蛋清蛋白水解物的制备、结构及其生物活性的研究
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摘要
我国是世界上最大的蛋品生产国,而我国目前的禽蛋加工状况是蛋品加工水平低、技术陈旧,由于我国的某些饮食习惯和一些行业的特殊需求,蛋黄的需求量较大,在生产过程中,蛋清被当成废弃物丢弃,既造成了蛋清资源的极大浪费,又造成了环境的污染。酶法水解蛋清蛋白质制备活性肽能有效地利用我国丰富的蛋清资源,将产生巨大的经济和社会效益。另外对具有抗凝血酶生物活性的蛋清活性肽鲜见报道,因此开展蛋清蛋白活性肽的基础研究工作,具有一定理论意义。
本论文以鸡蛋清为原料,以酶膜生物反应器制备具有抗凝血酶和抗氧化生物活性的蛋清蛋白水解物(EWPH),主要研究内容如下:
建立了一种体外测定抗凝血酶活性的方法。建立的新方法具有成本低、灵敏度高、重复性好等优点。
研究了酶解前蛋清热处理对水解度(DH)的影响,结果发现在酸性条件下热处理的蛋清对酶的抑制作用没有显著降低,而碱性条件下热处理可显著去除蛋清对蛋白酶的抑制作用。圆二色性测定发现热处理后蛋清形成了以β-折叠和无规卷曲为主的二级结构,蛋清经热处理引起的蛋白质的二级结构变化对DH的影响不大。蛋清水解度DH的提高主要是因为蛋清抑制蛋白酶作用的丧失引起的。
通过静态吸附和解吸实验,确定了DA201-C大孔树脂为EWPH的最佳脱盐树脂,对等量吸附焓变的测定发现DA201-C大孔树脂吸附EWPH是一种吸热的物理吸附过程。
通过比较水解蛋清蛋白的能力,筛选得到Alcalase和Protease N两种蛋白酶。Protease N水解物和Alcalase水解物的氨基酸组成相似,氨基酸评分比天然蛋清高,游离氨基酸含量很少,然而Protease N水解物比Alcalase水解物具有更高的抗氧化和抗凝血酶活性,最终确定了Protease N为蛋清蛋白水解用酶。
EWPH的疏水性与抗氧化活性的关系不大,与抗凝血酶活性具有一定相关性。具有最高抗氧化活性的EWPH的相对分子质量分布范围主要在249~4100之间。EWPH的氨基酸的极性大小和带电荷性质同抗凝血酶活性紧密相关。经过胃肠道消化酶的处理后,EWPH的抗氧化和抗凝血酶活性保持较好。
利用人工神经网络对EMBR的酶解过程进行了控制和仿真,实现了对EMBR制备活性肽的预测和模拟。EMRB抗凝血酶产物的氨基酸评分为113.25,第一限制氨基酸为苏氨酸,游离氨基酸含量为0.39%(w/w),产物中2~8个氨基酸组成的肽的相对含量接近80%。
采用大孔树脂吸附层析、凝胶层析、半制备RP-HPLC、分析型RP-HPLC分离得到四个具有抗凝血酶活性的组分。采用串联质谱从四个组分里分析得到17个肽的氨基酸序列,它们富含疏水氨基酸和碱性氨基酸。对其中的Leu-Val-Phe-Lys活性肽进行了合成,证明其具有抗凝血酶活性,抑制类型为可逆抑制。运用分子对接理论研究了肽Leu-Val-Phe-Lys的构效关系,发现对接后的配体结构与凝血酶空腔中的亲水-疏水区域形成了较好的匹配。肽链上增加疏水性侧链有助于增加配体分子对接过程中的脱溶作用以及疏水相互作用。结合能计算表明,增加肽链上的正电荷将有助于与凝血酶活性腔的氨基酸残基形成静电匹配,增强两者之间的结合力。
China has been the largest country of egg production in the world. At present, however, the level of egg processing in China is very low and the production technology is obsolescent. Because of some dietary habits and the special needs of some industries, large consumption of egg yolk has been on demand, while egg white has been regarded as the waste, which is not only a waste of egg white resources, but a source of environmental pollution.
Emzymatic hydrolysis of chicken egg white to produce egg white protein hydrolysates (EWPH) are investigated in this thesis, the contents are as following:
Microplate reader was used to establish an in vitro method for the determination of antithrombin activity. The new method has some advantages, like low cost, high sensitivity and good repeatability.
The contribution of the heat treatment of egg white before enzymatic hydrolysis to degree of hydrolsis (DH) was studied. The results showed that the heat treatment of egg white under acidic conditions did not significantly reduce the inhibition on the proteases, while the heat treatment under alkaline conditions could significantly eliminate the inhibition of egg white on the proteases. Circular dichroism measurement found the formation of aβ-sheet and random coil-based secondary structure in egg white proteins after heat treatment. The structural changes caused by the heat treatment of egg white proteins contributed little to the DH growth. The DH growth in egg white hydrolysis was ascribed to the elimination of inhibition of egg white on the proteases.
