融合松脂醇-落葉松脂醇還原酶及裂環落葉去氫酶將松脂醇連續轉化為羅漢松脂醇

Abstract

木酚素 (lignans)，為苯丙醇 (phenylpropanoid) 之二聚體衍生物，在生理學上具有植物雌激素功能 (phytoestrogen) 以及抗癌活性，存在於全麥食品、芝麻、亞麻籽。其中，裂環落葉松脂醇 (secoisolariciresinol) 及羅漢松樹脂醇 (matairesinol) 在哺乳類結腸中，可被轉換為屬於哺乳類木酚素 (mammalian lignans) 之腸內脂 (enterolactone) 及腸內二脂 (enterodiol)，而血清及尿液中高濃度腸內脂對於治療乳癌、骨質疏鬆症、及大腸癌都有正面效果。為建立木酚素高效率轉化系統，利用選殖自台灣八角蓮木酚素生合成酵素基因，松脂醇-落葉松脂醇還原酶 (pinoresinol-lariciresinol reductase, PLR) 以及裂環落葉松脂醇去氫酶(secoisolariciresinol dehydrogenase, SDH)，以非對稱重疊擴增聚合酶鏈鎖反應 (asymmetric overlap extension by polymerase chain reaction, AOE-PCR) 將上述酵素之基因藉由蛋白質連接序列 (linker) 連接形成雙功能性蛋白質融合基因，並於大腸桿菌 (Escherichia coli) 中表現。經聚丙烯醯胺膠體電泳 (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 及西方點墨法 (Western blot) 蛋白質分析，選定經 0.01 mM IPTG 誘導12 小時，重組融合蛋白質可以於大腸桿菌中表現。於 pH 8.0、22OC 進行胞外生物轉化反應1小時，發現融合蛋白質 PLR-SDH，不僅同時具有兩種酵素活性，且反應效率高於重組 PLR 混和重組 SDH 之組別。PLR-SDH 之 matairesinol/pinoresinol總轉換率為 49.82 %，單位蛋白質轉換率為 3.41 % /

Lignans, a class of dimeric phenylpropanoid derivatives, existing in whole-grain, sesame and flax seeds show anticancer activity and can react as phytoestrogen. Secoisolariciresinol and matairesinol, members of lignans, can be converted to enterolactone and enterodiol in mammalian proximal colon. Containing high concentration of enterolactone in serum and urine has positive effect for the treatment of breast cancer, osteoporosis and colon cancer. To establish a continuous bioconversion system for matairesinol formation from pinoresinol, pinoresinol reductase (plr) and secoisolariciresinol dehydrogenase (sdh) which were cloned from Podophyllum peltatum Hance were linked by asymmetric overlap extension by polymerase chain reaction (AOE-PCR), to form a bifunctional fusion gene (plr-sdh) and was expressed in Escherichia coli. The optimized conditions for recombinant fusion protein induction in E.coli were 0.01 mM IPTG, 12 h, 25oC determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. At pH 8.0, 22OC, 1 h reaction in vitro the results showed that the bioconversion efficiency of fusion protein PLR-SDH was higher than the mixture of individual rPLR and rSDH. The bioconversion rate of PLR-SDH was 49.82%, and 3.41% /