J. Biotechnol., 135, 181-189 (2008)
Vidinha, P., Augusto, V., Nunes, J., Lima, J. C., Cabral, J. M. S., Barreiros S.
Cutinase, an esterase from Fusarium solani pisi, was immobilized in sol-gel matrices of composition 1:5 tetramethoxysilane (TMOS):n-alkyltrimethoxysilane (n-alkylTMS). Fluorescence spectroscopy using the single tryptophan (Trp-69) residue of cutinase as a probe revealed that the polarity of the matrices decreased as their hydrophobicity increased up to the TMOS/n-butylTMS pair, which correlates with an increase in cutinase activity. Fluorescence emission was suppressed (a higher than two orders of magnitude reduction) in the TMOS/n-octylTMS matrix, suggesting a greater proximity of the tryptophan to a nearby disulfide bridge. When sol-gel matrices were prepared with added zeolite NaY, the fluorescence emission intensity maximum (lambda(max)) of the tryptophan did not change. And although the presence of the zeolite led to the recovery of fluorescence emission from the TMOS/n-octylTMS matrix, the corresponding lambda(max) fell in line with the values obtained for the matrices with lower n-alkyl chain lengths, indicating that the tryptophan does not sense the zeolite. On the other hand, the presence of the zeolite led to increases in cutinase activity in all the matrices. This suggests that the zeolite is in a position to affect the active site of the enzyme, located at the opposite pole of the enzyme molecule. Scanning electron microscopy and energy dispersive X-ray spectroscopy revealed that the zeolite particles were segregated to the pores of the matrices. Optical microscopy following the staining of the protein with a fluorescent dye showed that the enzyme was distributed throughout the material, and tended to accumulate around zeolite particles. By promoting the accumulation of the enzyme at the pores of the material, the zeolite should improve the accessibility of the enzyme to the substrates and lead to a higher enzymatic activity. Data obtained for sol-gel matrices with epoxy or SH groups provided further evidence that cutinase responded to changes in the chemical nature of the precursors.
Keywords: cutinase; sol-gel; fluorescence; tryptophan; zeolite; immobilization; SEM; EDS; optical microscopy
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