DA201-C macroporous absorption resin (MAR) was chosen as the best desalination resin through the static absorption and the desorption experiments. The static adsorption rule of EWPH on DA201-C MAR was found. The investigation of the isosteric adsorption enthalpy led to the founding that adsorption of EWPH on DA201-C MAR was a physical adsorption process.
By comparing the hydrolysis abilities of proteases, Alcalase and Protease N were chosen as enzymes with high capabilities to hydrolyze egg white proteins. The amino acid compositions of the hydrolysate from Protease N were similar to those from Alcalase. The amino acid scores of the hydrolysates of Alcalase and Protease N were higher than that of native egg white. The hydrolysates of Protease N had higher antithrombin activity and antioxidant activity than those of Alcalase. Protease N was eventually identified as the enzyme for egg white protein hydrolysates production.
It was found that there existed significant relevance between the hydrophobic property and the antioxidant activity of EWPH. The molecular weight distribution of the fraction with the highest antioxidant activity was in the range of 249 to 4100 Da. The antithrombin activity of EWPH was closely related to the polarity and the charge nature of the amino acids in EWPH. After the gastrointestinal tract digestive enzymes treatment, EWPH reserved the antioxidant and the antithrombin activities well.
Artificial neural network (ANN) was used to control and simulate the hydrolysis process of EMBR. The BP network could be used as a tool for simulating hydrolysis process. The EMRB product with highest antithrombin activity had an amino acid score of 113.25, with the first limiting amino acid of threonine. The free amino acid content was 0.39% (w/w). Two to eight amino-acid based peptides accounted for the relative content of nearly 80%.
Four fractions with antithrombin activity were obtained by macroporous resin chromatography, gel filtration chromatography, semi-preparative RP-HPLC, analytical RP-HPLC. The amino acid sequences of 17 peptides were gained using MS/MS analysis. It was found that the peptides were rich in hydrophobic amino acids and positive charged amino acids. The peptide with the sequence of Leu-Val-Phe-Lys was syntheticly produced and the antithrombin activity was verified. The inhibition type of the bioactive peptide on thrombin was reversible. The theoretical analysis using molecular docking showed that after the docking the ligands matched very well with a water drainage region in the cavity of active site of thrombin. The increased hydrophobic side chains on peptide chain could help the increase of the dissolution and hydrophobic interaction in the process of molecular docking. Binding energy calculation showed that the increase of positive charge in peptide chain contributed to the formation of electrostatic match with the active cavity of thrombin, thus enhancing the binding force between the both.
本论文以鸡蛋清为原料,以酶膜生物反应器制备具有抗凝血酶和抗氧化生物活性的蛋清蛋白水解物(EWPH),主要研究内容如下:
建立了一种体外测定抗凝血酶活性的方法。建立的新方法具有成本低、灵敏度高、重复性好等优点。
研究了酶解前蛋清热处理对水解度(DH)的影响,结果发现在酸性条件下热处理的蛋清对酶的抑制作用没有显著降低,而碱性条件下热处理可显著去除蛋清对蛋白酶的抑制作用。圆二色性测定发现热处理后蛋清形成了以β-折叠和无规卷曲为主的二级结构,蛋清经热处理引起的蛋白质的二级结构变化对DH的影响不大。蛋清水解度DH的提高主要是因为蛋清抑制蛋白酶作用的丧失引起的。
通过静态吸附和解吸实验,确定了DA201-C大孔树脂为EWPH的最佳脱盐树脂,对等量吸附焓变的测定发现DA201-C大孔树脂吸附EWPH是一种吸热的物理吸附过程。
通过比较水解蛋清蛋白的能力,筛选得到Alcalase和Protease N两种蛋白酶。Protease N水解物和Alcalase水解物的氨基酸组成相似,氨基酸评分比天然蛋清高,游离氨基酸含量很少,然而Protease N水解物比Alcalase水解物具有更高的抗氧化和抗凝血酶活性,最终确定了Protease N为蛋清蛋白水解用酶。
EWPH的疏水性与抗氧化活性的关系不大,与抗凝血酶活性具有一定相关性。具有最高抗氧化活性的EWPH的相对分子质量分布范围主要在249~4100之间。EWPH的氨基酸的极性大小和带电荷性质同抗凝血酶活性紧密相关。经过胃肠道消化酶的处理后,EWPH的抗氧化和抗凝血酶活性保持较好。
利用人工神经网络对EMBR的酶解过程进行了控制和仿真,实现了对EMBR制备活性肽的预测和模拟。EMRB抗凝血酶产物的氨基酸评分为113.25,第一限制氨基酸为苏氨酸,游离氨基酸含量为0.39%(w/w),产物中2~8个氨基酸组成的肽的相对含量接近80%。
采用大孔树脂吸附层析、凝胶层析、半制备RP-HPLC、分析型RP-HPLC分离得到四个具有抗凝血酶活性的组分。采用串联质谱从四个组分里分析得到17个肽的氨基酸序列,它们富含疏水氨基酸和碱性氨基酸。对其中的Leu-Val-Phe-Lys活性肽进行了合成,证明其具有抗凝血酶活性,抑制类型为可逆抑制。运用分子对接理论研究了肽Leu-Val-Phe-Lys的构效关系,发现对接后的配体结构与凝血酶空腔中的亲水-疏水区域形成了较好的匹配。肽链上增加疏水性侧链有助于增加配体分子对接过程中的脱溶作用以及疏水相互作用。结合能计算表明,增加肽链上的正电荷将有助于与凝血酶活性腔的氨基酸残基形成静电匹配,增强两者之间的结合力。
China has been the largest country of egg production in the world. At present, however, the level of egg processing in China is very low and the production technology is obsolescent. Because of some dietary habits and the special needs of some industries, large consumption of egg yolk has been on demand, while egg white has been regarded as the waste, which is not only a waste of egg white resources, but a source of environmental pollution.
