本文關(guān)鍵詞： 普瑞巴林 (S)-2-羧乙基-3-氰基-5-甲基已酸乙酯 假單胞菌 對(duì)映選擇性 靜息細(xì)胞　出處：《浙江大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：普瑞巴林(Pregabalin),(S)-3-氨甲基-5-甲基己酸,是一種用于抗神經(jīng)痛和癲癇癥的輔助治療的手性藥物,其國(guó)際市場(chǎng)需求呈現(xiàn)較快增長(zhǎng)之勢(shì)。開展生物催化法制備普瑞巴林的研究,篩選拆分制備普瑞巴林的微生物,具有重要的應(yīng)用前景和價(jià)值。 (S)-2-羧乙基-3-氰基-5-甲基己酸乙酯((S)-CCMAE)是合成普瑞巴林的關(guān)鍵手性中間體。本文首先以外消旋體2-羧乙基-3-氰基-5-甲基己酸乙酯(CCMAE)為底物,從土壤中篩選得到一株具有自主知識(shí)產(chǎn)權(quán)的能夠?qū)τ尺x擇性水解(R)-CCMAE的假單胞菌(Pseudomonas) CGMCC No.4184。對(duì)其產(chǎn)酶條件及培養(yǎng)基進(jìn)行了優(yōu)化,使酶活從15.92 U/L提高到33.43 U/L。隨后對(duì)假單胞菌CGMCCNo.4184靜息細(xì)胞水解拆分底物的反應(yīng)條件進(jìn)行了研究,確定了較優(yōu)反應(yīng)條件為：30～35℃,pH 7.5-8.5。并成功利用微生物靜息細(xì)胞轉(zhuǎn)化法制備了ee值達(dá)99.8%的(S)-CCMAE,這與目前報(bào)道的生物拆分工藝均有所不同。研究還發(fā)現(xiàn)Tween-80和TritonX-100可明顯提高對(duì)映選擇率E值和酶活。最后利用海藻酸鈉-PVA復(fù)合載體法制備了假單胞菌CGMCC No.4184的固定化細(xì)胞,對(duì)其技術(shù)參數(shù)和反應(yīng)條件作了優(yōu)化,并利用所制得的固定化細(xì)胞制備ee值99%以上的(S)-CCMAE。該固定化細(xì)胞能夠較好地進(jìn)行重復(fù)利用。 本研究中篩選獲得的高選擇性R型水解酶可能是一種新酶,后續(xù)的基因克隆和表達(dá)、酶學(xué)性質(zhì)研究工作正在進(jìn)行中,為進(jìn)一步改善酶的催化性能和拓展應(yīng)用范圍打下了良好的基礎(chǔ)。
[Abstract]:Pregabalinine is a chiral drug used in the adjuvant treatment of neuralgia and epilepsy. The research on the preparation of PreBahrain by biocatalysis and the screening and resolution of microbes in the preparation of PreBahrain have important application prospect and value. Sh 2 carboxyethyl 3 cyano 5 methyl hexanoic acid ethyl Ester (S0. CCMAE). It is a key chiral intermediate for the synthesis of PreBahrain. Firstly, the racem-2-carboxyethyl-3-cyano-5-methyl-caproate ethyl ester (CCMAE) was used as the substrate. A Pseudomonas pseudomonas strain with independent intellectual property rights capable of enantioselective hydrolysis of RCMAE was obtained from soil. CGMCC No. 4184. the conditions of producing enzyme and the medium were optimized. The enzyme activity was increased from 15.92 U / L to 33.43 UL. The reaction conditions of CGMCCNo.4184 resting cell hydrolysis and resolution of substrate were studied. The optimum reaction conditions were determined as follows: 1: 30 ~ 35 鈩，

本文編號(hào)：1454114