Microscopic and thermal characterization studies of heterogeneous biocatalysis: polyurethane film immobilized enzymes and degradation of algal cells.

Abstract

Heterogeneous biocatalysis, associated with either solid-state biocatalyst systems or solid-state substrate systems, has garnered a lot of interest for chemical synthesis, bioseparation, and biosensing. In the pursuit of high efficiency, the characterization of the physical structure of solid-state biocatalysts and solid-state substrates is necessary to provide detailed information and deepen the understanding for the heterogeneous biocatalytic systems. In this study, microscopic and thermal analytical methods were utilized to characterize the physical structure of solid-state biocatalysts and solid-state substrates. Particularly, we chose the versatile polymer polyurethane (PU) to immobilize enzymes as model solid-state biocatalysts. Two important physical properties, enzyme distribution and glass transition temperature (Tg), were analyzed. It was found that several preparation parameters, including hydrophobic/hydrophilic properties of resin and cross-linker for PU film, weight ratio of starting materials, and mixing speed, showed significant influences on enzyme distribution and Tg. Using microscopic and thermal characterization methods, we further studied the integrity of enzyme-containing PU film against water. The enzyme was found prone to leak from the PU film, indicating incompleteness of cross-linking with PU. Several approaches (e.g., increasing the reaction time and using a hydrophilic cross-linker) were explored in this work to promote better cross-linking between the enzyme and PU matrix. The hydrophilic cross-linker (Bayhydur 302) demonstrated 70% improvement in retaining the enzyme against 24 hours of washing compared to a hydrophobic cross-linker (DESMOPHEN N3600). Moreover, algae cells were studied as model solid-state substrates. We report a novel enzymatic method to disrupt the cell wall and release lipids from the algae cells. Microscopic analysis indicated that the cell wall of Chlamydomonas reinhardtii (C.R.) algae was disrupted after being treated by a three-step method, which included one hour of incubation in 4 M of lithium chloride, eight hours of hydrolytic reaction in enzyme solution, and one cycle of freeze/thaw process. Protease Subtilisin Carlsberg was found effective in catalyzing the degradation of C.R. cell wall due to its unspecific activity toward peptide bonds.