Division of Biochemistry, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
Biotechnol J. 2011 Apr;6(4):463-9. doi: 10.1002/biot.201000357. Epub 2011 Mar 7.
An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.
本文介绍了一种用于合成应用的 His（6）-标签酶的提取/固定化方法。通过修饰氧化硅珠以容纳金属离子，在 Co（II）吸附后，酶被固定在珠上。这些珠子成功地用于从周质制剂中直接提取南极假丝酵母脂肪酶 B（CalB），具有至少 58%的活性产率，创建了一种快速的一步提取-固定化方案。这种方法称为 HisSi 固定化，已用五种不同的酶[南极假丝酵母脂肪酶 B（CalB）、枯草芽孢杆菌脂肪酶 A（BslA）、枯草芽孢杆菌酯酶（BS2）、荧光假单胞菌酯酶（PFE）和马铃薯环氧化物水解酶 1（StEH1）]进行了评估。固定化 CalB 在有机溶剂（环己烷和乙腈）中的转酯化反应和水缓冲液中的酯水解中有效使用。对于其余的酶，在有机溶剂中可以显示出一些活性，而未固定化的酶则被发现无活性。本工作中提出的方案提供了一种简便的固定化方法，利用常见的 His（6）-标签，提供了在特定位置结合蛋白质的特定和定义的手段，适用于广泛的酶。
