A comprehensive study of protein–mesoporous–macroporous silica interactions by extended canonical variate analysis of Raman spectra

Abstract

Understanding the protein–support interactions is of major importance when manufacturing bionanomaterials to a certain application. These interactions can be the cause for enhanced properties or denaturation phenomena in the target protein. Raman spectroscopy was applied to a bionanomaterial comprehending the protein β‐galactosidase immobilized by physical adsorption into a mesoporous–macroporous silica material, with a nanoporous network consisting of 9‐nm mesopores and 200‐nm macropores. Raman spectra of the bionanomaterial evidenced a complex amount of differences related to the Raman shifts, intensities, band enlargement, appearance of new bands, and overlapping, in comparison with the silica support and the protein spectra. To help in the analysis of the Raman spectra and in the inspection of possible protein–support interactions, ECVA (extended canonical variate analysis) was used as a chemometric complementary tool, dividing the spectra into four segments: 1 (3,100 to 2,800 cm−1), 2 (1,800 to 1,500 cm−1), 3 (1,500 to 1,200 cm−1), and 4 (1,200 to 900 cm−1). Major alterations in the Amide I band (1,800 to 1,500 cm−1) and the amino acid band regions demonstrated possible structure alterations to a non‐native form of the protein β‐galactosidase. Also, other minor alterations were observed in other spectral regions (3,100 to 2,800 cm−1,1,500 to 1,200 cm−1, and 1,200 to 900 cm−1) also representative of protein structure alteration due to protein–support interactions.

Immobilization of Kluyveromyces lactis β‐Galactosidase on Meso‐macroporous Silica: Use of Infrared Spectroscopy to Rationalize the Catalytic Efficiency

“…This preliminary analysis gave us some evidence that a different conformational state may be present in the MMS_β‐gal samples in comparison to the L‐β‐gal‐KL due to the shifts observed in the amides I and II. These claims are also supported by our previous finds by using Raman spectroscopy where disruption in the amide I band regions were encountered [12] . The next step was to further explore and perform a secondary structure analysis for which the amide I was chosen, as it is more sensitive to secondary structure conformation [18] .…”

“…The IR data confirmed that the MMS carrier is rich on hydrophilic silanol groups (−Si−OH) and hydrophobic siloxane groups (−Si−O−Si−). Also, the presence of C−H groups is verified both by the IR and the Raman data, [12] being related to the presence of impurities from the surfactant (Pluronic P123) used in the MMS synthesis. [11] Moreover, since the immobilization was performed at the pH of 6.5, the MMS having an isoelectric point between 2.5 to 3.0 makes the surface negatively charged.…”

“…[ 16 , 19 ] The appearance of β‐sheet elements in the immobilized samples was also verified in the same system using Raman spectroscopy and associated to protein aggregation. [12] The curve fitting of the amide I band also provides the peak areas for each assigned band (according to Table S1) which were used for the calculation of the overall percentage of the secondary structure as represented in Table 2 .…”

“…These claims are also supported by our previous finds by using Raman spectroscopy where disruption in the amide I band regions were encountered. [12] The next step was to further explore and perform a secondary structure analysis for which the amide I was chosen, as it is more sensitive to secondary structure conformation. [18] In Figure 3 is depicted the curve fittings of the amide I region for each sample composition using the Lorentzian function.…”

“…To answer this, the enzyme‐MMS interactions, and the changes in the enzyme conformation upon immobilization were studied by infrared spectroscopy, and later correlated to the bioactivity performance. In an earlier study, [12] we analysed the same system by Raman spectroscopy and checked alterations in both the amino acid side chains and amide I band vibrations, concluding that a non‐native state is present at the MMS_β‐gal final product. In this study, an additional objective is to complement this information with the infrared analysis.…”