Imaging the distribution and secondary structure of immobilized enzymes using infrared microspectroscopy.
Synchrotron infrared microspectroscopy (SIRMS) was used for the first time to image the distribution and secondary structure of an enzyme (lipase B from Candida antarctica, CALB) immobilized within a macroporous polymer matrix (poly(methyl methacrylate)) at 10 microm resolution. The beads of this catalyst (Novozyme435) were cut into thin sections (12 microm). SIRMS imaging of these thin sections revealed that the enzyme is localized in an external shell of the bead with a thickness of 80-100 microm. Also, the enzyme was unevenly distributed throughout this shell. Furthermore, by SIRMS-generated spectra, it was found that CALB secondary structure was not altered by immobilization. Unlike CALB, polystyrene molecules of similar molecular weight diffuse easily throughout Novozyme435 beads. Scanning electron micrograph (SEM) images of the Novozyme435 beads showed that the average pore size is 10 times larger than CALB or polystyrene molecules, implying that there is no physical barrier to enzyme or substrate diffusion throughout the bead. Thus, the difference between polystyrene and enzyme diffusivity suggests that protein-matrix and protein-protein interactions govern the distribution of the enzyme within the macroporous resin.
Mei
Ying
Y
NSF I/UCRC for Biocatalysis and Bioprocessing of Macromolecules, Polytechnic University, Six MetroTech Center, Brooklyn, New York 11201, USA.
Miller
Lisa
L
Gao
Wei
W
Gross
Richard A
RA
D002175:Candida / Q000201:enzymology*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000648:ultrastructure*; D005656:Fungal Proteins; D007091:Image Processing, Computer-Assisted; D008049:Lipase / Q000737:chemistry* / Q000648:ultrastructure*; D008855:Microscopy, Electron, Scanning; D019904:Polymethyl Methacrylate; D017433:Protein Structure, Secondary; D012680:Sensitivity and Specificity; D013055:Spectrophotometry, Infrared
D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D019904:Polymethyl Methacrylate; C093667:Novozyme 435; D008049:Lipase