毕赤酵母表达重组猪肾氨基酰化酶Ⅰ的纯化与鉴定  

Purification and Characterization of Recombinant Porcine Aminoacylase Ⅰ Expression in Pichia pastoris

作　　者：刘志刚[1] 杨波[1] 胡征[1] 邓贝[1] 

机构地区：[1]湖北工业大学生物工程学院,湖北武汉430068

出　　处：《食品与发酵科技》2012年第4期95-99,共5页Food and Fermentation Science & Technology

基　　金：湖北省教育厅基金项目(Q20081411);湖北工业大学高层次人才基金项目(20062016)的资助

摘　　要：运用毕赤氏酵母对猪源重组氨基酰化酶Ⅰ(pACY1)进行了表达,并对其酶学参数进行了测定。构建毕赤氏酵母工程菌GS115-pacy1,通过甲醇诱导表达后,进行了Phenyl-Sepharose及Q-Sepharose两步色谱纯化;对纯化后重组pACY1的动力学参数进行测定。重组pACY1纯化后呈均一性,其纯度为98.6%;酶学参数测定结果表明,重组酶的比活力(Vmax)为280U/mg,米氏常数(Km)为0.91mM,接近天然酶对应的287U/mg和0.84mM;而酵母重组酶Tm值为74℃,高于天然酶的70℃。毕赤氏酵母表达重组pACY1的酶活性与天然酶相当,且其热稳定性优于天然酶。这为氨基酸拆分产业提供了更加优质而丰富的酶制剂。Recombinant porcine Aminoacylase Ⅰ （pACY1） was expressed in Pichia pastoris, and had been charac-tered. The recombinant strain GS115-pacyl was constructed. Recombinant pACY1 were purified from its culture after methanol inducing by Phenyl-Sepharose and Q-Sepharose. Then the kinetic parameters of the purified enzymes were determined. The purified enzyme was homogenicity. Ⅰts purity was 98.6%. Specific activity （Vmax） and Michaelis eonstant（Km） of it were 280U/mg and 0.91mM, similar to 287U/mg and 0.84mM of natural porcine enzyme individ-ually. The Tm value of recombinant pACY1 was 74℃, higher than 70℃ of natural enzyme. The activity of recombi-nant enzyme expressed in Pichia pastoris was similar with natural enzyme, but the heat stablity of former was better than the latter＇s. Ⅰt will provide abundant and high-grade enzyme for chiral aimino acid industry.

关 键 词：氨基酰化酶Ⅰ 毕赤氏酵母 表达纯化 热稳定性 

分 类 号：TQ926[轻工技术与工程—发酵工程]