The interactions between biomolecules and solid surfaces are complex phenomena. Understanding the nature of these interactions can allow engineering highly efficient systems for bioseparation and biocatalysis. However, there is still a lack of understanding of these fundamental interactions due to the complexity and fragility of biomolecules, especially proteins. The overall goal in this research is to improve the current understanding of these interactions as functions of the properties of mobile phases and stationary phases by investigating adsorption isotherms, adsorption thermodynamics, and biocatalytic activity of immobilized proteins.
Mesoporous silica and alumina were used as stationary phases (adsorbents). In particular, mesostructured cellular foam (MCF) silica, which has an open 3-dimensional pore structure with superior physical properties, was used to immobilize biomolecules. The surface chemistry of the synthesized MCF silica was engineered to control the immobilization of biomolecules by grafting functional groups, including charge-terminated (amine-terminated, mecapto-terminated) and hydrophobic-terminated groups (methyl-terminated) groups, to the surface.
The interactions between biomolecules and prepared adsorbents were also investigated using flow microcalorimetry (FMC) to reveal the adsorption mechanisms during the immobilization of biomolecules at different levels of pH and ionic strength and several functionalized solid surfaces. Adsorption thermodynamics and mechanism can be modulated by changing ionic strength by adding a neutral salt (sodium sulfate) and by changing the pH. Biomolecule adsorption is a complex phenomenon, exhibiting multiple heat events. However, similar thermograms were observed for the interactions between protein and MCF silicas in most cases. Also, the driving force for protein adsorption was investigated by using semi-empirical analysis.
Adsorption energetics were affected significantly by surface modification. Also, the FMC data along with batch adsorption isotherm revealed that aminopropyl-grafted MCF silica has the property of ion-exchanger. The energetics of different biomolecules (tryptophan, lysozyme, and bovine serum albumin) was investigated in different pH. The number of distinct exothermic peaks corresponded to the number of binding sites of biomolecules. And the magnitude of net heat of adsorption was increased according to increasing molecular weight at pH 5.2. However, the energetics of bovine serum albumin was significantly changed from pH 5.2 to pH4. It is attributed to the conformational change of protein as the function of pH.
The biocatalytic activity of immobilized enzyme (lipase from Pseudomonas fluorescens) was investigated on different functionalized MCF silicas to understand the effect of surface modification. The amine-terminated long functional group showed the most positive effect to increase the catalytic efficiency (kcat/Km) of immobilized enzyme due to the orientation change and the mixed interactions (charged interactions and hydrophobic) interactions. The biocatalytic activity of immobilized enzyme (lipase from Pseudomonas cepacia) was further extended by using functionalized aluminas as enzyme supporting materials. The measurement of the biocatalytic activity of immobilized enzymes showed that the effect of surface modification has important roles for the activation of immobilized enzyme activity and the optimal enzyme supporting material need to be selected.
This study provides insights into the natures and mechanisms of biomolecule immobilization on mesoporous silica to design highly efficient systems for biomolecule immobilization for bioseparation and immobilized enzyme technology for biocatalysis.