Kamiya Noriho, Doi Satoshi, Tanaka Yusuke, Ichinose Hirofumi, Goto Masahiro
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan; Center for Future Chemistry, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.
J Biosci Bioeng. 2007 Sep;104(3):195-9. doi: 10.1263/jbb.104.195.
Covalent and site-specific protein immobilization catalyzed by microbial transglutaminase (MTG) was investigated using recombinant Escherichia coli alkaline phosphatase (AP) tagged with a glutamyl donor substrate peptide (MLAQGS) of MTG. A polystyrene surface physically coated with beta-casein or bovine serum albumin (BSA) was employed as an MTG-specific surface displaying reactive lysine residues. MTG-mediated protein immobilization through catalytic epsilon-(gamma-glutamyl)lysine bond formation between the peptide tag of recombinant APs and beta-casein- or BSA-coated surface was verified by the detection of AP activity on the surface. It was found that the length and the insertion position of the peptide tag did not significantly affect the efficacy of enzymatic immobilization of the recombinant APs. On the other hand, pH and ionic strength in the reaction media had crucial effects on the immobilization yields. Interestingly, the optimum pH range of MTG-mediated protein immobilization differed markedly from that for an MTG-catalyzed reaction in aqueous solution. The results suggest that the concentration of reactive species due to electrostatic interaction between the enzyme-substrate intermediate and the protein-adsorbed surface is a key factor governing MTG catalysis at a solid surface.
利用标记有微生物转谷氨酰胺酶（MTG）的谷氨酰供体底物肽（MLAQGS）的重组大肠杆菌碱性磷酸酶（AP），研究了MTG催化的共价和位点特异性蛋白质固定化。使用物理包被有β-酪蛋白或牛血清白蛋白（BSA）的聚苯乙烯表面作为展示反应性赖氨酸残基的MTG特异性表面。通过检测表面上的AP活性，验证了MTG通过重组AP的肽标签与β-酪蛋白或BSA包被表面之间催化ε-（γ-谷氨酰）赖氨酸键形成介导的蛋白质固定化。发现肽标签的长度和插入位置对重组AP的酶促固定化效率没有显著影响。另一方面，反应介质中的pH和离子强度对固定化产率有至关重要的影响。有趣的是，MTG介导的蛋白质固定化的最佳pH范围与MTG在水溶液中催化反应的最佳pH范围明显不同。结果表明，酶-底物中间体与蛋白质吸附表面之间静电相互作用产生的反应性物种浓度是控制MTG在固体表面催化的关键因素。
