【作者】 陈丹；

【导师】 罗晓春；

【作者基本信息】 华南理工大学 ， 生物化学与分子生物学， 2014， 硕士

【摘要】 米曲霉（Aspergilus.oryzea）是是豆豉、豆酱以及酱油等传统发酵食品的重要生产菌株。米曲霉具有丰富的酶系，在生长的过程中能够向周围分泌丰富的蛋白酶和淀粉酶，在酶制剂生产工业中也具有广泛的应用。蛋白水解是提高蛋白利用效率和获得生物活性肽的有效手段。由于疏水氨基酸的暴露，蛋白在水解的过程往往会产生一些不想要的苦味，大大限制了其应用。羧肽酶（Carboxypeptidases，CPs）是一种外肽酶，因其能够从蛋白多肽的C端切掉影响苦味的疏水性氨基酸，所以具有一定的脱苦作用。本文在课题组前期研究的基础上，利用毕赤酵母表达系统，对米曲霉的丝氨酸羧肽酶O基因（ocpO）成功进行了克隆表达，随后对其进行了纯化和酶学性质的分析，最后通过观察其对大豆蛋白（Soy Protein Isolate, SPI）和酪蛋白（Casein）的碱性蛋白酶（Alcalase）水解物的进一步水解作用来评价其脱苦效应。主要结论如下：（1）克隆了米曲霉羧肽酶O的基因（ocpO），并将其利用毕赤酵母表达系统进行表达，结果成功表达出了带糖基化的蛋白酶，大小约为74KDa，酶产量为20.4nKat/ml。（2）通过分子筛和阴离子柱层析，将表达的重组米曲霉羧肽酶O（rOcpO）进一步纯化，去糖基化后分子量约为56KDa。通过测定其酶学性质，发现重组米曲霉羧肽酶O为酸性蛋白酶；最适pH为4，当pH处于6-7时仍然还有80%的酶活；最适温度约为40℃，具有良好的热稳定性，在60℃时保温1小时仍剩余63%的酶活；苯甲基磺酰氟（Phenylmethanesulfonyl fluoride，PMSF）能够部分抑制其活性，进一步证明其为丝氨酸羧肽酶。rOcpO的最终比酶活为24.9mKat/kg。（3）rOcpO对大豆蛋白和酪蛋白碱性蛋白酶水解液有明显的脱苦作用，并且水解度有明显提高。更多还原

【Abstract】 Aspergillus oryzae is an important fungus in the production of traditional fermentedfoods, such as fermented soybeans, soybean paste and soy sauce. It can secret abundantproteases and amylases. Hydrolysis is an efficient way to improve protein utility efficiencyand produce bioactive peptides, while with the exposure of hydrophobic amino acids duringthe protein hydrolysis, the hydrolysates are usually bitter tasted, which inhibits their utility infood industry. Carboxypeptidases(CPs) can remove hydrophobic amino acids from C terminalof peptides and have debitterring effects in protein hydrolysates. In this paper, we cloned acarboxypeptidase O (OcpO) from Aspergillus oryzae and expressed it in Pichia pastoris.Results showed that the expressed recombinant OcpO(rOcpO) was a glycosylated protein inPichia pastoris with a molecular weight of74KDa. The enzyme yield was20.4nKat/ml.rOcpO was purified by Sephadex G-75and anion-exchanging chromatography. The molecularweight of deglycosylated rOcpO was about56KDa. rOcpO was an acidic protease with theoptimal pH4and was stable in the pH range of3-6; its optimal temperature was about40℃,it retained63%enzyme activity after1h incubation at60℃; PSMF can efficiently inhibit itsactivity, which confirmed it t be a serine-type carboxypeptidase. rOcpO can significantlyimprove the hydrolysis degree of Alcalase hydrolyzed soybean protein and casein, and theirbitterness was greatly reduced.更多还原