| Summary: | A few α-glucan debranching enzymes (DBEs) of the large glycoside hydrolase family 13 (GH13), also known as the α-amylase family, have been shown to catalyze transglycosylation as well as hydrolysis. However, little is known about their acceptor and donor preferences. Here, a DBE from barley, limit dextrinase (<i>Hv</i>LD), is used as a case study. Its transglycosylation activity is studied using two approaches; (i) natural substrates as donors and different <i>p</i>-nitrophenyl (pNP) sugars as well as different small glycosides as acceptors, and (ii) α-maltosyl and α-maltotriosyl fluorides as donors with linear maltooligosaccharides, cyclodextrins, and GH inhibitors as acceptors. <i>Hv</i>LD showed a clear preference for pNP maltoside both as acceptor/donor and acceptor with the natural substrate pullulan or a pullulan fragment as donor. Maltose was the best acceptor with α-maltosyl fluoride as donor. The findings highlight the importance of the subsite +2 of <i>Hv</i>LD for activity and selectivity when maltooligosaccharides function as acceptors. However, remarkably, <i>Hv</i>LD is not very selective when it comes to aglycone moiety; different aromatic ring-containing molecules besides pNP could function as acceptors. The transglycosylation activity of <i>Hv</i>LD can provide glycoconjugate compounds with novel glycosylation patterns from natural donors such as pullulan, although the reaction would benefit from optimization.
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