重组氨肽酶底物特异性及协同水解玉米蛋白质

Study on Substrate Specificity of Aminopeptidase from Recombinant Bacillus subtilis and the Cooperative Hydrolysis of Corn Protein

摘要:

以9种不同的氨基酸-对硝基苯胺为底物,考察重组枯草芽孢杆菌氨肽酶对底物的特异性。结果显示,该氨肽酶对由疏水氨基酸组成的Leu-pNA水解活力最强,其次是两种由碱性氨基酸组成的Arg-pNA、Lys-pNA,对其它几种由疏水氨基酸组成的Ala-pNA、Ile-pNA、Val-pNA、Phe-pNA、Pro-pNA表现出较弱的水解活力,而对酸性氨基酸组成的Glu-pNA则无水解活力。在此基础上考察其与内切蛋白酶协同水解玉米蛋白质的效果,以水解度为指标进行单因素试验,明确氨肽酶与碱性蛋白酶复配水解玉米蛋白质的效果较佳。双酶水解产物中新增游离氨基酸含量分别为碱性蛋白酶和氨肽酶单独水解的4.89倍、6.05倍;水解产物中多肽相对分子质量多在1 000 Da以下,其中500~1 000 Da的寡肽占14.57%,180~500 Da的小肽质量分数为68.42%,水解度达65%。这些结果表明,氨肽酶和碱性蛋白酶协同水解蛋白质原料具有良好的应用前景。

Abstract:

The substrate specificity of aminopeptidase from the recombinant Bacillus subtilis was investigated by employing nine types of p-nitroanilide derivatives of amino acids as substrates. The results indicated that the aminopeptidase shows the highest acitivity for Leu-pNA,whose amino acid moiety is hydrophobic leucine. The enzyme also shows high hydrolytic activity to Arg-pNA and Lys-pNA,whose amino acid moieties are basic amino acids. Followed are Ala-pNA,Ile-pNA,ValpNA,Phe-pNA and Pro-pNA,which contain hydrophobic amino acid moiety. However,Glu-pNA which contains acidic amino acid moiety can hardly be hydrolyzed. Then the hydrolysis of corn protein was carried out by the combination of aminopeptidase and endoprotease. With the hydrolysis degree as index by using the single factor experiment,aminopeptidase combined with alkaline protease obtained high efficiency:(1) The content of free amino acid was 4.89 times and 6.05 times than that of alkaline protease aminopeptidase was used alone,respectively.(2)The molecular weight of the peptides was mostly less than 1 000 Da,14.57% is within 500 ~1 000 Da,and 68.42% is within 180~500 Da.(3)The hydrolysis degree was 65%. These results suggested that aminopeptidase combined with alkaline protease hydrolyzes protein raw material.