利用Rhodosporidium toruloides BCRC 21888 及 Serratia marcescens BCRC10948做為生物催化劑生產L-phenylephrine

Abstract

L-form phenylephrine (L-PE) is an effectively decongestant and commonly incorporated into cold and allergy drugs. The gene encoding a protein with ketone reductase activity was cloned for the asymmetric synthesis of L-PE from its precursor, 2-[benzyl(methyl)amino]-1-(3-hydroxyphenyl) ethanone (BMAHPE) and 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE). Both D-form and L-form of 3-{(1S)-2-[benzyl(methyl)amino]-1- hydroxyphenyl}phenol (BMA-PE) was prepared by chemical synthesis for the chirarity of the BMA-PE from bioconversion . It was founded that the BMA-PE from S. marcescens BCRC10948 was L-form. Inducer, the 2-phenylethanol、acetophenone and 1-phenylethanol were added in the culture medium of S. marcescens BCRC10948 for the conversion of HPMAE to PE, and the conversion ratio was 78.6 % ,77.5 % , and 73.2 % were obtained,respectively. Ribosyldihydronicotinamide dehydrogenase 1 from the genomic library of S. marcescens BCRC10948 could convert BMAHPE to BMA-PE with conversion ratio of 0.0094 % in the reaction mixture containing 5 mM BMAHPE, 0.1 M Na-phosphate buffer (pH 7.0) and 2 % glucose at 30℃ for 24 h. AKR7-2(aldo-keto reductase)、ADH3(alcohol dehydrogenase)、SMR 3(reductase)、SMR 44 and SMR 48 also possesed the enzymatic activity to convert BMAHPE to BMA-PE with conversion ratio below 0.013 %. Two gene product, SDR72 and SDR10 , showed capability of converting the HPMAE to D-PE and L-PE, respectively, with conversion ratio was 83 % and 68 % in the reaction mixture containing 10 mM HPMAE, 0.1 M sodium phosphate buffer (pH 7.0) and 2 % glucose at 30℃ for 24 h. It is the first reported that L-PE could be converted from HPMAE by SDR10 enzyme from S. marcescens BCRC10948.

L-form phenylephrine（L-PE；苯腎上腺素）為一化學合成藥物，屬於不對稱化合物（chiral compound），廣泛的添加於感冒藥及抗過敏藥物中。本研究擬選殖能將2-[benzyl(methyl)amino]-1-(3-hydroxyphenyl) ethanone (BMAHPE) 轉換成L-form 3-{(1S)-2-[benzyl(methyl)amino]-1- hydroxyphenyl}phenol (BMA-PE)的基因或將1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) 轉換成L-PE的基因，進而利用微生物催化方式生產L-PE。本研究先利用化學法合成D-form及L-form的BMA-PE標準品，並使用Cyclobond I 2000 AC管柱分析，證實Serratia marcescens BCRC10948能將BMAHPE轉換成L-form BMA-PE及將HPMAE轉換成L-PE。當在培養基內加入2-phenylethanol、acetophenone及1-phenylethanol時，菌體對HPMAE的轉換率會從21.3 % 提升到78.6 %、77.5 % 及73 %。在S. marcescens BCRC10948基因庫中，發現ribosyldihydronicotinamide dehydrogenase 1基因之產物具有轉換BMAHPE的活性，惟在含有5 mM BMAHPE、0.1 M sodium phosphate buffer (pH 7.0)及2 % glucose反應液內，30℃反應24小時後，轉換率只有0.0094 %。在S. marcescens dehydrogenase及reductase基因產物中，AKR7-2(aldo-ketone reductase)、ADH3(alcohol dehydrogenase)、SMR 3、SMR 44及SMR 48具有轉換BMAHPE成BMA-PE的活性，轉換率最高為0.013 % 。在轉換HPMAE成PE部分，發現基因產物SDR72及SDR10具有轉換活性，其分子量分別為29 kDa及27.5 kDa，在含有10 mM HPMAE、0.1 M Na-phosphate buffer (pH 7.0)及2 % glucose、30℃反應24小時後，轉換率分別為83 % 及68 %， SDR72的轉換產物為D-PE，而SDR10的轉換產物為L-PE。本研究結果顯示SDR10能將HPMAE轉換為L-PE，為首次發現可將HPMAE轉換成L-PE的基因，在工業應用上有其潛力，在基礎研究上也有其價值。