A Polymer Platform Composed of Structurally Well-defined Stereocomplex Structures for Efficient Enzymatic Reactions

Abstract

Previously we reported that p-galactosidase immobilized on the structurally well-defined poly(methyl methacrylate) (PMMA) stereocomplex films fabricated by layer-by-layer (LbL) assembly of isotactic (it) and syndiotactic (st) PMMAs on solid substrates were highly active when compared to the enzyme immobilized on the single-component films, and that a slight difference of polymer surface structures strongly affects activities of immobilized enzymes, even though polymers have the same chemical component (Chem. Mater., 19, 2174-2179 (2007)). In this paper, we demonstrated activities of other enzymes immobilized on the PMMA films. Activities of immobilized alkaline phosphatase (ALP) were measured by following hydrolysis rate of fluorescence substrate 4-methylumbelliferyl phosphate (4-MUP). Initial velocity of ALP hydrolysis reaction immobilized on the complex film was approximately 2-fold faster than those on the single-component it-PMMA and atactic (at) PMMA films. Michaelis-Menten analyses revealed that catalytic efficiency (kcat/Km) of ALP was strongly dependent on conformational regulation of PMMA chains at film surfaces. Quartz crystal microbalance (QCM) analyses revealed that immobilization amount of ALP on the complex was greater than those on the single-component films. Similar dependencies were also observed in the case of cellulases that degrade polymeric cellulose under acidic condition. Secondary structural analyses using an attenuated total reflection infrared (ATR-IR) spectroscopy revealed that structural denaturation of the enzyme after immobilization processes was well-suppressed on the complex film, although the enzymes on the single-component PMMA films were relatively denatured. We propose here that PMMA stereocomplex films are a generally convenient platform for immobilization of functional proteins and could be used under various conditions without loss of their activities.