The above analysis is also confirmed by the

The above analysis is also confirmed by the behavior of the amide A band around 3300cm−1, illustrated in Fig. 3 for the three samples under study, compared with that of the unprocessed lyophilized lipase (pressed into a KBr pellet). This band is due to the stretching of the N–H bond. It is exclusively localized on the NH group and is therefore insensitive to the conformation of the polypeptide backbone. However, the frequency of the amide A vibration depends on the strength of the hydrogen bond [34]. In particular, N–H bonds in α-helices and β-sheets absorb around 3300cm−1, while in random-coil structure the p2x7 antagonist band shifts towards 3400cm−1 due to the rupture of the H-bonds [41]. Going from ML1 to ML3, a component at 3440cm−1 representing H-unbound random coil structures progressively loses intensity, while that at 3300cm−1 increases, substantiating the decreasing degree of unfolding in ML2 and ML3. This trend outlined the efficacy of m-DOPA in protecting lipase during the MAPLE process. One reason for this is by acting as a radiation absorber, lowering the laser energy required for the deposition. Furthermore, thanks to the two hydroxyl groups of the chatecol side chain, m-DOPA provided hydrogen bonds with the polypeptide that can protect lipase from denaturing [42], helping to preserve its native form, both during the freezing phase of the MAPLE process and during the dehydration phase occurring in the target-to-substrate journey. In fact it was found that the addition of hydrogen bonding donor amino acids exerted a protective effect on protein secondary structure upon freeze-drying in a concentration-dependent manner [31]. The stabilization was supposed to proceed via the water replacement hypothesis, a mechanism which involves the formation of hydrogen bonds between a protein and an excipient at the end of the drying process that satisfy hydrogen bonding requirements of polar groups on the surface of the polypeptide. The excipient concentration was higher than the one utilized in this work (15–71% w/w vs. 10% w/w) and the efficacy of the addition increased with concentration. This opens the possibility of increasing m-DOPA concentration in order to obtain a better protective effect on the secondary structure of lipase. Preliminary catalytic essays were carried out using as biocatalyst the sample M-L2 in the hydrolysis of soybean oil. Reaction products were qualitatively characterized by reverse RP-TLC comparing the results to those obtained using as biocatalyst 15mg of CRL lyophilized powder. The chromatograms are shown in Fig. 5. For comparison, the chromatogram of untreated soybean oil is also reported. Reverse phase separation enabled the resolution of the reacted mixture into triglycerides (TGs), diglycerides (DGs), monoglycerides (MGs) and free fatty acids (FFA). The order of retention on the chromatographic layer of the sample components follows the adsorption/partition type mechanisms. The MGs, being the more polar, exhibit the least retention and hence migrate up towards the solvent front followed by the FFA, DGs and finally TGs [43]. The chromatogram of Fig. 4(a) and (b) showed the residual oil spots below that of the hydrolysis products that, being more polar, were less retained by apolar stationary phase. Furthermore, in the chromatogram of Fig. 4(b), there is a halo that traveled farther the FFA and that is due to MGs reaction intermediates. The chromatogram of the oil in Fig. 4(c), on the other hand, was much more elongated in the TG region and did not show a neat spot representing the fatty acids. The oil chromatogram appears elongated due to the variation in saturation and chain length of the component fatty acids [43]. Instead the stain left behind the TGs spot is due to some apolar compound present in the soybean oil and its appearance does not depend on the reaction, since it is always present both in the oil and in the reacted mixtures. By observing the different areas and intensity of the burnt spot representing the fatty acids, and considering the presence of MGs reaction intermediates in the chromatogram of the reaction catalyzed by M-L3, it is evident that the reaction catalyzed by the MAPLE sample occurred to a smaller degree. Nevertheless it did occur, and the smaller quantity of fatty acids obtained could depend on the smaller quantity of catalyst used (about 1mg after MAPLE deposition).