利用Serratia quinivorans BCRC 14811之short-chain dehydrogenase/reductase立體選擇性生產phenylephrine

Abstract

本研究以微生物轉換法來進行L-PE的合成，希望篩選到能將前驅物1-(3-hydroxyphenyl)-2-(methylamino)ethanone(HPMAE)不對稱還原成L-PE的酵素。利用S. quinivorans BCRC 14811菌體可將HPMAE轉換成L-PE (e.e >99%)，轉換率為15%。在培養過程中加入2-phenylethanol及acetophenone，轉換率可由原來的15%提升為88%及83%。利用pQE30做為表現載體以及對HPMAE敏感的Escherichia coli NovaBlue作為宿主，建構S. quinivorans BCRC 14811的基因庫，使用含有1~10 mM HPMAE之Luria-Bertani平板進行篩選。然而，並沒有得到任何具有HPMAE還原能力的轉型株。本研究由S. quinivorans BCRC 14811基因組序列中，選殖到short-chain dehydrogenase/reductase (SDR)基因，將此基因選殖並表現於E. coli，發現表現SDR蛋白之E. coli BL21(DE3)(pET30a-sdr-39729)轉型株能專一地將HPMAE還原成D-PE(e.e >99%)。由於反應受質HPMAE在室溫下久置容易分解，以10 mM HPMAE作為反應受質時，轉換率為89%，將HPMAE的濃度提高為60 mM時，轉換率可達94%。利用IMAC進行SDR酵素純化並分析酵素活性，發現SDR可利用NADPH或NADH做為輔酶(cofactor)，比活性分別為257 U/mg及285 U/mg。

In order to avoid involved in the chemical synthesis method, the present study was designed to use a biotransformation approach to produce L-PE from 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE). We found that S. quinivorans BCRC 14811 could convert HPMAE to L-PE with 15% of yield. Addition of 2-phenylethanol and acetophenone in the culture medium could increase conversion yield from 15% to 88% and 83%, respectively. A genomic library of S. quinivorans BCRC 14811 was constructed for the screening of clones capable of converting HPMAE to PE using pQE30 as cloning vector and HPMAE-sensitive Escherichia coli NovaBlue as host cell. Luria-Bertani plate containing 1 to 10 mM HPMAE were used as the selection medium. However no positive clone was obtained. Short-chain dehydrogenase / reductase (SDR) was cloned from S. quinivorans BCRC 14811 by PCR. When the sdr gene was expressed in E. coli BL21(DE3), the recombinant E. coli cell can convert 10 mM HPMAE to 8.9 mM D-PE with a yield of about 89% and 60 mM HPMAE to 56.2 mM D-PE with a conversion yield of 94%. The SDR was purified by immobilized metal affinity chromatography. Enzyme activity assay demonstrated that the SDR protein could uses NADPH and NADH as cofactors, which exhibit a specific activities of 257 U/mg and 285 U/mg, respectively.