Directed evolution of a ?-N-acetylhexosaminidase from Haloferula sp. for lacto-N-triose II and lacto-N-neotetraose synthesis from chitin

In our previous study, a beta-N-acetylhexosaminidase (HaHex74) from Haloferula sp. showing high human milk oligosaccharides (HMOs) synthesis ability was identified and characterized. In this study, HaHex74 was further engineered by directed evolution and site-saturation mutagenesis to improve its transglycosylation activity for HMOs synthesis. A mutant (mHaHex74) with improved transglycosylation activity (HaHex74-Asn401Ile/ His394Leu) was obtained and characterized. mHaHex74 exhibited maximal activity at pH 5.5 and 35 degrees C, respectively, which were distinct from that of HaHex74 (pH 6.5 and 45 degrees C). Moreover, mHaHex74 showed the highest LNT2 conversion ratio of 28.2% from N,N'-diacetyl chitobiose (GlcNAc2), which is 2.2 folds higher than that of HaHex74. A three-enzyme cascade reaction for the synthesis of LNT2 and LNnT from chitin was per-formed in a 5-L reactor, and the contents of LNT2 and LNnT reached up to 15.0 g L-1 and 4.9 g L-1, respectively. Therefore, mHaHex74 maybe a good candidate for enzymatic synthesis of HMOs.