大肠杆菌焦磷酸酶的表达及其酶学性质研究  

Expression and enzymatic properties of pyrophosphatase in Escherichia coli

作　　者：王赞丞 王璐 樊俊萍 陈雨点 陈婷婷 刘露露 李勇昊 WANG Zancheng;WANG Lu;FAN Junping;CHEN Yudian;CHEN Tingting;LIU Lulu;LI Yonghao(School of Chemistry and Chemical Engineering,Chongging University of Science and Technology,Chongging 40133l,China)

机构地区：[1]重庆科技大学化学化工学院,重庆401331

出　　处：《中国酿造》2024年第9期65-71,共7页China Brewing

基　　金：国家自然科学基金项目(22378033);重庆市自然科学基金项目(CSTB2022NSCQ-MSX0544);重庆市教委科学技术研究项目(KJQN202301546);重庆科技大学研究生科研创新项目(YKJCX2320503)。

摘　　要：该研究通过聚合酶链式反应(PCR)扩增克隆大肠杆菌(Escherichia coli)Rosetta(DE3)的焦磷酸酶(PPase)基因,采用无缝克隆技术构建焦磷酸酶表达载体pET-28a-ppa,并转化至感受态E.coliRosetta(DE3),构建重组菌株E.coli PPa.采用单因素试验对其诱导表达条件进行优化,并进一步采用镍离子磁珠纯化重组PPas,研究其酶学性质。结果表明,成功构建重组菌株E.coliPPa,当异丙基-β-D硫代半乳糖苷(IPTG)诱导浓度、诱导温度和诱导时间分别为0.4 mmolL、20℃和8h时,重组PPase活性达到最高,为129.05 IU/mL,是优化前的3.14倍。重组PPase的理论分子质量为19.7kDa;最适反应温度和pH分别为55℃和9,在温度10~30℃及pH 7.0~9.0范围内稳定;金属离子K^(+)、Mg^(2+)和Na^(+)可极显著性提高该酶活性(P<0.01),而Mn^(2+)和Zn^(2+)对该酶有极显著抑制作用(P<0.01);重组PPase的动力学参数米氏常数(K_(m)值)为3.58 μmol/mL、最大反应速率(V_(max)值)为314.66μmol/(mL·min),催化常数(K_(cat)值)为87300.56S^(-1),半失活时间为10 min。该研究为后续工业化生产PPase及对该酶的理性设计提供依据。The pyrophosphatase(PPase)gene of Escherichia col Roseta(DE3)was amplified and cloned by polymerase chain reaction(PCR),the py rophosphatase expression vector pET-28a-ppa was constnucted by seamless cloning techmigue,transformed into competence E.coli Rosetta(DE3) and the recombimant strain E.coliPPase was constncted.The induced expression conditions were optimized by single factor tests,recombinant PPas was purified by nickel ion magnetic beads,and the enzymatic properties were studied.The results showed that the recombiant stram E,col'Ppa wa sucesfiuly constructed.The recombinant PPase activity reached the highest level(129.05IU/ml)under the conditions:isopropyl-β-d-thiogalactoside(PTG)induction concentration,induction temperature,and induction time were 20℃,0.4 mmolL.and 8 h,respectively,which was 3.14 times higher than that before optimization.The theoretical molecwlar mass ofrecombinant PPase was 19.7 kDa.The optimal reaction temperature and pH were 55℃ and 9℃,respectively.and stable in the range of temperaure 10-30℃and pH 7.0-9.0.Metal ionsK^(+)、Mg^(2+)and Na^(+)could sienificantly enhance the enzyme activity(P<0.01),while Mg^(2+)andZn^(2+)significanty imhibited the enzyme activity(P<0.01).The kinetic parameters of the recombinant PPas were as folows:Michaelis constant(K_(m)valve)3.58 umolml,maximum reaction rate(V_(max)value)314.66 umol(ml·min).catalvic number(K_(cat)value)87300.56S^(-1).and half imactivation time 10 min.This study provided a basis for the subsequent industrial production of PPase and rational desigt ofthe enzyne.

关 键 词：焦磷酸酶 大肠杆菌 诱导表达 酶学性质 

分 类 号：Q819[生物学—生物工程]