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文章摘要
甘油激酶的表达纯化及在酮糖合成中的应用
Expression and Purification of Glycerol Kinase and Its Application for Ketose Synthesis
  
DOI:
中文关键词: 甘油激酶  醛缩酶  磷酸二羟基丙酮  一釜多酶法  酮糖
英文关键词: glycerol kinase,aldolase,dihydroxyacetone phosphate,one-pot multienzymatic reaction, ketose
基金项目:
作者单位
吴晓茹 江南大学 生物工程学院江苏 无锡 214122 
李子杰 江南大学 生物工程学院江苏 无锡 214122 
中西秀树 江南大学 生物工程学院江苏 无锡 214122 
高晓冬 江南大学 生物工程学院江苏 无锡 214122 
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中文摘要:
      研究了大肠杆菌MG1655来源的甘油激酶(GK)的表达及其在酮糖合成中的应用。本研究从大肠杆菌MG1655扩增了甘油激酶的基因片段glpK,连接到表达载体pET-28a上,在大肠杆菌Rosetta(DE3)中进行IPTG诱导表达,利用Ni2+亲和层析柱纯化目的蛋白。利用该甘油激酶可以催化成本较低的底物甘油生成L-3-磷酸甘油,再经过磷酸甘油氧化酶(GPO)的催化生成磷酸二羟基丙酮(DHAP),生成的DHAP可以在不同的DHAP依赖性醛缩酶的催化下与受体D-甘油醛合成酮糖-1-磷酸,最终用酸性磷酸酶脱掉磷酸生成一系列酮糖。结果表明,该酶成功地用于“一釜多酶法”合成体系中,以便宜的底物甘油为前体,合成出包括稀有糖在内的一系列酮糖,生成的产物、产率及比例用TLC、HPLC进行检测。该方法的建立将为其他稀有糖及其衍生物的合成提供理论依据。
英文摘要:
      In this paper,the expression of glycerol kinase(GK) from Escherichia coli MG1655 and its application for ketose synthesis were investigated. The gene glpK encoding GK was amplified and cloned into the expression plasmid pET-28a. The recombinant plasmid was transformed into the E.coli Rosetta(DE3) to express the protein induced by IPTG. GK enzyme was purified by Ni2+ chelating affinity column chromatography. The GK enzyme can catalyze cheap substrate glycerol to produce L-glycerol 3-phosphate which is converted into DHAP by glycerol phosphate oxidase(GPO).The generated DHAP can be coupled with the acceptor D-glyceraldehyde catalyzed by different DHAP-dependent aldolases to produce ketose-1-phosphate,which can be dephosphorylated with acid phosphatase(AP) to get a series of ketoses. The results demonstrated that a series of ketoses including rare sugars were successfully synthesized using one-pot multienzymatic reaction with the cheap substrate glycerol as the precursor. The product yields and ratios were detected by TLC and HPLC. This approach could provide the theory basis for the synthesis of more other rare sugars and their derivatives.
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