β-N-Acetylhexosaminidases for Carbohydrate Synthesis via Trans-Glycosylation

Abstract

β-N-acetylhexosaminidases (EC 3.2.1.52) are retaining hydrolases of glycoside hydrolase family 20 (GH20). These enzymes catalyze hydrolysis of terminal, non-reducing N-acetylhexosamine residues, notably N-acetylglucosamine or N-acetylgalactosamine, in N-acetyl-β-D-hexosaminides. In nature, bacterial β-N-acetylhexosaminidases are mainly involved in cell wall peptidoglycan synthesis, analogously, fungal β-N-acetylhexosaminidases act on cell wall chitin. The enzymes work via a distinct substrate-assisted mechanism that utilizes the 2-acetamido group as nucleophile. Curiously, the β-N-acetylhexosaminidases possess an inherent trans-glycosylation ability which is potentially useful for biocatalytic synthesis of functional carbohydrates, including biomimetic synthesis of human milk oligosaccharides and other glycan-functionalized compounds. In this review, we summarize the reaction engineering approaches (donor substrate activation, additives, and reaction conditions) that have proven useful for enhancing trans-glycosylation activity of GH20 β-N-acetylhexosaminidases. We provide comprehensive overviews of reported synthesis reactions with GH20 enzymes, including tables that list the specific enzyme used, donor and acceptor substrates, reaction conditions, and details of the products and yields obtained. We also describe the active site traits and mutations that appear to favor trans-glycosylation activity of GH20 β-N-acetylhexosaminidases. Finally, we discuss novel protein engineering strategies and suggest potential “hotspots” for mutations to promote trans-glycosylation activity in GH20 for efficient synthesis of specific functional carbohydrates and other glyco-engineered products.

Rational Enzyme Design without Structural Knowledge: A Sequence‐Based Approach for Efficient Generation of Transglycosylases

“…Previously, GH20 hexosaminidases have been extensively used for transglycosylation [69–71] . Herein, we focus on Bb HI from Bifidobacterium bifidum [72,73] .…”

Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

“…Although equivalent positions in the sequence alignment, these results may reflect the different relative orientation of the side chains in the 3D structures (not solved for Bh H1). Finally, Trp373 in LnbB was also mutated, but to the best of our knowledge, the equivalent position has not been studied in other GH20 enzymes [ 32 ]. Although close to the sugar ring in subsite -1, W373F retained 15% of the wt hydrolase activity and had almost no effect on TG activity.…”

A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities

“…For GH20 NAGases, production of higher N-acetyl COSs from shorter N-acetyl COSs were often catalyzed by an auto-condensation reaction (a special case of transglycosylation, which involves only one substrate (acts both donor and acceptor) [24]. For example, a GH20 NAGase from A. oryzae was used to catalyze the formation of (GlcNAc) 3 and (GlcNAc) 4 from (GlcNAc) 2 [29].…”

“…With the cooperative action of NAGase and chitinase, chitin can be hydrolyzed into GlcNAc. Moreover, some GH20 NAGases can also be used to synthesize highvalue GlcNAc-containing products and N-acetyl COSs by transglycosylation and reverse hydrolytic reactions [19][20][21][22][23][24]. These excellent features make GH20 NAGases receive increased attention.…”

“…NAGases can be found in a wide variety of organisms including bacteria, fungal, insects, plants, and animals [24][25][26][27]. However, studies about the GH20 NAGases with transglycosylation and reverse hydrolysis activities are mainly derived from fungal sources [28][29][30][31][32][33][34].…”