N-糖酰胺酶(N-glycosidase,PNGase)是一种糖苷水解酶,主要功能为释放糖肽或糖蛋白上的N-糖链,在食品、生物医药等领域中具有重要的应用价值。目前,商业化的PNGase在使用过程中具有一定的局限性,因此,开发性能优良的新型PNGase具有研究意义。该研究在细菌Dyella caseinilytica中利用编码PNGase的基因,通过在大肠杆菌中进行重组表达,成功获得一种新型PNGase,命名为PNGase Dc,该酶分子质量为63.01 kDa。之后对其酶学特性、功能和应用潜力进行分析。酶学特性结果表明,PNGase Dc的最适pH和温度分别为2.0和37 ℃,酶促反应无金属离子依赖性;此外,该酶稳定性优良,在4 ℃下保存45 d仍能维持最高活性;分子对接结果表明,PNGase Dc与底物N′N-二乙酰壳二糖的相互作用力以氢键和范德华力为主;定点突变证明,Asp102和Glu236是影响重组PNGase Dc活性的关键氨基酸;PNGase Dc对标准糖蛋白辣根过氧化物酶和牛乳铁蛋白均具有良好的去糖基化效果;最后,以拟南芥和小鼠血浆作为底物评估PNGase Dc应用潜力,证实PNGase Dc在动植物源糖蛋白的研究中具有一定的应用潜力。综上所述,该研究为动植物源糖蛋白的研究提供了一个新的工具酶PNGase Dc,并为该酶的开发利用奠定理论基础。
N-Glycosidase (PNGase) is a type of glycoside hydrolase whose main function is to release N-glycan chains from glycopeptides or glycoproteins, holding significant application value in fields such as food and biomedicine.Currently, commercial PNGases exhibit certain limitations in their application.Therefore, developing new PNGases with superior performance holds research value.This study successfully obtained a novel PNGase, named PNGase Dc, by expressing the gene encoding PNGase from Dyella caseinilytica in Escherichia coli.The molecular weight of PNGasse Dc was 63.01 kDa.Its enzymatic characteristics, functionality, and potential applications were then analyzed.The enzymatic characterization results indicated that the optimal pH and temperature for PNGase Dc are 2.0 and 37 ℃, respectively, and its catalytic reaction did not depend on metal ions.The enzyme also demonstrated excellent stability, retaining its activity even after storage at 4 ℃ for 45 days.Molecular docking analysis indicated that PNGase Dc interacts with its substrate N′N-diacetylchitobiose through hydrogen bonds and van der Waals forces.Site-directed mutagenesis identified Asp102 and Glu236 as key amino acids affecting the enzyme′s activity.PNGase Dc exhibited effective deglycosylation on standard glycoproteins, including horseradish peroxidase and lactoferrin.Lastly, PNGase Dc′s application potential was evaluated.Arabidopsis thaliana and mouse plasma was used as substrates, confirming its promising applicability in the study of glycoproteins from both animal and plant sources.In summary, this research introduced a new enzymatic tool, PNGase Dc, for the study of glycoproteins derived from animal and plant sources, thereby laying a theoretical foundation for the enzyme′s development and utilization.
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