酪蛋白非磷肽酶膜耦合法制备ACE抑制肽
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摘要
酪蛋白磷酸肽(CPPs)是具有结合矿物质作用的活性肽,人们对它的研究开发较早,已经实现工业化生产,但是伴随其生产产生的副产物CNPPs未得到足够的重视和研究开发。由于CNPPs中的疏水性氨基酸含量较高,是ACE抑制肽的良好来源,特别是肽段相对分子质量小、含有特定疏水性氨基酸时具有较高的ACE抑制活性,因此本研究采用酶膜耦合法处理CNPPs制备ACE抑制肽,通过对CNPPs的控制水解,制备和分离相对分子质量较小的酶解产物,循环利用水解酶,建立一种制备ACE抑制肽的新方法。
首先,对CNPPs的氨基酸组成和相对分子质量分布进行测定,结果表明,CNPPs疏水性氨基酸的摩尔百分比和疏水性值分别为54.67%和7.47,相对分子质量大于1500Da的肽占相当大一部分比例。CNPPs经1K膜超滤分离后,相对分子质量小于1K Da滤过液的ACE抑制率为50.40%,远高于相对分子质量大于1K Da的肽的ACE抑制率18.82%。结合CNPPs和ACE抑制肽的组成和结构特点,有对CNPPs进一步研究的必要。
然后,对CNPPs酶膜耦合法制备ACE抑制肽水解用酶进行筛选。对几种蛋白酶制备的ACE抑制肽的抑制活性、得率和酶活力保留率的测定结果表明,Protease N制备ACE抑制肽的抑制率大于55%,酶活力保留率大于90%,优于其它几种蛋白酶。ACE抑制肽得率较高,仅次于AS1398中性蛋白酶。综合考虑,Protease N是适合酶膜耦合法制备ACE抑制肽的水解用酶。
接着研究了CNPPs酶膜耦合反应的条件。以ACE抑制肽得率和酶残余活力为指标,确定了各反应条件对CNPPs酶膜耦合反应影响的主次顺序为:压力>进料速度>温度>加酶量。最后得出CNPPs酶膜耦合反应较佳条件为:压力12psi,进料速度145mL/min,温度47.5℃,加酶量6000 U/g蛋白质。在此优化条件下对CNPPs酶膜耦合反应,达稳定状态后,滤液肽浓度为2.5mg/mL,膜通量为50mL·min~(-1)·m~(-2),反应器中肽浓度为5.7 mg/mL,蛋白酶活力每小时下降8%左右。
最后对ACE抑制肽进行脱盐并对产品性质进行测定。大量无机盐的存在使ACE抑制肽的品质受到影响,本文选择DA201-C大孔吸附树脂可以完全脱除ACE抑制肽中的盐分。用85%(v/v)乙醇静态解吸,蛋白质回收率大于85%,所得ACE抑制肽脱盐率大于95%。IC50值由脱盐前0.86mg/mL降到脱盐后0.12mg/mL。经体外消化稳定性实验发现ACE抑制肽具有稳定性。
Casein phosphopeptides(CPPs)have been reported as the typical functional peptides for mineral binding and extensive researches have already been carried out. Casein non-phosphopeptides (CNPPs), by-products for industrial production of CPPs, however, have not been paid enough attention. Extensive studies showed that, CNPPs have high content of hydrophobic amino acids and are good sources of ACE inhibitory peptides, which could help modulate blood pressure. ACE inhibitory activity will be more significant when the peptide molecular weight is small and the structure consists of certain hydrophobic amino acids. Therefore, protease was screened and controlled hydrolysis of CNPPs and separation of the hydrolysates were carried out in an enzymatic membrane reactor to obtain high ACE inhibitory peptides.
