| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 88 === Protein can supply several physical properties on processing industries. Various peptides with low molecular weight were produced through protein hydrolysis. Production of bioactive peptides through microbial fermentation or protease hydrolysis is prevalent now. Besides having characteristics of easier digestion, better taste, and lower allergy, the resulted peptide products also contain functions in immunity, lipid metabolic, and blood pressure regulation etc.
In order to develop and substantiate the production of bioactive peptides from soybean proteins, following four bioactive assay systems have been established in this study: (a). Inhibition-activity of angiotensin I converting enzyme (ACE). (b). Enhancement of activity through 3T3-L1 lipid cell differentiation. (c). Enhancement of the human absorption of calcium.
This thesis is aimed to produce and purify the bioactive peptides from soybean protein through protease hydrolysis. The analysis of physiological activities was also useful in analyzing the peptides. The goal is to study the peptide composition and analyze the physiological activities by the systems mentioned above. In this thesis, the soybean protein was digested by using various protease on the market and the subtilisin YaB developed by protein engineering in this laboratory, and the resulted peptides were fractionated by gel filtration Fractogel HW-50(s) column chromatography and reverse phase HPLC. The rate of protease digestion and the optimum condition of hydrolysis process were studied.
The bioactivity of each peptide fraction was assayed using these assay systems stated above. Peptides with molecular weight of 1,000 to 10,000 Da were produced from casein protease-digested soybean and purified by either gel filtration or anion exchange chromatography after Ca2+-ethanol precipitation. The peptides which contain high acidic amino acids ( aspartate, glutamate ), serine and threonine, or histidine are known to have high affinity to Ca2+. In this research, we have obtained such kind of peptides from trypsin and pepsin digested soybean. The peptide with high histidine content was shown to have high affinity for calcium binding.
The physiological activities investigated in this project include the blood pressure regulation by ACE enzyme activity analysis, improving the lipid metabolism by 3T3-L1 lipid cells differentiation analysis. All these items are common concerns to the majority. The bioactive peptides developed in this project will be marked as the products containing physiological activities. The bioactive peptides were purified and the structure and action mechanism will be investigated.
縮寫-全文對照表
圖表目次
中文摘要
英文摘要
第一章 前言
第一節 蛋白質水解物之應用
第二節 黃豆蛋白質
第三節 Subtilisin YaB及其突變酵素G124A簡介
一、Subtilisin
二、Subtilisin YaB
三、Subtilisin YaB之酵素活性
四、Subtilisin YaB的應用價值
五、Subtilisin G124A
第四節 胜之生理活性
一、鈣吸收促進活性
二、脂肪細胞分化調節活性
三、ACE酵素抑制活性
第五節 研究構想與目的
第二章 實驗材料與方法
第一節 實驗材料
一、蛋白質來源
二、酵素來源
三、菌株來源、培養基及培養液
四、細胞株來源、培養基及培養液
五、管柱
六、藥品及儀器
第二節 實驗方法
一、Subtilisin YaB及G124A修飾型酵素之生產純化
二、蛋白質水解液之製備及酵素分解過程之探討
三、鈣結合性胜之生產純化
四、3T3-L1前驅脂肪細胞分化調節活性分析系統之建立
五、篩選具ACEI活性之胜
第三章 結果與討論
第一節 Subtilisin YaB 酵素及其突變酵素G124A之生產純化
一、Subtilisin YaB 之純化
二、G124A突變酵素之純化
第二節 酵素水解反應條件之探討及比較
一、Trypsin等八種蛋白對黃豆蛋白、酪蛋白分解率之比較
二、黃豆蛋白水解之探討結果及所產生胜分布之比較
三、蛋白水解液以 HW-50(s) 分子篩管柱層析分析結果
第三節 鈣吸收促進活性胜之生產純化
一、黃豆蛋白質之水解
二、鈣結合性胜之管柱層析分析及其胺基酸組成特性之比較
第四節 3T3-L1前驅脂肪細胞分化調節活性分析系統之建立
一、3T3-L1細胞經分化處理後之形態變化
二、3T3-L1脂肪細胞處理以各胜區分之變化比較
第五節 ACE酵素抑制活性之測定
第四章 參考文獻
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