糖巨肽蛋白源DPP-4抑制肽的制备、鉴定及抑制机理研究
发布时间：2021-05-14 18:20
近年来,由食物衍生的生物活性肽被开发出来并用于治疗糖尿病。本研究旨在探讨糖巨肽作为天然来源DPP-Ⅳ抑制剂的潜力。为了研究源自糖巨肽的DPP-4抑制肽,首先,收集新鲜的小鼠小肠粘膜分泌物和上皮细胞以分析DPP-4酶的活性,建立DPP-4抑制剂筛选体系,然后,我们使用糖巨肽（CGMP20）作为原料并用胰蛋白酶和木瓜蛋白酶消化。为了更有效地获得源自糖巨肽来源的DPP-4抑制肽,进行单因素实验以确定四个因子的中心点。依次测定DPP-4抑制活性,然后对二次非线性回归拟合进行响应面分析。回归模型具有良好的相关性（R2=0.6876）,测试的准确度为13.477。根据该模型预测的最佳条件是组合温度:温度（40℃）,时间（1h）,加酶量（4500U/g pro）,蛋白质浓度（4%）。通过木瓜蛋白酶酶解并使用半制备液相色谱分离纯化水解物。收集到的七个级分中的一个显示出显著的DPP-Ⅳ抑制活性。通过HPLC串联质谱（HPLC-MS/MS）分析这些级分。最高活性的片段序列是NQDKTEIPT f（130-140）和NQDKTEIPTIN f（140-150）,其IC50值为563.8...
【文章来源】：中国农业科学院北京市
【文章页数】：82 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
Abbreviation
CHAPTER 1 Background and objectives
1.1 General introduction
1.2 Milk Proteins
1.2.1 Overview of bioactive peptides
1.2.2 Milk-derived bioactive peptides
1.2.3 Enzymatic hydrolysis by digestive enzymes
1.2.4 Microbial fermentation
1.3 Bioactivities from peptides and health significance
1.3.1 Antidiabetic functionalities of milk protein-derived bioactive peptides
1.3.2 Effect of milk protein on insulin secretion
1.3.3 Antidiabetic applications of bioactive peptides
1.3.4 In vitro antidiabetic effect of peptides
1.4 Objectives of the study
1.4.1 The Specific Objectives of this Research are:
CHAPTER 2 Effects of microwave and ultrasound pretreatments on preparation of Glycomacropeptide withtrypsin as a source DPP-IV inhibitory peptide
2.1 Introduction
2.2 Materials and methods
2.2.1 Materials and reagents
2.2.2 Instruments equipments
2.2.3 Determination of protein content
2.2.4 Protease activity assay
2.2.5 Trichloroacetic acid precipitation -Determination of short peptide content by forinolmethod
2.2.6 Determination of the degree of hydrolysis of enzymatically digested samples by the o-Phthalaldehyde method (OPA method)
2.2.7 Microwave and ultrasonic pretreatments
2.2.8 Enzyme hydrolysis and degree of hydrolysis
2.2.9 Assay of the DPP-IV-Inhibitory Activity
2.2.10 Sttistical analysis
2.3 Results and discussions
2.3.1 Protein content and enzyme activity
2.3.2 Effects of Microwaves and Ultrasound on degree of hydrolysis
2.3.3 Effect on Enzymatic hydrolysis of GMP with different temperature, time, proteinconcentration and enzyme concentration
2.3.4 Effects of substrate concentration on DPP-V inhibitory activity
2.3.5 Effects of enzyme concentration, hydrolysis temperature and hydrolysis time on DPP-4inhibitory activity
2.4 Conclusion
CHAPTER 3 Response surface optimization of dipeptidyal peptidase (DPP-IV) Inhibition ofglycomacropeptides hydrolysates
