Christian Thörn

Enzymes Immobilized in Mesoporous Silica: a Physical-Chemical Perspective

Mesoporous materials as support for immobilized enzymes have been explored extensively during the last two decades, primarily not only for biocatalysis applications, but also for biosensing, biofuels and enzyme-controlled drug delivery. The activity of the immobilized enzymes inside the pores is often different compared to that of the free enzymes, and an important challenge is to understand how the immobilization affects the enzymes in order to design immobilization conditions that lead to optimal enzyme activity. This review summarizes methods that can be used to understand how material properties can be linked to changes in enzyme activity. Real-time monitoring of the immobilization process and techniques that demonstrate that the enzymes are located inside the pores is discussed by contrasting them to the common practice of indirectly measuring the depletion of the protein concentration or enzyme activity in the surrounding bulk phase. We propose that pore filling (pore volume fraction occupied by proteins) is the best standard for comparing the amount of immobilized enzymes at the molecular level, and present equations to calculate pore filling from the more commonly reported immobilized mass. Methods to detect changes in enzyme structure upon immobilization and to study the microenvironment inside the pores are discussed in detail. Combining the knowledge generated from these methodologies should aid in rationally designing biocatalyst based on enzymes immobilized in mesoporous materials.

A comparison of lipase and trypsin encapsulated in mesoporous materials with varying pore sizes and pH conditions

Immobilized enzymes have an advantage over enzymes free in solution in that they are easily recovered after completed reaction. In addition, immobilization often gives enhanced stability. Entrapment of an enzyme in the pores of a mesoporous material is an attractive procedure since the enzyme is immobilized without any covalent bonding to a support which may be detrimental to the catalytic performance. The objective of this work is to compare the encapsulation and catalytic performance of lipase from Mucor miehei and trypsin from bovine pancreas, two hydrolases with rather dissimilar properties and structures. We also demonstrate the importance of the pore dimensions and the pH for proper function of the encapsulated enzyme. Mesoporous silica particles (SBA-15) with three different pore sizes (50 Å, 60 Å and 89 Å) were synthesized and hexagonal structures with narrow pore size distributions were confirmed with TEM, SAXS and N(2)-adsorption. Lipase and trypsin were encapsulated separately in the silica particles and the results indicate distinct differences between the two enzymes, both in loading capacity and catalytic activity. For trypsin the encapsulation rate and the loading capacity were large with a maximum reached at pH 7.6. The largest product yield was obtained with the particles with 60 Å pores, however, the yield was significantly lower than with free trypsin. For lipase optimal encapsulation rate and loading capacity were reached with the particles with 89 Å pores at pH 6.0 but were low compared to trypsin. However, the catalytic activity of the encapsulated lipase was more than twice as large as for free lipase, which can be explained by an interfacial activation of lipase at the silica surface.