大肠杆菌磷酸甘油酸脱氢酶突变体的构建及抗反馈抑制效应  

Construction and characterization of Escherichia coli D-3-phosphoglycerate dehydrogenase mutants with feedback-inhibition relief

作　　者：邓辉[1,2] 陈存武[1] 孙传伯[1] 韦传宝[1,2] 

机构地区：[1]皖西学院生物与制药工程学院,安徽六安237012 [2]六安市蛋白质分离与纯化研究中心,安徽六安237012

出　　处：《生物工程学报》2016年第4期468-477,共10页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2012AA021500);国家重点基础研究发展计划(973计划)(No.2012CB720805);国家自然科学基金(Nos.81274021;H2801)资助~~

摘　　要：磷酸甘油酸脱氢酶(D-3-phosphoglycerate dehydrogenase,PGDH,EC 1.1.1.95)为L-丝氨酸合成途径的关键酶,其编码基因为ser A,其活性受到合成产物L-丝氨酸的反馈抑制调控。为解除丝氨酸的反馈抑制,采用定点突变技术把编码PGDH酶344位组氨酸或346位天冬氨酸或364位天冬氨酸的密码子定点突变为丙氨酸密码子。改造后的ser AFbr被连到表达载体pT7-7上,并转入大肠杆菌Escherichia coli BL21(DE3)中进行表达,破壁回收粗酶液,通过DEAE阴离子柱纯化PGDH突变体,并对其酶活性和IC_(50)值进行了测定。结果,野生型PGDH酶IC_(50)值为7μmol/L,而PGDH双突变体N346A/H344A催化活性与野生型相近,在丝氨酸浓度为160 mmol/L时,其酶活仍保持未添加丝氨酸时酶活的96%,基本解除反馈抑制。3-Phosphoglycerate dehydrogenase（PGDH, EC 1.1.1.95） is the key enzyme in L-serine biosynthesis and its coding gene is ser A. PGDH is feedback inhibited by L-serine. In order to relieve the feedback-inhibition of PGDH by L-serine, H344 or D346 or D364 were chosen for site directed mutagenesis. The mutants were generated by the standard Quik Change mutagenesis, further subcloned into expression vector pT7-7 and transformed into Escherichia coli BL21（DE3） cells. The recombinant cells were collected after cultured in LB media post induced by isopropyl beta-Dthiogalactopyranoside. The enzymes were purified by anion exchange chromatography, and SDS-PAGE showed that the purified enzymes were homogenous. Enzyme characterization indicated that the mutant enzyme showed similar activity, optimal temperature, and optimal p H as that of the wild-type enzyme. Moreover, feedback inhibition study showed that the activity of the double mutant（N346A/H344A） could remain 96% in the presence of serine up to 160 mmol/L, whereas the activity of the wild-type enzyme remains only 50% in the presents of serine of 7 μmol/L, thus successfully relieving the feedback inhibition of PGDH with its activity remained.

关 键 词：大肠杆菌磷酸甘油酸脱氢酶 突变体的构建 解除反馈抑制 

分 类 号：Q78[生物学—分子生物学]