New biocatalytic systems with enhanced enzymatic performances have been rationally designed based on the coordination and self-assembly of beta-galactosidase with its Mg2+ allosteric effector in the presence of organic linkers. Optical microscopy and scanning electron microscopy confirm the formation of spherical metal-enzyme hybrid microspheres with the particle diameter in a range of 5-35 mu m. The immobilized beta-galactosidase exhibits substantially enhanced catalytic activity and efficiency compared with free enzyme in solution because of the Mg2+-induced allosteric effect that assists protein folding. In particular, the Mg/beta-galactosidase hybrid microsphere prepared using 1,3,5-benzenetricarboxylic acid as the organic linker reveals the highest content of Mg(2+ )in comparison to other organic linkers, and as a result displays the best catalytic performances with an enzymatic activity of 43.8 U/mg and a k(cat)/K-m value of 95.1 s(-1) M-1. These values are 4.0- and 2.6-fold higher than free beta-galactosidase. Moreover, the incorporation of organic linkers is also favorable to maintain the thermal and chemical stability of enzyme microspheres due to the confinement effect. This suggests that the allosteric and confinement effects play essential roles in the enhancement of allosteric enzyme performances.