Alpha chymotrypsin coated clusters of Fe(3)O(4) nanoparticles for biocatalysis in low water media

BACKGROUND: Enzymes in low water containing non aqueous media are useful for organic synthesis. For example, hydrolases in such media can be used for synthetic purposes. Initial work in this area was carried out with lyophilized powders of enzymes. These were found to have poor activity. Drying (removing bulk water) by precipitation turned out to be a better approach. As enzymes in such media are heterogeneous catalysts, spreading these precipitates over a large surface gave even better results. In this context, nanoparticles with their better surface to volume ratio provide obvious advantage. Magnetic nanoparticles have an added advantage of easy separation after the reaction. Keeping this in view, alpha chymotrypsin solution in water was precipitated over a stirred population of Fe(3)O(4) nanoparticles in n-propanol. This led to alpha chymotrypsin activity coated over clusters of Fe(3)O(4) nanoparticles. These preparations were found to have quite high transesterification activity in low water containing n-octane. RESULTS: Precipitation of alpha chymotrypsin over a stirred suspension of Fe(3)O(4) nanoparticles (3.6 nm diameter) led to the formation of enzyme coated clusters of nanoparticles (ECCNs). These clusters were also magnetic and their hydrodynamic diameter ranged from 1.2- 2.6 microns (as measured by dynamic light scattering). Transmission electron microscopy (TEM), showed that these clusters had highly irregular shapes. Transesterification assay of various clusters in anhydrous n-octane led to optimization of concentration of nanoparticles in suspension during precipitation. Optimized design of enzyme coated magnetic clusters of nanoparticles (ECCN 3) showed the highest initial rate of 465 nmol min(-1) mg(-1)protein which was about 9 times higher as compared to the simple precipitates with an initial rate of 52 nmol min(-1) mg(-1) protein. Circular Dichroism (CD)(with a spinning cell accessory) showed that secondary structure content of the alpha Chymotrypsin in ECCN 3 [15% α-helix, 37% β-sheet and 48% random coil] was identical to the simple precipitates of alpha chymotrypsin. CONCLUSION: A strategy for obtaining a high activity preparation of alpha chymotrypsin for application in low water media is described. Such high activity biocatalysts are useful in organic synthesis.