【摘要】：手性氨基酸、氨基醇、氨基腈等手性胺是多種化合物合成的關(guān)鍵中間體,廣泛應(yīng)用于活性藥物成分、精細(xì)化工品、農(nóng)藥以及染料等領(lǐng)域。手性胺合成的方法包括化學(xué)和酶法立體選擇性�；�、氧化拆分與不對稱氫轉(zhuǎn)移。其中,氨基酸脫氫酶(Amino acid dehydrogenase,AADH)不對稱還原潛手性酮酸或酮制備手性胺具有高立體選擇性、高效率、反應(yīng)溫和以及環(huán)境友好等優(yōu)勢,是合成手性胺的重要途徑之一。本研究通過基因挖掘策略,獲得了來自克勞氏芽孢桿菌(Bacilllus clausii)和解淀粉芽孢桿菌(Bacilllus amyloliquefaciens)的兩個有較高催化活性的氨基酸脫氫酶BcAADH和BaAADH。(1)對BcAADH的酶學(xué)性質(zhì)研究發(fā)現(xiàn):其還原胺化的最適pH為9.5,氧化脫氨的最適pH為10.5;該酶的最適反應(yīng)溫度為30℃,在30、40和50℃條件下的半衰期分別為315、86.6和4.49 h。BcAADH可高效還原芳基酮酸類底物,對苯甲酰甲酸的比活力為17.7 U?mg?1,kcat/Km為5.02 s 1 mmol·L 1。將其與來自巨大芽孢桿菌(Bacillus megaterium)的葡萄糖脫氫酶(Glucose dehydrogenase,GDH)共表達(dá),對共表達(dá)重組菌合成L-苯甘氨酸的反應(yīng)條件進(jìn)行了優(yōu)化。在6小時(shí)內(nèi)可將400 mmol·L 1苯甲酰甲酸完全轉(zhuǎn)化為L-苯甘氨酸,e.e.99.9%,底物與酶量的比值高達(dá)60 g·g 1,環(huán)境因子和酶消耗數(shù)分別為4.7和0.018。(2)對BaAADH的酶學(xué)性質(zhì)研究發(fā)現(xiàn):其還原胺化和氧化脫氨的最適pH分別為8.5和10.5;最適反應(yīng)溫度為45℃,在30、40和50℃的半衰期分別為462、117.6和6.17 h。BaAADH對苯甲酰甲酸的比活力為44.5 U·mg 1,kcat/Km為9.8 s 1·mmol·L 1。對五種AADH進(jìn)行雙突變顯示,僅BaAADHK68S/N261L具有不對稱還原烷基酮類底物的活力。進(jìn)一步通過同源建模、分子對接和定向進(jìn)化,使BaAADHK68S/N261L具有還原胺化苯乙酮的催化活性。構(gòu)建其底物結(jié)合口袋的定點(diǎn)飽和突變文庫,基于2,4-二硝基苯肼法對構(gòu)建的突變文庫進(jìn)行高通量篩選。最終獲得突變體BaAADHK68S/N261L/L74N和BaAADHK68S/N261L/L76N,對苯乙酮的比活力分別為75.6和46.8 mU·mg 1。綜上所述,利用BcAADH可以綠色和高效地制備L-苯甘氨酸;通過定向進(jìn)化研究鑒定獲得BaAADH不對稱還原胺化苯乙酮的關(guān)鍵位點(diǎn)L74和L76。本研究為手性非天然氨基酸的合成提供了高效的生物催化劑,為指導(dǎo)氨基酸脫氫酶的分子改造和合成手性芳基胺提供了突變熱點(diǎn)并奠定了理論基礎(chǔ)。
[Abstract]:Chiral amino acids, amino alcohols, amino nitriles and other chiral amines are key intermediates in the synthesis of various compounds. They are widely used in the fields of active drug components, fine chemicals, pesticides and dyes. The methods of chiral amine synthesis include chemical and enzymatic stereoselective acylation, oxidative resolution and asymmetric hydrogen transfer. Among them, the asymmetric reduction of amino acid dehydrogenase (Amino acid dehydrogenase,AADH) to chiral ketonic acids or ketones has the advantages of high stereoselectivity, high efficiency, mild reaction and environmental friendliness. It is one of the important ways to synthesize chiral amines. In this study, through the strategy of gene mining, Two amino acid dehydrogenase (BcAADH) and two amino acid dehydrogenase (BaAADH.) with high catalytic activity from Bacillus Claus (Bacilllus clausii) and Bacillus amylase (Bacilllus amyloliquefaciens) were obtained and their enzymatic properties on BcAADH were studied. It was found that the reductive amidation of the two amino acid dehydrogenase (BcAADH) and BaAADH. (1) to BcAADH. The optimal pH is 9.5. The optimum pH for oxidative deammoniation was 10.5; The optimum reaction temperature was 30 鈩，

本文編號：2434659