【摘要】：β-苯丙氨酸變位酶屬于MIO-依賴氨基變位酶(MIO-dependent aminomutase),可催化α-苯丙氨酸轉(zhuǎn)化為β-苯丙氨酸,是目前最有效的抗癌藥物—紫杉醇的重要前體物質(zhì)。隨著癌癥在全球范圍內(nèi)發(fā)病率的逐年提升,β-苯丙氨酸需求量也日益增加。目前,在生產(chǎn)中大量獲取β-苯丙氨酸的方法主要是化學(xué)拆分法和Mannich反應(yīng)法等,這些方法步驟復(fù)雜,產(chǎn)物難以純化,生產(chǎn)成本昂貴還會(huì)產(chǎn)生有毒有害的副產(chǎn)物。與這些方法相比,酶催化法有著高效、快捷、安全的巨大優(yōu)勢(shì)。β-苯丙氨酸變位酶作為可以高效催化生成β-苯丙氨酸的生物催化劑受到了越來越多的關(guān)注。本研究根據(jù)β-苯丙氨酸變位酶PAM基因序列設(shè)計(jì)合成上下游引物,利用PCR技術(shù)擴(kuò)增了目的基因,并將其和表達(dá)載體pET-28a連接,構(gòu)成了表達(dá)質(zhì)粒pET-28a-pam。將表達(dá)質(zhì)粒轉(zhuǎn)入大腸桿菌表達(dá)菌株BL21中,篩選陽(yáng)性菌株,并通過酶切檢測(cè)在菌株內(nèi)是否成功轉(zhuǎn)入目的基因條帶,得到一株基因重組菌。通過對(duì)IPTG誘導(dǎo)劑的單因素試驗(yàn)確定了最佳誘導(dǎo)劑含量以及最佳誘導(dǎo)時(shí)間,并對(duì)重組菌進(jìn)行誘導(dǎo)表達(dá)。以SDS-PAGE手段檢測(cè)了重組菌的誘導(dǎo)表達(dá)并對(duì)重組酶進(jìn)行了純化。對(duì)重組酶的基本酶學(xué)性質(zhì)pH、溫度以及熱穩(wěn)定性進(jìn)行了研究,確定了重組酶的最佳催化pH為9.0,最佳催化溫度為50℃,并對(duì)重組酶的熱穩(wěn)定性進(jìn)行了驗(yàn)證,發(fā)現(xiàn)其熱穩(wěn)定性較好。利用單因素試驗(yàn)和響應(yīng)面法對(duì)重組菌的產(chǎn)酶條件進(jìn)行了優(yōu)化,確定了最優(yōu)發(fā)酵條件為種齡:12.8 h,初始pH:6.7,發(fā)酵時(shí)間:25.2 h。在最優(yōu)發(fā)酵條件下,重組菌產(chǎn)PAM最大酶活可達(dá)11.15 U/mL,較優(yōu)化前提高了 128.9%。在3 L發(fā)酵罐中對(duì)重組菌進(jìn)行擴(kuò)大培養(yǎng)。利用單因素試驗(yàn)確定了最佳溶氧為30%。分別比較了未添加任何補(bǔ)料的發(fā)酵方式,培養(yǎng)20 h時(shí)流加30 mL補(bǔ)料培養(yǎng)基的發(fā)酵方式以及培養(yǎng)30 h時(shí)流加40 mL補(bǔ)料培養(yǎng)基的發(fā)酵方式。確定了補(bǔ)料發(fā)酵方式優(yōu)于未進(jìn)行補(bǔ)料的發(fā)酵方式,并且對(duì)比得到在20 h時(shí)進(jìn)行流加補(bǔ)料的發(fā)酵方式優(yōu)于在30 h時(shí)進(jìn)行補(bǔ)料發(fā)酵的方式。以此方式進(jìn)行擴(kuò)大培養(yǎng)發(fā)酵所得最高酶活為16.63 U/mL,與搖瓶發(fā)酵相比提高了 49.1%,較優(yōu)化前的菌株產(chǎn)酶活性共提高了 241.5%。
[Abstract]:尾 -phenylalanine translocation enzyme (尾 -phenylalanine) belongs to MIO- dependent aminotranslocation enzyme (MIO-dependent aminomutase), which can catalyze the conversion of 偽 -phenylalanine to 尾 -phenylalanine. It is an important precursor of paclitaxel, the most effective anticancer drug. With the increasing incidence of cancer worldwide, the demand for 尾-phenylalanine is increasing. At present, the main methods of obtaining 尾 -phenylalanine in production are chemical resolution method and Mannich reaction method. These methods are complex, the product is difficult to purify, and the production cost is expensive, and the toxic and harmful by-products will be produced. Compared with these methods, enzyme catalytic method has a great advantage of high efficiency, rapidity and safety. 尾 -phenylalanine translocation enzyme has attracted more and more attention as a biocatalyst that can efficiently catalyze the production of 尾 -phenylalanine. In this study, the upstream and downstream primers were designed and synthesized according to the sequence of 尾 -phenylalanine translocation enzyme PAM gene. The target gene was amplified by PCR technique and ligated with the expression vector pET-28a to form the expression plasmid pET-28a-pam.. The expression plasmid was transferred into Escherichia coli expression strain BL21 to screen positive strain, and a recombinant strain was obtained by enzyme digestion to detect whether the recombinant strain was successfully transferred into the target gene band. The optimal inducer content and the best induction time were determined by single factor test of IPTG inducer, and the recombinant bacteria were induced to express. The induced expression of the recombinant strain was detected by SDS-PAGE and the recombinant enzyme was purified. The basic enzymatic properties, pH, temperature and thermal stability of the recombinant enzyme were studied. The optimum catalytic pH of the recombinant enzyme was 9. 0 and the optimum catalytic temperature was 50 鈩，

本文編號(hào)：2203081