摘要
用双引物法对GI基因进行体外定点突变 ,构建了突变体Q2 0L和G2 47D。含突变基因的重组表达质粒pTKD GIQ2 0L及 pTKD GIG2 47D在E .coliK38菌株中表达。纯化的突变酶与野生型酶相比 :(1)GIQ2 0L的最适反应温度下降 5℃ ,热稳定性为野生型酶的 78% ,对底物的亲和性增强 ;(2 )GIG2 47D的酶活提高约 33% ,最适pH下降 0 6个单位 ,但热稳定性降低。初步分析认为 ,Gln 2 0位于α0～α1螺旋之间 ,其亲水侧链被Leu的疏水侧链取代后 ,分子表面增强的疏水作用 ,反而不利于蛋白质的稳定 ,使GIQ2 0L的热稳定性降低。Gly2 47是酶活性中心β 折叠 (2 42～ 2 47aa)的最后一个残基。引入电负性极强的Asp后 ,可能改变分子的静电场分布 ,影响了活性部位的电荷传递过程 ,使GIG2 47D酶活提高。引入的电荷 ,可能改变活性中心可解离基团的 pKa ,使其最适pH下降。另外Asp2 47的侧链在周围空间结构中显得过于拥挤 ,易与其他侧链产生排斥 ,由此影响到 β 折叠的稳定性 ,接近亚基结合面的Asp2 47,可能进一步影响到亚基间相互作用的稳定性 ,最终导致酶热稳定性的降低。GI酶活和最适pH的改善更利于工业生产。
The mutants of Q20L and G247D of glucose isomerase (GI) were constructed by in vitro site directed mutagenesis of GI gene with double primersmethod. The recombinant plasmids pTKD GIQ20L and pTKD GIG247D were expressed in E.coli K38 strain. The comparison experiments of mutant enzymes with wild type GI showed that: (1) the optimum temperature of GIQ20L was decreased by 5℃. Its thermostability was only 78% half time of the wild type. But its substrate affinity was enhanced. (2) The specific activity of GIG247D was increased by 33%,and the optimum pH was lowered by 0.6 unit. However,the thermostability of GIG247D was decreased. We supposed,based on the above facts and 0.19nm resolution crystal structure of SM33GI,that Gln20 locates between α0 helix and α1 helix,the substitution of hydrophobic side chain of Leu for hydrophilic side chain of Gln may enhance the hydrophobic interaction of the molecular surface,leading to the decrease of the stability and thermostability of GIQ20L. Gly247 which is the last amino acid of a β sheet from 242 to 247 residues locates in the active core of GI. After replacement,Asp247 which has strong nagetive electricity may change the electrostatic distribution and influence the charge transfer processes of the active core. So the specific activity of GIG247D was increased. The introduced charge could alter the pKa of dissociable groups and make the optimum pH lower. In addition,the side chain of Asp247 seems to be very crowded in the surrounding space conformation and is easy to exclude with the other side chains,therefore influences the stability of β sheet. Furthermore,Asp247 is in the vicinity of the interface of subunits,so it could interfere with the stability of the interaction between subunits. Thus,the GIG247D decreased the thermostability of SM33GI. The higher enzyme activity and the lower optimum pH will be very useful for industrial production of GI.
出处
《生物工程学报》
CAS
CSCD
北大核心
2000年第4期469-473,共5页
Chinese Journal of Biotechnology
基金
国家高技术研究发展计划项目资助!( 863 13 0 13 0 2 0 4)
关键词
葡萄糖异构酶
酶活
最适PH
高果糖浆
定点突变
Molecular modeling,protein engineering,glucose isomerase,enzyme activity,optimum pH,substrate affinity
