Recombinant Selective Β4-Galactosidase in the Synthesis of Galectin Ligands

β-Galactosidases (EC 3.2.1.23), which in vivo catalyze hydrolysis of glycosidic bonds, can efficiently act in transglycosylation mode with suitable acceptors. A commercially produced enzyme, Biolacta® (Daiwa Kasei, Shiga, Japan) contains four isoforms of β-galactosidase from Bacillus circulans ATCC 31382 (BgaD, glycoside hydrolase family 2), which originate by endogenous C-terminal cleavage of the enzyme precursor [1]. BgaD has a higher transglycosylation activity and higher thermostability than other known β-galactosidases. Its isoforms can be distinguished by molecular weight as well as by different pH optimum. They also exhibit varying transglycosylation abilities and regioselectivity.

In this work, the longest β-galactosidase isoform A (BgaD-A; 189 kDa) was recombinantly produced in E. coli BL21-Gold(DE3) and kinetically characterized. This enzyme was used for the synthesis of functionalized LacNAc (Galβ4GlcNAc) epitope in a one step reaction. LacNAc disaccharide is a typical epitope for galectins [2]. The recombinant enzyme also enabled galactosylation of more complex acceptors, such as functionalized chitooligomers. Thus, we obtained LacNAc epitope on nature-like carbohydrate linkers (Fig. 1), suitable for multivalent presentation. The carbohydrate linkers were prepared by mutant Tyr470Asn β-N-acetylhexosaminidase from Talaromyces flavus with suppressed hydrolytic activity. The present recombinant β-galactosidase is unique due to its exclusive β4-selectivity and high synthetic yield and can well replace the more expensive glycosyltransferases. The azide at C-1 will be used for multivalent conjugation.