酶法制备玉米七肽糖基化产物及其对乙醇脱氢酶激活活性的影响研究

Enzymatic preparation of glycosylated corn heptapeptide and effect on alcohol dehydrogenase activating activity

中文关键词:  糖肽  酶法糖基化  结构鉴定  乙醇脱氢酶激活  玉米

英文关键词:glycopeptide  enzymatic glycosylation  structural identification  alcohol dehydrogenase activation  corn

中文摘要:

      目的 对玉米七肽(Ser-Ser-Asn-Cys-Gln-Pro-Phe, SSNCQPF)进行糖基化制备和分离纯化, 并对其进行乙醇脱氢酶(alcohol dehydrogenase, ADH)激活活性验证。方法 通过谷氨酰胺转氨酶(transglutaminase, TGase)介导, 以玉米七肽作为酰基供体, D-氨基葡萄糖(D-glucosamine, GlcN)为酰基受体合成玉米糖肽的糖基化反应, 并分离纯化玉米糖肽单体[SSNCQ(GlcN)PF]。使用超高效液相色谱-静电场轨道阱质谱仪(ultra performance liquid chromatography-quadrupole exactive orbitrap mass spectrometry, UPLC-QE Orbitrap MS)进行面积归一化法测定糖肽纯度, 采用核磁共振波谱仪(nuclear magnetic resonance, NMR)测定糖肽的氢谱(1H NMR)和碳谱(13C NMR), 并结合二级质谱进行结构定性分析。最后用酶标仪(microplate reader spectrometry, MRS)对比测定玉米七肽及其糖肽的ADH激活活性, 以验证玉米七肽糖基化修饰对其ADH激活活性的影响。结果 玉米糖肽经过分离、纯化和冻干后, 其纯度达到99.11%, 并且通过二级质谱、核磁氢谱和碳谱的分析, 确定氨基葡萄糖连接在玉米七肽谷氨酰胺残基的氨基上。在摩尔浓度为2.5 mmol/L时, 糖肽的ADH激活率比玉米七肽高9.89%。结论 与玉米肽相比, 采用TGase酶介导玉米七肽和氨基葡萄糖合成的糖肽, 具有更高的ADH激活活性, 为玉米肽糖基化在食品工业中的应用提供参考。

英文摘要:

      Objective To prepare and purify glycosylated corn heptapeptide (Ser-Ser-Asn-Cys-Gln-Pro-Phe, SSNCQPF), and verify its activation activity on alcohol dehydrogenase (ADH). Methods The glycosylation reaction of corn glycopeptide was mediated by transglutaminase (TGase) with maize heptapeptide as the acyl donor and D-glucosamine (GlcN) as the acyl acceptor, and the maize glycopeptide monomer [SSNCQ(GlcN)PF] was isolated and purified. The purity of the glycopeptide was determined by area normalization method using ultra performance liquid chromatography-quadrupole exactive orbitrap mass spectrometry (UPLC-QE Orbitrap MS). The 1H NMR and 13C NMR of glycopeptides were determined by nuclear magnetic resonance (NMR) spectrometry and combined with secondary mass spectrometry for qualitative structural analysis. Finally, the ADH activation activities of maize heptapeptide and its glycopeptide were comparatively determined by microplate reader spectrometry (MRS) to verify the effect of glycosylation modification of maize heptapeptide on ADH activation activity. Results After separation, purification, and freeze-drying, the purity of corn peptide was determined to be 99.11%. Furthermore, it was confirmed that the glucosamine was connected to the amino group of the glutamine residue in the corn heptapeptide by the analysis of secondary mass spectrometry, 1H NMR and 13C NMR. At a molar concentration of 2.5 mmol/L, the ADH activation rate of glycopeptide was 9.89% higher than that of corn heptapeptide. Conclusion Compared with corn heptapeptide, the glycopeptide synthesized using TGase enzyme-mediated corn heptapeptide and glucosamine exhibits higher bioactivity in ADH activating ability, providing valuable insights for the potential application of glycosylated corn peptides in the food industry.