Tanaka Yusuke, Tsuruda Yukito, Nishi Motohiro, Kamiya Noriho, Goto Masahiro
Department of Applied Chemistry, Graduate School of Engineering and Center for Future Chemistry, Kyushu University, 744 Motooka, Fukuoka, 819-0395, Japan.
Org Biomol Chem. 2007 Jun 7;5(11):1764-70. doi: 10.1039/b701595j. Epub 2007 May 9.
The factors affecting enzymatic protein immobilization with microbial transglutaminase (MTG) were explored. As model proteins, enhanced green fluorescent protein (EGFP) and glutathione S-transferase (GST) were chosen and tagged with a neutral Gln-donor substrate peptide for MTG (Leu-Leu-Gln-Gly, LLQG-tag) at their C-terminus. To create a specific surface, displaying reactive Lys residues, to be cross-linked with the Gln residue in the LLQG-tag of target proteins by MTG catalysis, a polystyrene surface was physically coated with beta-casein. Both recombinant proteins were immobilized onto the beta-casein-coated surface only in the presence of active MTG, indicating that those proteins were enzymatically immobilized to the surface. MTG-mediated protein immobilization markedly depends on the pH and ionic strength of the reaction media. The optimal pH range of MTG-mediated immobilization of both recombinant proteins was around 5, at which point the MTG-catalyzed reaction in aqueous solution is not normally preferred. By utilizing a pH-dependent change in EGFP fluorescence, we found that the apparent pH at the surface is likely to be lower than bulk pH, this difference is not attributed to an optimal pH shift in MTG-mediated immobilization. On the other hand, lower yields of protein immobilization at higher ionic strength suggest that electrostatic interaction is a key factor governing MTG catalysis at a solid surface. The results of this study indicate that, in enzymatic catalysis at a solid surface, the concentration of substrates at the surface can enhance the catalytic efficiency, and this could alter the pH dependence of enzymatic catalysis.
研究了影响微生物转谷氨酰胺酶（MTG）固定化酶蛋白的因素。作为模型蛋白，选择了增强型绿色荧光蛋白（EGFP）和谷胱甘肽S-转移酶（GST），并在其C末端用MTG的中性Gln供体底物肽（Leu-Leu-Gln-Gly，LLQG标签）进行标记。为了创建一个展示反应性Lys残基的特定表面，以便通过MTG催化与目标蛋白的LLQG标签中的Gln残基交联，在聚苯乙烯表面物理包被β-酪蛋白。仅在活性MTG存在的情况下，两种重组蛋白才被固定在β-酪蛋白包被的表面上，这表明这些蛋白是通过酶法固定在表面上的。MTG介导的蛋白固定化明显取决于反应介质的pH值和离子强度。两种重组蛋白的MTG介导固定化的最佳pH范围约为5，而在该pH值下，MTG在水溶液中的催化反应通常不受青睐。通过利用EGFP荧光的pH依赖性变化，我们发现表面的表观pH可能低于本体pH，这种差异并非归因于MTG介导固定化中的最佳pH偏移。另一方面，在较高离子强度下蛋白固定化产率较低，这表明静电相互作用是控制MTG在固体表面催化的关键因素。本研究结果表明，在固体表面的酶催化中，表面底物浓度可以提高催化效率，并且这可能会改变酶催化的pH依赖性。