3.1 Introduction
3.2 Materials and methods
3.2.1 Materials
3.2.2 Experimental design of glycomacropeptide Concentrate Hydrolysate by CentralComposite (CCD)
3.2.3 RSM and Generation of the Optimum GMP Hydrolysate.
3.2.4 Enzyme hydrolysis and degree of hydrolysis
3.2.5 Determination of DPP-IV Inhibitory Activity
3.2.6 Statistical Analysis
3.3 Results and discussion
3.3.1 Hydrolysates Generated Within the Experimental Design
3.3.2 The Effects of different Factors affecting on the DPP-IV Inhibitory Activity of TheHydrolysate
3.3.3 Optimization and validation
3.4 Conclusion
CHAPTER 4 Dipeptidyl peptidase-IV inhibitory peptides generated from Papain-Treated hydrolysis of aglycomacropeptide (cGMP20) protein
4.1 Introduction
4.2 Materials and methods
4.2.1 Materials
4.2.2 Preparation of the glycomacropeptide (GMP) hydrolysate
4.2.3 Separation and Purification of DPP-IV Inhibitory Peptides
4.2.4 Identification of DPP-IV Inhibitory Peptides by RP-HPLC-ESI-MS/MS
4.2.5 Assay of the DPP-IV-Inhibitory Activity
4.2.6 Peptide Synthesis
4.2.7 Peptide-cutter tool predicts enzymatic cleavage sequence
4.3 Results and discussion
4.3.1 DPP-4 inhibitory activity of GMP with different molecular weight ranges
4.3.2 Identification of peptide sequences
4.3.3 DPP-IV inhibitory activity of synthetic peptides
4.3.4 Peptide-cutter tool predicts enzymatic cleavage sequence
4.4 Conclusion
CHAPTER 5 Effect of Glycomecropeptide (cGMP20) peptides on DPP-IV and GLP-1 in H716 cells
5.1 Introduction
5.2 Materials and methods
5.2.1 Materials and reagents
5.2.2 Instruments and equipments
5.2.3 Test design
5.2.4 cell culture
5.2.5 Deermination the number of cells
5.2.6 Cytotoxicity experiment
5.2.7 Treatment of different concentrations of glycomacropeptide peptides NCI-H716 cells
5.2.8 Extraction of DPP-4 and GLP-1 in cells
5.2.9 Determination of intracellular DPP-4 activity
5.2.10 Determination of GLP-1 secretion
5.2.11 Statistical analysis
5.3 Results and discussion
5.3.1 DPP-4 half inhibition rate of linagliptin and active peptide
5.3.2 Cell proliferation assay to determine optimal culture concentration
5.3.3 Cytotoxicity test to determine the optimum culture concentration of the sample to betested
5.3.4 Effect of sample to be tested on intracellular DPP-IV secretion
5.3.5 Effect of sample to be tested on intracellular GLP-1 secretion
5.4 Conculusion
CHAPTER 6 Overall conclusions
6.1 Innovation points
6.2 Outlook
References
致谢 (Acknowledgement)
作者简历 (Resume)
本文编号：3186109
【文章来源】：中国农业科学院北京市
【文章页数】：82 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
Abbreviation
CHAPTER 1 Background and objectives
1.1 General introduction
1.2 Milk Proteins
1.2.1 Overview of bioactive peptides
1.2.2 Milk-derived bioactive peptides
1.2.3 Enzymatic hydrolysis by digestive enzymes
1.2.4 Microbial fermentation
1.3 Bioactivities from peptides and health significance
1.3.1 Antidiabetic functionalities of milk protein-derived bioactive peptides
1.3.2 Effect of milk protein on insulin secretion
1.3.3 Antidiabetic applications of bioactive peptides
1.3.4 In vitro antidiabetic effect of peptides
1.4 Objectives of the study
1.4.1 The Specific Objectives of this Research are:
CHAPTER 2 Effects of microwave and ultrasound pretreatments on preparation of Glycomacropeptide withtrypsin as a source DPP-IV inhibitory peptide
2.1 Introduction
2.2 Materials and methods
2.2.1 Materials and reagents
2.2.2 Instruments equipments
2.2.3 Determination of protein content
2.2.4 Protease activity assay
