treated with enzyme solution was studied. It was found that the reaction rate in microchannel is much faster than the micro test tube. Mass transfer is also much more efficient in microchannel.
Facile preparation of indoxyl- and nitrophenyl glycosides of lactosamine and isolactosamine
Boettcher, Stephan,Thiem, Joachim
, p. 10856 - 10861 (2014/03/21)
The synthesis of the novel indoxyl glycosides of N-acetyl-lactosamine (X-LacNAc) and N-acetyl-isolactosamine (X-LNB) is reported employing glycosyl chlorides in a facile phase transfer glycosylation, followed by mild decarboxylation and finally deacetylat
Fluorescence activated cell sorting as a general ultra-high-throughput screening method for directed evolution of glycosyltransferases
Yang, Guangyu,Rich, Jamie R.,Gilbert, Michel,Wakarchuk, Warren W.,Feng, Yan,Withers, Stephen G.
supporting information; experimental part, p. 10570 - 10577 (2010/09/17)
Glycosyltransferases (GTs) offer very attractive approaches to the synthesis of complex oligosaccharides. However, the limited number of available GTs, together with their instability and strict substrate specificity, have severely hampered the broad application of these enzymes. Previous attempts to broaden the range of substrate scope and to increase the activity of GTs via protein engineering have met with limited success, partially because of the lack of effective high-throughput screening methods. Recently, we reported an ultra-high-throughput screening method for sialyltransferases based on fluorescence-activated cell sorting (Aharoni et al. Nat. Methods 2006, 3, 609-614). Here, we considerably improve this method via the introduction of a two-color screening protocol to minimize the probability of false positive mutants and demonstrate its generality through directed evolution of a neutral sugar transferase, β-1,3-galactosyltransferase CgtB. A variant with broader substrate tolerance than the wild-type enzyme and 300-fold higher activity was identified rapidly from a library of >107 CgtB mutants. Importantly, the variant effected much more efficient synthesis of G M1a and asialo GM1 oligosaccharides, the building blocks of important therapeutic glycosphingolipids, than did the parent enzyme. This work not only establishes a new methodology for the directed evolution of galactosyltransferases, but also suggests a powerful strategy for the screening of almost all GT activities, thereby facilitating the engineering of glycosyltransferases.
