异丁香酚单加氧酶活性聚集体的构建及其应用研究

发布时间：2019-04-26 10:00

【摘要】：香草醛素有“食品香料之王”的美誉,广泛在食品、烟草、饮料、酒类、化工、医药和各类化妆用品等行业内使用,是一种性能比较稳定、香味纯正独特、留香持久的优良香料和食品添加剂。通过微生物法转化底物生成的香草醛即是生物香草醛,有着化学合成香草醛无法比拟的优点,符合欧盟和美国等的食品安全标准,反应条件温和,对环境污染程度小。异丁香酚单加氧酶是转化异丁香酚生成香草醛的唯一关键酶,此酶存在底物抑制和产物抑制以及酶纯化困难等问题。为解决此类问题,本论文通过对异丁香酚单加氧酶进行改造,结合具有两亲性自组装短肽和自切割功能特性的Mxe内含肽,用于目标蛋白的表达和制备;通过米曼模式研究其重组蛋白酶动力学特性。采用CLEAs技术探究固定化的异丁香酚单加氧酶的转化能力。本论文的方法和主要结论如下:(1)利用基因工程技术构建重组质粒pET(30a)-IEM-Mxe-ELK16,结合具有两亲性的自组装短肽和具有介导自切割特性的内含肽,诱导表达重组蛋白。经SDS-PAGE分析:融合蛋白的分子量在75 kDa左右,与实验预期相符合,说明重组质粒构建成功。通过分析重组前后酶动力学数据可得:IEM-Mxe-ELK16的Vmax、Km和Kcat分别是原始IEM的11.3、11.7和7.2倍,说明重组质粒pET(30a)-IEM-Mxe-ELK16酶与底物异丁香酚结合效率降低,因此可在一定程度上解除底物抑制,使得最大反应速率增加。条件优化结果为当发酵培养细胞OD600为0.7左右时,加入IPTG至终浓度0.2 mg/L,200rpm,30℃诱导8 h,离心收集细胞,加入9 mL 0.05 mol/L甘氨酸-氢氧化钠缓冲溶液(pH10.5)配制成100 g/L的细胞悬浊液,150 mmol/L异丁香酚,1 m L DMSO,25℃,200rpm转化48 h,所获得香草醛浓度最高可达1.80 g/L。(2)对Mxe内含肽介导的自切割体系进行了初步的优化,其最佳自切割条件为:4℃,切割缓冲液(20 mmol/L Tris-HCl,0.5 mol/L NaCl,1 mmol/L EDTA二钠,pH 8.5)重悬10 OD/m L的大肠杆菌裂解液沉淀,切割24 h。(3)通过CLEAs技术对异丁香酚单加氧酶融合蛋白进行了初步的固定化研究,制备IEM-Mxe-ELK16交联酶的最佳交联时间和交联剂浓度为100 mmol/L戊二醛交联2h;交联酶在50℃-100℃的范围内的具有良好的热稳定性,反复利用7次之后相对酶活保留50%以上;交联酶的最佳转化条件为:0.025 mol/L碳酸钠-氢氧化钠缓冲溶液(pH10.5),10%(v/v)DMSO,100 mmol/L异丁香酚,30℃,200 rpm,转化60 h,所得香草醛浓度为0.96 g/L。
[Abstract]:Vanillin is widely used in food, tobacco, beverage, wine, chemical industry, medicine and various kinds of cosmetics and other industries because of its good reputation as "king of food and spices". It is a kind of stable performance, pure and unique fragrance, and has been widely used in the fields of food, tobacco, beverage, wine, chemical industry, medicine and all kinds of cosmetics. A long-lasting bouquet of spices and food additives. Vanillin produced by microbial transformation of substrate is biovanillin, which has the advantage of chemical synthesis of vanillin, which accords with the food safety standards of European Union and USA. The reaction conditions are mild and the pollution to the environment is small. Isoeugenol monooxygenase is the only key enzyme to convert isoeugenol to vanillin. This enzyme has many problems such as substrate inhibition and product inhibition as well as difficulty in enzyme purification. In order to solve this problem, the isoeugenol monooxygenase was modified to combine the amphiphilic self-assembled short peptide and the self-cleavage function of Mxe to express and prepare the target protein. The kinetic characteristics of recombinant protease were studied by Miman model. The conversion ability of immobilized isoeugenol monooxygenase was studied by CLEAs technique. The methods and main conclusions of this paper are as follows: (1) the recombinant plasmid pET (30a)-IEM-Mxe-ELK16, was constructed by using genetic engineering technology to bind the amphiphilic self-assembled short peptide and the endopeptide which mediates the self-cleavage. The recombinant protein was induced to express. SDS-PAGE analysis showed that the molecular weight of the fusion protein was about 75 kDa, which was consistent with the expectation of the experiment, indicating that the recombinant plasmid was successfully constructed. The enzyme kinetic data before and after recombination showed that the Vmax,Km and Kcat of IEM-Mxe-ELK16 were 11.3, 11.7 and 7.2 times higher than those of the original IEM, respectively, indicating that the binding efficiency of recombinant plasmid pET _ (30a)-IEM-Mxe-ELK16 to isoeugenol was lower than that of the original plasmid pET _ (30a)-IEM-Mxe-ELK16. Therefore, the substrate inhibition can be relieved to a certain extent, and the maximum reaction rate can be increased. The optimal conditions were as follows: when the OD600 of the cultured cells was about 0.7, the cells were induced at 0.2 mg/L,200rpm,30 鈩，

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