【作者】 徐金玲；

【导师】 吴琼英；

【作者基本信息】 江苏科技大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 蚕蛹是蚕茧缫丝的副产物,由于色泽与口感较差,因而限制了它在食品和药品工业中的应用。本文旨在利用酶法制备蚕蛹蛋白ACE(血管紧张素转化酶)抑制肽,以扩大其生产应用范围。通过SDS-PAGE(聚丙烯酰胺凝胶电泳)、氨基酸组成分析、DSC(差示扫描量热仪分析)等方法研究了蚕蛹蛋白及其分离蛋白组分的理化性质;以酶解产物的ACE抑制活性为评价指标,采用响应曲面法对酶法制备蚕蛹蛋白ACE抑制肽的工艺进行了优化;并采用超滤、DEAE-52和Sephadex G -50柱层析等方法对蚕蛹蛋白ACE抑制肽进行了分离纯化,初步探讨了蚕蛹蛋白ACE抑制肽对ACE的作用机制。干蚕蛹中,粗蛋白的含量为57.61 %,其中,水溶性蛋白和碱溶性蛋白是蚕蛹主要的蛋白组分。蚕蛹水溶性蛋白和碱溶性蛋白的氨基酸种类齐全,且必需氨基酸占总氨基酸的质量分数分别为41.24 %和39.16 %,明显高于WHO推荐的氨基酸组成模式(>36 %);蚕蛹水溶性蛋白有2次热变性过程,起始变性温度分别为45.98℃和88.87℃,蚕蛹碱溶性蛋白有1次热变性过程,其变性起始温度为180.37℃。蚕蛹水溶性蛋白和碱溶性蛋白的Alcalase酶解产物均具有较强的ACE抑制活性,IC50(半抑制浓度)分别为0.121和0.113 mg/mL。以酶解产物对ACE的抑制率为评价指标,从Alcalase、α-胰凝乳蛋白酶、胰蛋白酶中筛选出Alcalase做为蚕蛹蛋白制备ACE抑制肽的水解用酶,并采用响应面试验设计方法对酶法制备蚕蛹蛋白ACE抑制肽的工艺进行优化。确定最优工艺参数为:pH 9.0、酶解温度50.8℃、加酶量3500 U/g;在此条件下,得到的蚕蛹蛋白酶解产物对ACE的IC50值为0.102 mg/mL。蚕蛹蛋白酶解产物经超滤分离后,ACE抑制活性最强的是MW(分子量)<5 kDa组分,其次依次为5～10 kDa组分和MW>10 kDa组分。利用Lineweaver-Burk方程发现MW<5 kDa组分对ACE呈现一种竞争性抑制关系;此外,紫外光谱分析表明MW<5 kDa组分能够改变ACE的结构。蚕蛹蛋白ACE抑制肽的性能试验表明,蚕蛹蛋白ACE抑制肽具有良好的酸、热稳定性和抗肠道酶消化能力。蚕蛹蛋白ACE抑制肽经DEAE-52柱层析和Sephadex G -50柱层析纯化后,其活性得到极大的提高,它的IC50值为0.072 mg/mL,分子量分布为226.34～983.61 Da,由2～8肽组成,其中,分子量为474.63 Da的肽链是主要的活性成分。更多还原

【Abstract】 Silkworm pupae is a by-product of silk reeling industry. The protein source has poor utility for the applications of food and medicine industry due to its bad color and taste. The objective of this study was to prepare angiotensin-converting enzyme (ACE) inhibitory peptides from silkworm pupae protein by enzymatic hydrolysis. The physicochemical properties of silkworm pupae protein and its fractions were investigated by the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid analysis and differential scanning calorimetry analysis (DSC). According to ACE-inhibitory activity of hydrolysates, the enzymatic hydrolysis technology of ACE-inhibitory peptides from silkworm pupae protein was also optimized by response surface methodology. ACE-inhibitory peptides of silkworm pupae were isolated and purificated from hydrolysates of silkworm pupae protein by ultrafiltration, DEAE-52 ion-exchange chromatography, and Sephadex G-50 gel chromatography. Furthermore, the interaction mechanism of the ACE-inhibitory peptides and ACE was investigated.The content of silkworm pupae protein in silkworm pupae (Dry base) was about 57.61 %. The water-soluble protein and alkali-soluble protein were the main protein fractions in silkworm pupae protein, the two protein fractions had complete kinds of amino acids and were well-balanced. The water-soluble protein and alkali-soluble protein had higher ratio of essential to total amino acids (41.24 % and 39.16 %, respectively) than the pattern recommended by WHO (at least 36 %). Two endothermic denaturation transitions were observed in the DSC thermograms of silkworm pupae water-soluble protein. The denaturation temperature (Td) was observed at 45.98°C and 88.87°C. The alkali-soluble protein of silkworm pupae had a endothermic denaturation transition, and its Td was 180.37°C. The alcalase hydrolysates for the water-soluble protein and alkali-soluble protein of silkworm pupae had strong ACE-inhibitory activities (Their IC50 values were 0.121 and 0.113 mg/mL, respectively).Taking ACE inhibitory activity as index, alcalase was chosen from three commercial proteases (alcalase,α-chymotrypsin and trypsin), which was used to prepare ACE-inhibitory peptides from silkworm pupae protein. The hydrolysis process of alcalase was optimized by response surface methodology. The results showed that the optimal hydrolysis conditions were pH 9.0, temperature of enzymatic hydrolysis 50.8°C and enzyme/substrate (E/S) 3500 U/g. Under the optimal conditions, the IC50 value of alcalase hydrolysates of silkworm pupae protein was 0.102 mg/mL.The hydrolysates fractions of molecular weight (MW) <5 kDa, 5~10 kDa, and MW>10 kDa were obtained from hydrolysates of silkworm pupae protein by ultrafication. Their ACE-inhibitory activities decreased in the following order: MW <5 kDa, 5~10 kDa, and MW>10 kDa. The ACE inhibition pattern of MW <5 kDa fractions isolated from hydrolysates of silkworm pupae protein was investigated using Lineweaver–Burk plots, and found to be competitive. In addition, ultraviolet spectra revealed that the MW <5 kDa fractions could cause a change on molecular structure of ACE. The performance tests showed that ACE-inhibitory peptides obtained from silkworm pupae protein had good acidic, alkaline, and thermal stability and ability of anti-intestinal digestion.The activities of ACE-inhibitory peptides from silkworm pupae protein, which were purificated by DEAE-52 ion-exchange chromatography and Sephadex G-50 gel chromatography, were markedly increased. Their IC50 value was 0.072 mg/mL. The purificated ACE-inhibitory peptides were composed of dipeptide to octapeptide, and their molecular weights were 226.34 ~ 983.61 Da. Among them, the peptides of 474.63 Da were the main activity fractions.更多还原