本文選題：苯乙酮酸 + 生物催化��； 參考：《南京理工大學(xué)》2010年碩士論文

【摘要】： 苯乙酮酸(PGA)，，又稱苯甲酰甲酸,是應(yīng)用廣泛的有機(jī)合成中間體。本實(shí)驗(yàn)室從銅綠假單胞菌(Pseudomonas aeruginosa)中克隆得到扁桃酸消旋酶的編碼基因(mdlA)和扁桃酸脫氫酶基因(mdlB),己成功構(gòu)建了可將扁桃酸轉(zhuǎn)化為苯乙酮酸的重組表達(dá)菌株E.coli BL21 (DE3)/pEt30a (mdlA+mdlB),并且在IPTG誘導(dǎo)下有較高的表達(dá)量。本文在以上工作的基礎(chǔ)上,從該工程菌株的性質(zhì)出發(fā),對菌體培養(yǎng)條件進(jìn)行系統(tǒng)優(yōu)化,旨在獲得高菌體密度的同時保證有較高的蛋白表達(dá)量；除獲得高密度、高活力菌體外,提高苯乙酮酸產(chǎn)率的另一個重要方面就是轉(zhuǎn)化工藝條件的優(yōu)化。菌體只有在最適宜的轉(zhuǎn)化條件下,才能表現(xiàn)最佳的轉(zhuǎn)化活力,保證苯乙酮酸的產(chǎn)率實(shí)現(xiàn)最大化。具體實(shí)驗(yàn)設(shè)計及結(jié)果如下： 對重組菌的基本性質(zhì)和誘導(dǎo)培養(yǎng)條件進(jìn)行了研究。選用乳糖代替IPTG作為誘導(dǎo)劑,采用搖瓶培養(yǎng),相繼確定了乳糖最適添加濃度為5g／L、添加時間4h、誘導(dǎo)培養(yǎng)時間8h、誘導(dǎo)前培養(yǎng)溫度37℃,誘導(dǎo)培養(yǎng)溫度30℃等一系列適合該工程菌高效表達(dá)重組蛋白的誘導(dǎo)條件。 為獲得高密度、高活力菌體,采用統(tǒng)計學(xué)優(yōu)化方法對該重組菌的培養(yǎng)基組成及培養(yǎng)條件進(jìn)行優(yōu)化研究。首先,利用Plackett-Burman試驗(yàn)設(shè)計篩確定三個主要影響因素,即碳、氮源濃度和pH值；在此基礎(chǔ)上用最陡爬坡路徑逼近最大響應(yīng)區(qū)域；再以菌體轉(zhuǎn)化比活力(即蛋白表達(dá)量)為考察指標(biāo)進(jìn)行CCD試驗(yàn)設(shè)計,并對實(shí)驗(yàn)數(shù)據(jù)進(jìn)行回歸分析,通過求解回歸方程得到三個顯著因素的最優(yōu)取值,即碳源濃度6.34g／L,復(fù)合氮源濃度為13.56g／L,pH值為7.7。在此條件下,模型預(yù)測最高響應(yīng)值為18.5276 U／g。經(jīng)五批次培養(yǎng)驗(yàn)證實(shí)驗(yàn),預(yù)測值與驗(yàn)證試驗(yàn)平均值非常接近,表明該模型能很好地預(yù)測實(shí)際的培養(yǎng)條件。 在對菌體培養(yǎng)條件進(jìn)行優(yōu)化,成功獲得高密度、高活力菌體后,為進(jìn)一步提高重組菌轉(zhuǎn)化苯乙酮酸的能力,對重組菌轉(zhuǎn)化條件進(jìn)行了詳細(xì)的研究。最終確定轉(zhuǎn)化反應(yīng)最適條件為：底物濃度為20g/L,細(xì)胞濃度為10g/L,磷酸體系濃度67mM, pH6.0,37℃,200rpm。在此條件下,轉(zhuǎn)化比活力又有所提高,可達(dá)到20.5273U／g,轉(zhuǎn)化反應(yīng)進(jìn)行8h即完成轉(zhuǎn)化。 通過上述對培養(yǎng)條件和轉(zhuǎn)化條件的優(yōu)化實(shí)驗(yàn),當(dāng)?shù)孜锉馓宜釢舛葹?%時,重組菌在8h可完全轉(zhuǎn)化,與未經(jīng)優(yōu)化的原始條件相比,在底物濃度提高了1倍的同時轉(zhuǎn)化反應(yīng)時間縮短了40h。
[Abstract]:Acetophenone acid PGA, also known as benzoyl formic acid, is a widely used intermediate in organic synthesis. From Pseudomonas aeruginosa), the coding gene of mandelic acid racemase was cloned from Pseudomonas aeruginosa) and the gene of mandelic dehydrogenase (MDLBN) was cloned from Pseudomonas aeruginosa. A recombinant expression strain, E.coli BL21 DE3 / pEt30a, was successfully constructed to transform mandelic acid into phenylketonic acid. And there was a high expression level induced by IPTG. On the basis of the above work and the properties of the engineering strain, the culture conditions of the strain were systematically optimized to obtain high bacterial density and ensure high protein expression, in addition to obtaining high density and high activity bacteria in vitro. Another important aspect of improving the yield of acetophenone acid is the optimization of conversion process conditions. Only under the most suitable transformation conditions can the bacteria exhibit the best transformation activity and ensure the maximum yield of acetophenone acid. The experimental design and results are as follows: The basic properties and induced culture conditions of recombinant bacteria were studied. Lactose instead of IPTG was used as inducer. The optimum concentration of lactose was 5 g / L, the addition time was 4 h, the induction time was 8 h, and the culture temperature before induction was 37 鈩

本文編號：1887276