First, the amino acid composition and molecular weight distribution of CNPPs were determined. Results show that, molar percentage of hydrophobic amino acids and hydrophobicity values of CNPPs were 54.67% and 7.47 respectively. A big portion of peptides with molecular weight greater than 1500 Da exist in the CNPPs. CNPPs were separated by 1K membrane ultrafiltration, and ACE inhibition rate of peptides with molecular weight less than 1K Da was 50.40%. Meanwhile, peptides with molecular weight greater than 1K Da possessed ACE inhibition rate of 18.82%, which is much lower than that of small peptides. Thus, CNPPs could be adopted as abundant sources for have ACE inhibitory peptides due to its strong hydrophobic features.
Several proteases were screened for enzymatic hydrolysis of CNPPs. Considering the ACE inhibitory activity, the yield and enzyme activity retention rate after hydrolysis. Protease N was choosen for preparation of ACE inhibitory peptides, which provided ACE inhibition rate more than 55% and enzyme activity retention rate more than 90%. Both are better than the other several proteases. AS1398 neutral protease also brought very high ACE inhibitory activity, but lower than Protease N. However, the yield of its ACE inhibitory peptides is higher than Protease N. Therefore, Protease N was adopted to prepare the ACE inhibitory peptides.
The conditions of reaction coupling of enzymatic hydrolysis and membrane separation on CNPPs had been studied. ACE inhibitory peptides yield and the enzyme activity retention rate were considered when optimize the reaction and separation conditions. The significance of different factors were disclosed as: Pressure> feed speed> temperature> enzyme concentration. And optimized conditions of reaction were: pressure of 12psi, feed rate of 145mL/min, temperature of 47.5℃, enzyme concentration of 6000 U/g protein. Under the optimal conditions, the filtrate peptide concentration was 2.5mg/mL, membrane flux 50mL.min~(-1).m~(-2), reactor peptide concentration 5.7 mg/mL, enzyme activity retention rate decreased about 8% per hour.
Desalting of ACE inhibitory peptides was carried out and the properties of the products were determined. The existence of a large amount of inorganic salts deteriorated the quality of ACE inhibitory peptides, so DA201-C macroporous resin was adopted for salt removal from crude ACE inhibitory peptides. With 85% (v/v) ethanol static desorption, the protein recovery was more than 85% and more than 95% salt could be removed. IC50 value was reduced from 0.86mg/mL to 0.12mg/mL after desalting. In vitro digestion stability of ACE inhibitory peptides was reliable.
首先,对CNPPs的氨基酸组成和相对分子质量分布进行测定,结果表明,CNPPs疏水性氨基酸的摩尔百分比和疏水性值分别为54.67%和7.47,相对分子质量大于1500Da的肽占相当大一部分比例。CNPPs经1K膜超滤分离后,相对分子质量小于1K Da滤过液的ACE抑制率为50.40%,远高于相对分子质量大于1K Da的肽的ACE抑制率18.82%。结合CNPPs和ACE抑制肽的组成和结构特点,有对CNPPs进一步研究的必要。
然后,对CNPPs酶膜耦合法制备ACE抑制肽水解用酶进行筛选。对几种蛋白酶制备的ACE抑制肽的抑制活性、得率和酶活力保留率的测定结果表明,Protease N制备ACE抑制肽的抑制率大于55%,酶活力保留率大于90%,优于其它几种蛋白酶。ACE抑制肽得率较高,仅次于AS1398中性蛋白酶。综合考虑,Protease N是适合酶膜耦合法制备ACE抑制肽的水解用酶。
接着研究了CNPPs酶膜耦合反应的条件。以ACE抑制肽得率和酶残余活力为指标,确定了各反应条件对CNPPs酶膜耦合反应影响的主次顺序为:压力>进料速度>温度>加酶量。最后得出CNPPs酶膜耦合反应较佳条件为:压力12psi,进料速度145mL/min,温度47.5℃,加酶量6000 U/g蛋白质。在此优化条件下对CNPPs酶膜耦合反应,达稳定状态后,滤液肽浓度为2.5mg/mL,膜通量为50mL·min~(-1)·m~(-2),反应器中肽浓度为5.7 mg/mL,蛋白酶活力每小时下降8%左右。
最后对ACE抑制肽进行脱盐并对产品性质进行测定。大量无机盐的存在使ACE抑制肽的品质受到影响,本文选择DA201-C大孔吸附树脂可以完全脱除ACE抑制肽中的盐分。用85%(v/v)乙醇静态解吸,蛋白质回收率大于85%,所得ACE抑制肽脱盐率大于95%。IC50值由脱盐前0.86mg/mL降到脱盐后0.12mg/mL。经体外消化稳定性实验发现ACE抑制肽具有稳定性。
