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colloidal Coomassie blue. As shown in figure 2.5, only the supernatant from cultures on Spinello’s glue gave a ladder of caseinolytic activity bands, extending from ca. 20 kDa up to 250 kDa. Also a commercial preparation of collagenase purified from C. hystoliticum (tracks 7 and 8) was analysed by this technique along with the supernatant samples, producing an intense band at 120 kDa, that apparently coincides with the strongest activity bands in the supernatant from Spinello’s glue (track 2).

250 - 150 - 100 -

75 -

50

-37.5 - MW (kDa)

25 -

1 2 3 4 5 6 7 8 Figure 2.5. Casein zymograms

via SDS-PAGE. The first three samples are supernatants (300 X concentrated) of P. stutzeri grown on glucose (track 1), on Spinello’s glue (2) and on fresh animal glue (3). Tracks 4-6 are positive controls with purified trypsin (10, 5 and 1 µg, respectively, in tracks 4 to 6).

Tracks 7 and 8 are purified collagenase from C.

hystoliticum (20 µg in 7, 10 µg in 8).

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work detached from the walls of Pisa’s Monumental Cemetery could not be removed any longer from the face of the fresco, due to hardening and cross-linking of the animal glue during the 20 years of storage. No single enzyme, or concoctions of the most powerful proteases available on the market, could act on the layer of glue, and the fresco seemed to be irremediably lost. It is remarkable that P. stutzeri strain A29 could extensively digest this intricate mass of untreatable proteinaceous glue. For the in situ bioremediation process, the fresco had been entirely covered with a thin layer of hydrophilic cotton wool impregnated with the bacterial cell suspension at a high cell density (8.5 log CFU/mL). Contact times of 12-14 hours at a temperature of 30ºC had ensured almost full digestion (100% in the thinner areas, 80-90% in the thicker, up to 3 mm, layers), to the point that the gauze coating the fresco could be easily removed

With the present laboratory study, we wanted to investigate more in depth the molecular mechanism responsible for such a good performance. In this concern, we had to select optimal growth times for both control cells and cells grown on fragments of the original formaldehyde cross-linked glue, so as to ensure that all of the samples would be harvested, for analysis, not later than the late exponential growth phase, so as to avoid interferences in the following analysis due to potential cell lysis after reaching a growth plateau. In fact we started from the reasonable assumption that the bacterium, when attempting to digest such a recalcitrant protein substrate, had to secrete in the surrounding environment one or more proteolytic enzymes, whose synergic action would have been necessary for an effective degradation of the glue. Growth curves revealed that, although the onset of growth occurs at earlier times in the case of glucose, in presence of the aged-glue as the sole carbon source, after a short lag time, the growth rate approaches the one observed in the case of glucose, and a confluence plateau is reached by both cultures after about 10 hours. The fact that, after a short lag time, probably necessary for protease production and secretion, the growths proceed at similar rates with both glucose and the cross-linked glue, demonstrates the great ability of this strain to degrade the otherwise untreatable protein material, thus confirming its suitability for difficult biorestoration interventions. The time required for reaching the growth plateau (10 hours) is in full agreement with the incubation

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period necessary to achieve the gauze detachment during the in situ intervention (12-14 hours), thus this incubation time was selected for all our experiments.

In order to make sure that no contamination between secreted and cytoplasmic proteins occurred during cell culture preparation, LDH activity assay was carried out. In fact LDH is known to be a cell sap enzyme that should not be found in the culture supernatant. This assay definitely proved that no cell lysis took place during sample preparation, thus confirming the extracellular nature of the enzymes under study.

The entire sets of proteins secreted by P. stutzeri cells under the two different growth conditions were also analysed by 2-DE. Although numerous proteins appeared to be expressed and secreted exclusively in cultures with the aged glue, thus suggesting that a quite complex set of enzymatic activities might operate in a synergic way to achieve the purpose, nevertheless PMF analysis by MALDI-TOF MS didn’t lead to any protein identification. This failure had two concomitant causes: too little amount of material present in each spot, which in most cases prevented us from obtaining good spectra, and the few entries in the SWISS PROT and TrEMBL data bases for this species (Swiss Prot lists only 59 proteins, among which not a single protease, for P. stutzeri). Because not enough material for further gel analysis was available, we resorted to other classical biochemical studies, which required a much smaller amount of sample proteins.