Emzymatic hydrolysis of chicken egg white to produce egg white protein hydrolysates (EWPH) are investigated in this thesis, the contents are as following:
Microplate reader was used to establish an in vitro method for the determination of antithrombin activity. The new method has some advantages, like low cost, high sensitivity and good repeatability.
The contribution of the heat treatment of egg white before enzymatic hydrolysis to degree of hydrolsis (DH) was studied. The results showed that the heat treatment of egg white under acidic conditions did not significantly reduce the inhibition on the proteases, while the heat treatment under alkaline conditions could significantly eliminate the inhibition of egg white on the proteases. Circular dichroism measurement found the formation of aβ-sheet and random coil-based secondary structure in egg white proteins after heat treatment. The structural changes caused by the heat treatment of egg white proteins contributed little to the DH growth. The DH growth in egg white hydrolysis was ascribed to the elimination of inhibition of egg white on the proteases.
DA201-C macroporous absorption resin (MAR) was chosen as the best desalination resin through the static absorption and the desorption experiments. The static adsorption rule of EWPH on DA201-C MAR was found. The investigation of the isosteric adsorption enthalpy led to the founding that adsorption of EWPH on DA201-C MAR was a physical adsorption process.
By comparing the hydrolysis abilities of proteases, Alcalase and Protease N were chosen as enzymes with high capabilities to hydrolyze egg white proteins. The amino acid compositions of the hydrolysate from Protease N were similar to those from Alcalase. The amino acid scores of the hydrolysates of Alcalase and Protease N were higher than that of native egg white. The hydrolysates of Protease N had higher antithrombin activity and antioxidant activity than those of Alcalase. Protease N was eventually identified as the enzyme for egg white protein hydrolysates production.
It was found that there existed significant relevance between the hydrophobic property and the antioxidant activity of EWPH. The molecular weight distribution of the fraction with the highest antioxidant activity was in the range of 249 to 4100 Da. The antithrombin activity of EWPH was closely related to the polarity and the charge nature of the amino acids in EWPH. After the gastrointestinal tract digestive enzymes treatment, EWPH reserved the antioxidant and the antithrombin activities well.
Artificial neural network (ANN) was used to control and simulate the hydrolysis process of EMBR. The BP network could be used as a tool for simulating hydrolysis process. The EMRB product with highest antithrombin activity had an amino acid score of 113.25, with the first limiting amino acid of threonine. The free amino acid content was 0.39% (w/w). Two to eight amino-acid based peptides accounted for the relative content of nearly 80%.
Four fractions with antithrombin activity were obtained by macroporous resin chromatography, gel filtration chromatography, semi-preparative RP-HPLC, analytical RP-HPLC. The amino acid sequences of 17 peptides were gained using MS/MS analysis. It was found that the peptides were rich in hydrophobic amino acids and positive charged amino acids. The peptide with the sequence of Leu-Val-Phe-Lys was syntheticly produced and the antithrombin activity was verified. The inhibition type of the bioactive peptide on thrombin was reversible. The theoretical analysis using molecular docking showed that after the docking the ligands matched very well with a water drainage region in the cavity of active site of thrombin. The increased hydrophobic side chains on peptide chain could help the increase of the dissolution and hydrophobic interaction in the process of molecular docking. Binding energy calculation showed that the increase of positive charge in peptide chain contributed to the formation of electrostatic match with the active cavity of thrombin, thus enhancing the binding force between the both.
引文
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