Casein phosphopeptides(CPPs)have been reported as the typical functional peptides for mineral binding and extensive researches have already been carried out. Casein non-phosphopeptides (CNPPs), by-products for industrial production of CPPs, however, have not been paid enough attention. Extensive studies showed that, CNPPs have high content of hydrophobic amino acids and are good sources of ACE inhibitory peptides, which could help modulate blood pressure. ACE inhibitory activity will be more significant when the peptide molecular weight is small and the structure consists of certain hydrophobic amino acids. Therefore, protease was screened and controlled hydrolysis of CNPPs and separation of the hydrolysates were carried out in an enzymatic membrane reactor to obtain high ACE inhibitory peptides.
First, the amino acid composition and molecular weight distribution of CNPPs were determined. Results show that, molar percentage of hydrophobic amino acids and hydrophobicity values of CNPPs were 54.67% and 7.47 respectively. A big portion of peptides with molecular weight greater than 1500 Da exist in the CNPPs. CNPPs were separated by 1K membrane ultrafiltration, and ACE inhibition rate of peptides with molecular weight less than 1K Da was 50.40%. Meanwhile, peptides with molecular weight greater than 1K Da possessed ACE inhibition rate of 18.82%, which is much lower than that of small peptides. Thus, CNPPs could be adopted as abundant sources for have ACE inhibitory peptides due to its strong hydrophobic features.
Several proteases were screened for enzymatic hydrolysis of CNPPs. Considering the ACE inhibitory activity, the yield and enzyme activity retention rate after hydrolysis. Protease N was choosen for preparation of ACE inhibitory peptides, which provided ACE inhibition rate more than 55% and enzyme activity retention rate more than 90%. Both are better than the other several proteases. AS1398 neutral protease also brought very high ACE inhibitory activity, but lower than Protease N. However, the yield of its ACE inhibitory peptides is higher than Protease N. Therefore, Protease N was adopted to prepare the ACE inhibitory peptides.
The conditions of reaction coupling of enzymatic hydrolysis and membrane separation on CNPPs had been studied. ACE inhibitory peptides yield and the enzyme activity retention rate were considered when optimize the reaction and separation conditions. The significance of different factors were disclosed as: Pressure> feed speed> temperature> enzyme concentration. And optimized conditions of reaction were: pressure of 12psi, feed rate of 145mL/min, temperature of 47.5℃, enzyme concentration of 6000 U/g protein. Under the optimal conditions, the filtrate peptide concentration was 2.5mg/mL, membrane flux 50mL.min~(-1).m~(-2), reactor peptide concentration 5.7 mg/mL, enzyme activity retention rate decreased about 8% per hour.
Desalting of ACE inhibitory peptides was carried out and the properties of the products were determined. The existence of a large amount of inorganic salts deteriorated the quality of ACE inhibitory peptides, so DA201-C macroporous resin was adopted for salt removal from crude ACE inhibitory peptides. With 85% (v/v) ethanol static desorption, the protein recovery was more than 85% and more than 95% salt could be removed. IC50 value was reduced from 0.86mg/mL to 0.12mg/mL after desalting. In vitro digestion stability of ACE inhibitory peptides was reliable.
引文
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