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

作　　者：王哲 李冠龙 刘晓兰 WANG Zhe;LI Guan-Long;LIU Xiao-Lan(College of Food Engineering,Harbin University of Commerce,Harbin 150028,China;College of Food and Bioengineering,Qiqihar University Heilongjiang Key Laboratory of Corn Deep Processing Theory and Technology,Qiqihar 161006,China)

机构地区：[1]哈尔滨商业大学食品工程学院,哈尔滨150028 [2]齐齐哈尔大学食品与生物工程学院,黑龙江省玉米深加工理论与技术重点实验室,齐齐哈尔161006

出　　处：《食品安全质量检测学报》2024年第2期28-34,共7页Journal of Food Safety and Quality

基　　金：国家重点研究计划项目(2017YFD0400200);哈尔滨商业大学研究生创新基金项目(YJSCX2020-637HSD);中央支持地方高校改革发展资金高水平人才项目(2020GSP08);黑龙江省玉米主食工业化工程技术研究中心开放课题项目(SPKF202025);黑龙江省省属本科高校基本科研业务费项目(145209309)。

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(1 H NMR)和碳谱(^(13)C 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。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 1 H NMR and ^(13)C 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,1 H 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.

关 键 词：糖肽 酶法糖基化 结构鉴定 乙醇脱氢酶激活 玉米 

分 类 号：TS201.2[轻工技术与工程—食品科学]