2.2.5 Trichloroacetic acid precipitation -Determination of short peptide content by forinolmethod
2.2.6 Determination of the degree of hydrolysis of enzymatically digested samples by the o-Phthalaldehyde method (OPA method)
2.2.7 Microwave and ultrasonic pretreatments
2.2.8 Enzyme hydrolysis and degree of hydrolysis
2.2.9 Assay of the DPP-IV-Inhibitory Activity
2.2.10 Sttistical analysis
2.3 Results and discussions
2.3.1 Protein content and enzyme activity
2.3.2 Effects of Microwaves and Ultrasound on degree of hydrolysis
2.3.3 Effect on Enzymatic hydrolysis of GMP with different temperature, time, proteinconcentration and enzyme concentration
2.3.4 Effects of substrate concentration on DPP-V inhibitory activity
2.3.5 Effects of enzyme concentration, hydrolysis temperature and hydrolysis time on DPP-4inhibitory activity
2.4 Conclusion
CHAPTER 3 Response surface optimization of dipeptidyal peptidase (DPP-IV) Inhibition ofglycomacropeptides hydrolysates
3.1 Introduction
3.2 Materials and methods
3.2.1 Materials
3.2.2 Experimental design of glycomacropeptide Concentrate Hydrolysate by CentralComposite (CCD)
3.2.3 RSM and Generation of the Optimum GMP Hydrolysate.
3.2.4 Enzyme hydrolysis and degree of hydrolysis
3.2.5 Determination of DPP-IV Inhibitory Activity
3.2.6 Statistical Analysis
3.3 Results and discussion
3.3.1 Hydrolysates Generated Within the Experimental Design
3.3.2 The Effects of different Factors affecting on the DPP-IV Inhibitory Activity of TheHydrolysate
3.3.3 Optimization and validation
3.4 Conclusion
CHAPTER 4 Dipeptidyl peptidase-IV inhibitory peptides generated from Papain-Treated hydrolysis of aglycomacropeptide (cGMP20) protein
4.1 Introduction
4.2 Materials and methods
4.2.1 Materials
4.2.2 Preparation of the glycomacropeptide (GMP) hydrolysate
4.2.3 Separation and Purification of DPP-IV Inhibitory Peptides
4.2.4 Identification of DPP-IV Inhibitory Peptides by RP-HPLC-ESI-MS/MS
4.2.5 Assay of the DPP-IV-Inhibitory Activity
4.2.6 Peptide Synthesis
4.2.7 Peptide-cutter tool predicts enzymatic cleavage sequence
4.3 Results and discussion
4.3.1 DPP-4 inhibitory activity of GMP with different molecular weight ranges
4.3.2 Identification of peptide sequences
4.3.3 DPP-IV inhibitory activity of synthetic peptides
4.3.4 Peptide-cutter tool predicts enzymatic cleavage sequence
4.4 Conclusion
CHAPTER 5 Effect of Glycomecropeptide (cGMP20) peptides on DPP-IV and GLP-1 in H716 cells
5.1 Introduction
5.2 Materials and methods
5.2.1 Materials and reagents
5.2.2 Instruments and equipments
5.2.3 Test design
5.2.4 cell culture
5.2.5 Deermination the number of cells
5.2.6 Cytotoxicity experiment
5.2.7 Treatment of different concentrations of glycomacropeptide peptides NCI-H716 cells
5.2.8 Extraction of DPP-4 and GLP-1 in cells
5.2.9 Determination of intracellular DPP-4 activity
5.2.10 Determination of GLP-1 secretion
5.2.11 Statistical analysis
5.3 Results and discussion
5.3.1 DPP-4 half inhibition rate of linagliptin and active peptide
5.3.2 Cell proliferation assay to determine optimal culture concentration
5.3.3 Cytotoxicity test to determine the optimum culture concentration of the sample to betested
5.3.4 Effect of sample to be tested on intracellular DPP-IV secretion
5.3.5 Effect of sample to be tested on intracellular GLP-1 secretion
5.4 Conculusion
CHAPTER 6 Overall conclusions
6.1 Innovation points
6.2 Outlook
References
致谢 (Acknowledgement)
作者简历 (Resume)
本文编号：3186109
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