Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β-N-acetylhexosaminidases

Abstract

This paper describes the discovery and characterization of two novel β-N-acetylhexosaminidases HEX1 and HEX2, capable of catalyzing the synthesis of human milk oligosaccharides (HMO) backbone structures with fair yields using chitin oligomers as β-N-acetylglucosamine (GlcNAc) donor. The enzyme-encoding genes were identified by functional screening of a soil-derived metagenomic library. The β-N-acetylhexosaminidases were expressed in Escherichia coli with an N-terminal His6-tag and were purified by nickel affinity chromatography. The sequence similarities of the enzymes with their respective closest homologues are 59 % for HEX1 and 51 % for HEX2 on the protein level. Both β-N-acetylhexosaminidases are classified into glycosyl hydrolase family 20 (GH 20) are able to hydrolyze para-nitrophenyl-β-N-acetylglucosamine (pNP-GlcNAc) as well as para-nitrophenyl-β-N-acetylgalactosamine (pNP-GalNAc) and exhibit pH optima of 8 and 6 for HEX1 and HEX2, respectively. The enzymes are able to hydrolyze N-acetylchitooligosaccharides with a degree of polymerization of two, three, and four. The major findings were, that HEX1 and HEX2 catalyze trans-glycosylation reactions with lactose as acceptor, giving rise to the human milk oligosaccharide precursor lacto-N-triose II (LNT2) with yields of 2 and 8 % based on the donor substrate. In total, trans-glycosylation reactions were tested with the disaccharide acceptors β-lactose, sucrose, and maltose, as well as with the monosaccharides galactose and glucose resulting in the successful attachment of GlcNAc to the acceptor in all cases.

Loop Protein Engineering for Improved Transglycosylation Activity of a β‐N‐Acetylhexosaminidase

“…From inspection of the catalytic site models for HEX1 and HEX2 it was evident that the five amino acid stretches might be close to the active site, especially considering the likely positions of W354 in HEX1 and W394 in HEX2. A minimum of three of the microbial sources of the enzyme sequences (AAT89596.1, AGW40804.1, ABY25113.1) appear to be either plant or fish pathogens (Table ).…”

“…Recently, the biocatalytic synthesis of LNT2 in high yields by a recombinantly produced β‐ N ‐acetylhexosaminidase originating from Bifidobacterium bidifum was reported, but the reaction relied on p NP‐GlcNAc as a donor substrate, which released toxic p ‐nitrophenol ( p NP) as a by‐product of the reaction. In our group, we recently synthesized LNT2 successfully by using N , N ′‐diacetylchitobiose (chitobiose) as a donor and β‐lactose as an acceptor substrate by transglycosylation catalyzed by two novel β‐ N ‐acetylhexosaminidases of metagenomics origin, HEX1 and HEX2 (Scheme ) . Chitobiose (and other chitooligosaccharides) are advantageous donor substrates, as they can be obtained by chemical or enzymatic hydrolysis of chitin, which is a highly abundant natural polysaccharide present in the exoskeleton of, for example, crabs and shrimps.…”

Efficient and Regioselective Synthesis of β-GalNAc/GlcNAc-Lactose by a Bifunctional Transglycosylating β- N -Acetylhexosaminidase from Bifidobacterium bifidum

“…However, access to these trisaccharides at large scale remains a challenge. Nevertheless, GlcNAc␤1-3Lac has been synthesized by ␤-N-acetylhexosaminidase at a low yield of less than 10% (33,35,36,44), whereas the synthesis of GalNAc␤1-3Lac by this enzyme has not yet been reported.…”

“…Most of the reported ␤-N-acetylhexosaminidases with transglycosylation ability are obtained from fungi, especially those belonging to the genera Aspergillus (22)(23)(24)(26)(27)(28)(29), Penicillium (28,30), and Talaromyces (31,32), and only three are obtained from the bacteria Nocardia orientalis and Serratia marcescens YS-1 (33)(34)(35). Moreover, most ␤-N-acetylhexosaminidases with transglycosylation ability display low glycosyl transfer efficiency at 1% to 10% yield, as well as poor regioselectivity, resulting in isomer production with multiple glycosyl linkages that are difficult to isolate (33,35,36). Only two fungal ␤-N-acetylhexosaminidases can catalyze transfer of both GalNAc and GlcNAc residues for synthesis (24,25).…”

“…The ␤-Nacetylhexosaminidase from A. oryzae synthesizes GlcNAc␤1-3/1-6Gal␤1-4Glc via reverse hydrolysis by using GlcNAc and lactose (44). Two ␤-N-acetylhexosaminidases from the soil-derived metagenomic library form GlcNAc␤1-3Gal␤1-4Glc and GlcNAc␤1-4Gal␤1-4Glc using GlcNAc 2 as donor and lactose as acceptor (36). The ␤-N-acetylhexosaminidase from N. orientalis transfers GlcNAc from GlcNAc 2 to methyl-␤-lactoside to form GlcNAc␤1-3/1-6Gal␤1-4Glc-OMe and Gal␤1-4(GlcNAc␤1-6) Glc-OMe, as well as to pNP-␤-LacNAc to form GlcNAc␤1-3/1-6Gal␤1-4GlcNAc-␤-pNP and Gal␤1-4(GlcNAc␤1-6) GlcNAc-␤-pNP (33,35).…”