Recombinant Selective Β4-Galactosidase in the Synthesis of Galectin Ligands

Session: 
PS2 Poster session 2 Even numbers
Code: 
P96
Location (hall): 
Foyer
Start/end time: 
Tuesday, July 2, 2019 - 15:45 to 17:15
Michaela
Hovorková

Michaela Hovorková1, Vladimír Křen1, Pavla Bojarová1

1Laboratory of Biotransformation, Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-14220, Prague 4, Czech Republic

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.

Fig. 1 Cascade enzymatic synthesis of LacNAc epitope – ligand of galectins - on a chitooligomer linker. 

Acknowlegements

Support by grant projects LTC18038 and LTC18041 is gratefully acknowledged.

References: 
  1. Song, J.; Abe, K.; Imanaka, H.; Imamura, K.; Minoda, M.; Yamaguchi, S.; Nakanishi, K. Biosci. Biotechnol. Biochem. 2011, 75, 268−278.
  2. Laaf, D.; Steffens, H.; Pelantová, H.; Bojarová, P.; Křen, V.; Elling, L. Adv. Synth. Catal., 2017, 359, 4015-4024.

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