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首页> 外文会议>Asia-Pacific Chemical Reaction Engineering Symposium(APCRE'05); 20050612-15; Gyeongju(KR) >Comparison of whole cell biocatalytic reaction kinetics for recombinant Escherichia coli with periplasmic-secreting or cytoplasmic-expressing organophosphorus hydrolase
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Comparison of whole cell biocatalytic reaction kinetics for recombinant Escherichia coli with periplasmic-secreting or cytoplasmic-expressing organophosphorus hydrolase

机译：重组大肠杆菌与周质分泌或细胞质表达有机磷水解酶的全细胞生物催化反应动力学的比较。

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摘要

Organophosphate compounds are widely used in many pesticides (Paraoxon, Parathion, Coumaphos, and Diazinon) and chemical nerve agents (Sarin and Soman). Organophosphorus hydrolase (OPH) from Pseudomonas diminuta or Flavobacterium sp. is a homodimeric organophosphotriesterase that requires metal ion as a cofactor and can degrade a broad spectrum of toxic organophosphates. This enzyme can hydrolyse various phosphorus-ester bonds including P-O, P-F, P-CN, and P-S bonds. The application of OPH for bioremediation is of great interest due to its high turnover rate. Recombinant Escherichia coli expressing OPH can degrade a variety of organophosphates. The ability of E. coli to grow to much higher densities than P. diminuta or Flavobacterium enables the development of large-scale detoxification processes. However, recombinant E. coli cells produce low yields of OPH due to the low solubility of this protein. In addition, the E. coli cell membrane can be a substrate diffusion barrier affecting whole cell biocatalytic efficiency. Several strategies attempted to enhance OPH production yield or whole cell biocatalytic efficiency include fusion with a soluble partner to increase solubility, co-expression with Vitreoscilla hemoglobin and display on the cell surface. Periplasmic secretion of target proteins via translocation across the cytoplasmic membrane in Gram-negative bacteria such as E. coli can be a potential strategy for reducing the substrate diffusion barrier in whole cell biocatalyst systems. Previously, we have successfully shown that functional secretion of OPH molecules into the periplasmic space was achieved using the twin-arginine translocation (Tat) pathway. In particular, we have used the twin-arginine signal sequence of the E. coli enzyme trimethylamine N-oxide (TMAO) reductase (TorA) because OPH molecules require metal ion cofactors. Using this system, whole cell OPH activity was approximately 2.8-fold higher due to successful translocation of OPH into the periplasmic space. This study has shown that Tat-driven periplasmic secretion of OPH is a potential strategy to overcome traditional substrate diffusion limitations in whole cell biocatalyst systems. In the present work, for detail kinetic studies, we compared biocatalytic reaction kinetics for four types of whole cell biocatalyst systems; whole cells with periplasmic-secreting OPH under trc or T7 promoters and whole cells with cytoplasmic-expressing OPH under trc or T7 promoters.
机译：有机磷酸酯化合物被广泛用于许多农药（派拉信，对硫磷，库玛磷和二嗪农）和化学神经毒剂（沙林和索曼）。假单胞菌或黄杆菌属的有机磷水解酶（OPH）。是一种需要金属离子作为辅因子的同型二聚有机磷酸三酯酶，可以降解多种有毒的有机磷酸酯。该酶可以水解各种磷酯键，包括P-O，P-F，P-CN和P-S键。 OPH由于其高周转率而在生物修复中的应用备受关注。表达OPH的重组大肠杆菌可以降解多种有机磷酸酯。大肠埃希氏菌生长到比小食单胞菌或黄杆菌更高的密度的能力使得能够进行大规模的排毒过程。然而，重组大肠杆菌细胞由于该蛋白的低溶解性而产生低产量的OPH。另外，大肠杆菌细胞膜可以是影响全细胞生物催化效率的底物扩散屏障。尝试提高OPH产量或全细胞生物催化效率的几种策略包括与可溶性伴侣融合以增加溶解度，与玻璃体血红蛋白共表达并在细胞表面展示。通过在革兰氏阴性细菌（例如大肠杆菌）中跨细胞质膜转运而靶蛋白的周质分泌可能是减少全细胞生物催化剂系统中底物扩散障碍的潜在策略。以前，我们已经成功地证明了使用双精氨酸易位（Tat）途径实现了OPH分子向周质空间的功能性分泌。特别是，由于OPH分子需要金属离子辅因子，因此我们使用了大肠杆菌酶三甲胺N-氧化物（TMAO）还原酶（TorA）的双精氨酸信号序列。使用该系统，由于OPH成功转移到周质空间中，整个细胞的OPH活性大约高2.8倍。这项研究表明，OPH的Tat驱动的周质分泌是克服全细胞生物催化剂系统中传统底物扩散限制的一种潜在策略。在目前的工作中，为进行详细的动力学研究，我们比较了四种类型的全细胞生物催化剂系统的生物催化反应动力学。在trc或T7启动子下具有周质分泌OPH的全细胞，在trc或T7启动子下具有细胞质表达OPH的全细胞。

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