Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli
Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli
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DOI：
10.1186/1472-6750-10-33
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发表时间：
2010-04-21
影响因子：
3.5
通讯作者：
Piubelli, Luciano
中科院分区：
文献类型：
--
作者：
Volonte, Federica;Pollegioni, Loredano;Piubelli, Luciano
背景：胆固醇氧化酶是一种醇脱氢酶/氧化酶黄素蛋白，催化胆固醇的C（3）-OH脱氢。它有两个主要的生物技术应用，即测定血清（和食物）胆固醇水平和作为生物催化剂，为工业类固醇药物生产提供有价值的中间体。I型胆固醇氧化酶是那些含有FAD辅因子紧密但不共价结合蛋白质部分的胆固醇氧化酶，而II型成员含有共价结合的FAD。结果：设计了成熟胆固醇氧化酶基因的表达载体，优化了重组大肠杆菌的培养基组成，确定了重组大肠杆菌中胆固醇氧化酶活性蛋白表达的最佳培养条件和诱导条件。大肠杆菌细胞，同时实现了有价值的改进：BCO体积生产率从接近500增加到接近25000 U/L，其粗提物比活性从0.5增加到7.0 U/mg蛋白。有趣的是，在最佳表达条件下，近55%的可溶性重组BCO产生的共价FAD结合的形式，而含有非共价结合的FAD的蛋白质优先积累在不溶性包涵体。大肠杆菌或链霉菌属），表明在E.在大肠杆菌中表达。FAD结合的胆固醇氧化酶的改进的过量生产将支持其作为一种新的生物工具在生物技术应用中开发的发展。
Background: Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the overexpression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO).Results: Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant E. coli cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from similar to 500 up to similar to 25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies.Conclusions: Comparison of our results with those published on non-covalent (type I) COs expressed in recombinant form (either in E. coli or Streptomyces spp.), shows that the fully active type II BCO can be produced in E. coli at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.