Abstract
N-glycosylation alters the properties of different enzymes in different ways. Rhizopus homothallicus was first described as an environmental isolate from desert soil in Guatemala. A new gene encoding glucanase RhGlu16B was identified in R. homothallicus. It had high specific activity (9673 U/mg) when barley glucan was used as a substrate, and β-glucan is hemicellulose that is abundant in nature. RhGlu16B has only one N-glycosylation site in its Ala55-Gly64 loop. It was found that N-glycosylation increased its Tm value and catalytic efficiency by 5.1 °C and 59%, respectively. Adding N-glycosylation to the same region of GH16 family glucanases TlGlu16A (from Talaromyces leycettanus) increased its thermostability and catalytic efficiency by 6.4 °C and 38%, respectively. In a verification experiment using GH16 family glucanases BisGlu16B (from Bisporus) in which N-glycosylation was removed, N-glycosylation also appeared to promote thermostability and catalytic efficiency. N-glycosylation reduced the overall root mean square deviation of the enzyme structure, creating rigidity and increasing overall thermostability. This study provided a reference for the molecular modification of GH16 family glucanases and guided the utilization of β-glucan in hemicellulose.
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All data generated or analyzed during this study are included in this published article. The raw data retrieved during the current study are available from the corresponding author on reasonable request.
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The authors thank the Biotechnology Laboratory of Jiangsu University of Science and Technology for providing experimental equipment.
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This work was supported by the National Natural Science Foundation of China (21978121), and the Natural Science Foundation of Jiangsu Province (BK20190957).
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Conceptualization, JW and SY; methodology, ZZ and FZ; formal analysis, ZZ and YC; investigation, ZZ and HH; writing—original draft preparation, ZZ and FZ; writing—review and editing, ZZ, FZ, and SY; visualization, ZZ; supervision, JW. All authors have read and agreed to the published version of the manuscript.
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Zha, ZQ., You, S., Hu, YH. et al. Asn57 N-glycosylation promotes the degradation of hemicellulose by β-1,3–1,4-glucanase from Rhizopus homothallicus. Environ Sci Pollut Res 30, 8707–8721 (2023). https://doi.org/10.1007/s11356-022-19959-5
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DOI: https://doi.org/10.1007/s11356-022-19959-5