重组米曲霉丝氨酸羧肽酶O的性质鉴定及脱苦应用研究  

Characteristics of carboxypeptidase O from Asperigillus oryzae expressed in Pichia pastoris and its application in protein hydrolysis and debittering

作　　者：陈丹[1] 游子娟 黄伟谦[1] 马纳纳 罗晓春[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《广东农业科学》2015年第5期103-108,共6页Guangdong Agricultural Sciences

基　　金：广东省科技计划项目(2012B020311003)

摘　　要：为了研究羧肽酶在蛋白水解及对蛋白水解物脱苦中的重要作用,利用毕赤酵母表达系统对米曲霉（Asperigillus oryzae）羧肽酶O（carboxypeptidase O,Ocp O）进行表达。重组米曲霉羧肽酶O（r Ocp O）经过分子筛和离子柱纯化,对其进行脱糖基化及酶学性质的检测研究。结果表明,该蛋白酶是以糖蛋白的形式进行表达,分子量约为74 ku,脱糖基化后的相对分子质量约为56 ku,产量约为20.4 n Kat/m L。该蛋白酶是一种酸性蛋白酶,最适p H值为4.0,且在p H 3.0～6.0时保持稳定;最适温度约为40℃,具有良好的热稳定性,在60℃孵育1 h后仍能保持63%的酶活;PMSF能够显著抑制该酶酶活,证明其为丝氨酸羧肽酶。通过r Ocp O对大豆蛋白和酪蛋白的Alcalase蛋白酶水解物的进一步水解研究发现,r Ocp O水解对两种水解液的苦味均有明显减轻,并且水解度有明显升高。In order to study the importance of carboxypeptidase in protein hydrolysis and debittering, a truncated carboxypetidase O from Asperigillus oryzae was expressed in Pichia pastoris. The recombinant carboxypeptidase O（r Ocp O） was deglycosylated and characterized after purified through Sephadex G-75 and DEAE anionic-exchange chromatography. The results showed that r Ocp O was glycosylated of a molecular weight of about 74 ku and 56 ku after been deglycosylated, the enzyme yield was about 20.4 n Kat/m L. r Ocp O was an acid protease, its optimal p H was about 4.0, and it was stable in the p H range of 3-6; its optimal temperature was about 40℃, it retained 63% enzyme activity after 1 h incubation at 60℃; PMSF could efficiently inhibit its activity, which confirmed it was a serine-type carboxypeptidase. The bitterness of SPI and casein hydrolysates catalyzed by Alcalase significantly reduced after treated with r Ocp O and the degree of hydrolysis（DH） efficiently improved at the same time.

关 键 词：米曲霉 重组羧肽酶O 酶学性质 脱苦 

分 类 号：Q814.9[生物学—生物工程]