β-苯丙氨酸变位酶基因工程菌的构建及其产酶条件优化
发布时间：2018-08-25 13:57
【摘要】：β-苯丙氨酸变位酶属于MIO-依赖氨基变位酶(MIO-dependent aminomutase),可催化α-苯丙氨酸转化为β-苯丙氨酸,是目前最有效的抗癌药物—紫杉醇的重要前体物质。随着癌症在全球范围内发病率的逐年提升,β-苯丙氨酸需求量也日益增加。目前,在生产中大量获取β-苯丙氨酸的方法主要是化学拆分法和Mannich反应法等,这些方法步骤复杂,产物难以纯化,生产成本昂贵还会产生有毒有害的副产物。与这些方法相比,酶催化法有着高效、快捷、安全的巨大优势。β-苯丙氨酸变位酶作为可以高效催化生成β-苯丙氨酸的生物催化剂受到了越来越多的关注。本研究根据β-苯丙氨酸变位酶PAM基因序列设计合成上下游引物,利用PCR技术扩增了目的基因,并将其和表达载体pET-28a连接,构成了表达质粒pET-28a-pam。将表达质粒转入大肠杆菌表达菌株BL21中,筛选阳性菌株,并通过酶切检测在菌株内是否成功转入目的基因条带,得到一株基因重组菌。通过对IPTG诱导剂的单因素试验确定了最佳诱导剂含量以及最佳诱导时间,并对重组菌进行诱导表达。以SDS-PAGE手段检测了重组菌的诱导表达并对重组酶进行了纯化。对重组酶的基本酶学性质pH、温度以及热稳定性进行了研究,确定了重组酶的最佳催化pH为9.0,最佳催化温度为50℃,并对重组酶的热稳定性进行了验证,发现其热稳定性较好。利用单因素试验和响应面法对重组菌的产酶条件进行了优化,确定了最优发酵条件为种龄:12.8 h,初始pH:6.7,发酵时间:25.2 h。在最优发酵条件下,重组菌产PAM最大酶活可达11.15 U/mL,较优化前提高了 128.9%。在3 L发酵罐中对重组菌进行扩大培养。利用单因素试验确定了最佳溶氧为30%。分别比较了未添加任何补料的发酵方式,培养20 h时流加30 mL补料培养基的发酵方式以及培养30 h时流加40 mL补料培养基的发酵方式。确定了补料发酵方式优于未进行补料的发酵方式,并且对比得到在20 h时进行流加补料的发酵方式优于在30 h时进行补料发酵的方式。以此方式进行扩大培养发酵所得最高酶活为16.63 U/mL,与摇瓶发酵相比提高了 49.1%,较优化前的菌株产酶活性共提高了 241.5%。
[Abstract]:尾 -phenylalanine translocation enzyme (尾 -phenylalanine) belongs to MIO- dependent aminotranslocation enzyme (MIO-dependent aminomutase), which can catalyze the conversion of 伪 -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，
本文编号：2203081
[Abstract]:尾 -phenylalanine translocation enzyme (尾 -phenylalanine) belongs to MIO- dependent aminotranslocation enzyme (MIO-dependent aminomutase), which can catalyze the conversion of 伪 -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，
本文编号：2203081
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