In the first place, the general proteolytic activity in the culture supernatants was assessed by measuring the rate of coloured peptide release in solution in presence of an azo-derivative of β-casein (Azocasein). A general, easily degradable protein substrate was chosen in order to have a comprehensive picture of the panel of proteases present in the samples. This simple assay revealed that a considerable proteolytic activity (3.63 mg of substrate converted per mL of supernatant per hour) is secreted by P. stuzteri cells in the case of aged glue-cultures, but not when glucose or fresh animal glue were provided as the sole carbon sources. Because the glue employed for the fresco detachment was of animal origin, thus containing a large amount of collagen, the more particular collagenolytic activity was evaluated as well. Again, only the aged-glue sample showed a good proteolytic activity (2.31 mg/mL*h), as compared to pure Clostridiopeptidase A purified from C. hystoliticum. The two values of activity of

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3.63 and 2.31 mg/(mL*h) for, respectively, general and collagenolytic activities, suggest that the majority of the proteases secreted by this bacterial strain are collagenases, while a few components of the proteolytic machinery are not able to digest such a substrate. Also the supernatant obtained from a culture with fresh-glue showed the ability to partially digest Azocoll, but at a much lower extent (5,91 x 10^-3 mg/mL*h). This fact implies two consequences: first, the proteolysis of common animal glue doesn’t require the special set of enzymes secreted by P.

stutzeri strain A29 in order to grow on the Spinello’s glue; in the second place, and as a corollary of the previous consideration, in absence of the original glue it was impossible for us to simulate the original environmental conditions in which P.

stutzeri had produced such a unique set of proteases.

Although these data fully confirm the presence of proteolytic activities (at least against collagen and casein) essentially only in the supernatants of intact P.

stutzeri grown on the original glue from Spinello’s fresco, they could hardly give us any clue for the identification of such enzyme(s) (no data exist on proteases in P.

stutzeri strains). For this reason we resorted to a classical zymogram technique, a peculiar type of SDS-PAGE by which the various supernatants were analysed in a 10%T, 2.5%C polyacrylamide gel cast in presence of 0.1% casein as a substrate for proteases. With this technique, after electrophoretic separation in moderately denaturing conditions, the proteins are generally allowed to re-fold by overnight detergent exchange with Triton X-100. In this particular case, during incubation for surfactant exchange, the re-folding proteases also degrade the surrounding casein, thus creating a white band over a dark-blue background when the entire gel is stained with Coomassie blue. As shown in Fig. 2.5, only the supernatant obtained from a culture with Spinello’s glue gave a ladder of caseinolytic activity bands, extending from ca. 20 kDa up to 250 kDa. Interestingly, a commercial preparation of Collagenase from C. hystoliticum (tracks 7 and 8) gave an intense band at 120 kDa, which apparently coincides with the strongest activity band in the supernatant from Spinello’s glue (track 2). This suggests that the P. stutzeri collagenase might have some homology with the C. hystoliticum one. We do not know, at present, if the other major activity bands of lower Mr values, extending from 100 kDa down to as low as 20 kDa, represent novel types of proteolytic enzymes or merely some degradation products of the 120 kDa parental species, 50

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as reported for other types of collagenases from diverse bacterial species [6]. We believe, though, that at least some of these bands should represent unique enzyme activities, each endowed with specific proteolytic performance, and that only their combined action is able to attack the hardened glue till its complete digestion. In fact, when originally treated with purified enzyme preparations (including the same 120 kDa C. hystoliticum collagenase), the 20-year-aged glue could not be digested at all, as clearly demonstrated by the researchers who carried out the intervention [4].