In this study, a novel β-N-acetylhexosaminidase (AuHex92) was expressed in Escherichia coli. AuHex92 could utilize both p-nitrophenyl-N-acetylglucosamine (pNP-GlcNAc) and N-acetyl chitobiose [(GlcNAc)2] as donors. pNP-GlcNAc as the donor afforded an exceptional N-acetyl chitotriose [(GlcNAc)3] yield (74.5%), whereas (GlcNAc)2 gave a yield of 12.6%. AuHex92 featured a broad entrance, large internal volume, and good flexibility in its catalytic groove, which enabled utilization of both donors, while the hydrophobicity and π-π stacking interactions in the groove improved its transglycosylation activity, with these functional contributions further validated by site-directed mutagenesis. Molecular dynamics simulation results indicated that AuHex92-pNP-GlcNAc exhibited a stable, compact conformation, which enhanced its substrate affinity and transglycosylation activity. AuHex92-(GlcNAc)2 achieved both-donor usage by utilizing a flexible catalytic groove region to guide the entry of the larger (GlcNAc)2 donor. The unique biochemical properties and structural features of AuHex92 establish a foundation for producing (GlcNAc)3 in the food, chemical, and pharmaceutical industries.
Tian-Yu Wang, Xue-Jing Liu, Qing-Xuan Zhao et al.· Journal of Agricultural and...· 0 citations
Lignocellulose is an abundant renewable feedstock for biofuels and value-added bioproducts, yet its efficient bioconversion is hindered by the recalcitrant lignin barrier. While laccases show great potential for lignin modification and delignification, their limited thermostability restricts their application in high-temperature lignocellulose biorefinery processes. To address this constraint, an integrated computational engineering strategy was employed to rationally improve the thermostability and catalytic performance of a laccase from Bacillus aryabhattai TCCC 11368. The optimal variant, S281E/N387D, obtained through the combination of FireProt, PROSS, and supercharge-based engineering, exhibited enhanced thermal stability and catalytic efficiency. Its half-life at 60 °C increased 3.7-fold to 330 min compared with the wild type, accompanied by a 35.4% improvement in catalytic efficiency (kcat/Km). Structural analysis suggested that the improved performance may result from enhanced hydrogen-bond networks, strengthened electrostatic interactions, improved hydrophobic packing, and optimized substrate-binding interactions, which collectively contribute to its enhanced capability in lignocellulosic biomass saccharification. Under optimized conditions, enzymatic treatment of wheat straw using the S281E/N387D variant combined with cellulase yielded 8.63 mg/mL reducing sugars, representing a 10.86% increase over the wild-type laccase treatment. This study provides an effective computational framework for developing robust laccases and demonstrates their potential for improving lignocellulosic biomass conversion in biorefinery applications.
Lei Zhao, Dianqing Liu, Yi-Min Cai et al.· Bioresource Technology· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
