Characterization of a β-N-acetylhexosaminidase with transglycosylation activity from Metarhizium sp. A34

In applications of chitin, one of the most abundant resources on earth, human milk oligosaccharides with many health functions were synthesized by transglycosylation of β-N-acetylhexosaminidase. Synthesis of new transfer products can be expected by other β-N-acetylhexosaminidases in nature. A total of 38 microorganisms that secrete β-N-acetylhexosaminidases with transglycosylation activity were isolated from a soil screen. Using N,N'-diacetylchitobiose as the substrate, the transfer ratio increased with a decrease in substrate degradation when it was less than 60%. Metarhizium sp. A34 β-N-acetylhexosaminidase had high transglycosylation activity and showed a maximum production of the oligosaccharides against the substrate degradation where (GlcNAc)₅ and (GlcNAc)₄ were produced in addition to (GlcNAc)₃ . The maximum curve was attributed to a sequential reaction of transglycosylation followed by hydrolysis where oligosaccharides are an intermediate product and are hydrolyzed in a second step. The purified β-N-acetylhexosaminidase from Metarhizium sp. A34 had an optimal pH of 5 and was stable from pH 7 to 8. At pH 5, it had an optimal temperature of 40°C and was stable up to 30°C for 30 min. This enzyme had high thermostability up to 55°C when bound to the cell wall. The acceptor specificity for the transglycosylation reaction was enhanced for lower molecular weight sugar alcohols in the order of glycerin (C3), erythritol (C4), and xylitol (C5). The transfer product with glycerin was identified as 1-O-β-d-N-acetylglucosaminyl glycerin, which may prove useful as a starting material for new glycolipids in food applications. PRACTICAL APPLICATION: Metarhizium sp. A34 β-N-acetylhexosaminidase produced 1-O-β-d-N-acetylglucosaminyl glycerin through the transglycosylation. Chitin oligosaccharides of the donor are obtained by hydrolysis of chitin. 1-O-β-d-N-Acetylglucosaminyl glycerin may be useful to start material for the synthesis of new glycolipids. High thermostability of this enzyme is useful to prevention of contamination in the transglycosylation reaction.