by NMR relaxometry, EPR spectroscopy and X - Ray powder spectra Donatella Capitani, Maria Carmela Emanuele, AnnaLaura Segre, Corrado Fanelli, Anna Adele Fabbri, Donato Attanasio, Bonaventura Focher and Giorgio Capretti Keywords : Paper, Enzymatic Attack, NMR Relaxometry, EPR Spectroscopy. SUMMARY: Fungal growth on paper is one among the causes responsible for paper deterioration in archives and libraries. The study of paper deterioration is a complex phenomenon in particu- lar when it is due to the biotic action of fungi and bacteria. One of the major problems is the early detection of fungal activity and, following an enzymatic attack, the evaluation of actual per - manent damage on different types of paper. In this report we pre- sent a combined study based on NMR relaxometry, EPR (Electron Paramagnetic Resonance) spectroscopy, X-Ray powder spectra and chemical analysis. These methods altogether indicate that, in the case of an enzymatic attack by end0 glucanase, it is the amorphous fraction of paper to be attacked. In particular, NMR relaxometry is able to reveal extremely mild enzymatic attacks, not observable with any other method. ADDRESSES TO THE AUTHORS: Donatella Capitani, Maria Carmela Emanuele, AnnaLaura Segre (to whom correspon- dence should be addressed): lstituto di Strutturistica Chimica NMR Service of CNR - Area della Ricerca di Roma, MBlO - Monterotondo Staz. 00016 Rome, Italy. Corrado Fanelli, Anna Adele Fabbri: Dipartimento di Biologia Vegetale, Universita di Roma "La Sapienza". Donato Attanasio: 1st. Chimica dei Materiali del CNR - Area della Ricerca di Roma. Bonaventura Focher, Giorgio Capretti: Staz.Sperimentale Cellulosa Carta e Fibre Tessili-Milano. ct Fungal growth on paper is one among the causes responsible for paper deterioration in archives and librari- es. The composition of softwood and hardwood, main components of paper, is mainly characterized by cellulose (linear polymer of D-glucose connected by P-(1-4)-linka- ges) that exists in a crystalline state, as Ia and IP poly- morphs, and in amorphous states. The cellulose is about 40-50% of the wood composition, but also important are the hemicelluloses and lignin (Biermann, 1993). It is well known that many fungi active on paper deterioration pro- duce d~fferent hydrolytic enzymes such as cellulases and ligninases (Wood, Garcia Campayo, 1990; Coughlan et al. 1993; Fabbri et al. 1997, Jeffries, 1990; Zabel, Morrel, 1992). Such enzymatic activities present different operating mechanisms in the solubilization of different paper compo- nents. In fact it is known that degradation of cellulose due to different fungi involves the synergic action of enzymes defined esoglucanases (i.e. cellobiohydrolases), endoglu- canases (endo- l ,4-P-D-glucan-4-glucanohydrolases) and P-glucosidases (Wood, GarciaCampayo, 1994). On the other hand the biodeterioration of lignin, present in most commercial papers, needs a complex of enzymes activities such as lignin peroxidases, Mn-peroxidase and laccase (Jeffries, 1994). Ligninase and cellulase act in synergism to achieve a complete solubilization of paper components (Teen, Koivula, 1995). It appears evident therefore that the study of paper dete- rioration is a complex phenomenon especially when it is due to the biotic action of fungi and bacteria, difficult to be compared with abiotic factors such as light, metals, oxy- gen, humidity, UV radiation and temperature. One of the major problems is the early detection of fun- gal activity and, following an enzymatic attack, the evalua- tion of actual permanent damage on different types of paper. To be as near as possible to the real systems we used a purified enzyme with a well defined activity. No model compounds were studied, such as pure cellulose or lignin, the study was performed only on paper. In general, the methods used for the evaluation of paper degradation require a large amount of material and are time consuming; moreover, to detect significant evidences of biotic and abiotic paper degradation (Gumagual et al. 1992; 1993), a large number of data is required often follo- wed by statistical methods. Another important point regards the enzymatic action of endo-glucanases. The enzymatic action on amorphous area of cellulose by endo-glucanase has been reported (Gascoigne, Gascoigne, 1960; Wood, MC Crae, 1972), however it is not yet clear if endo-glucanase activity can work with hydrolytic action together with eso-glucanase (cellobiohydrolase activity) to attack crystalline cellulose (Wood, Garcia Campayo, 1990; 1994). In this paper we will show how an early detection of biotic attack can be studied by EPR and NMR methods. The study was performed after the attack of cellulase enzy- mes. It is worth to note that both resonance spectroscopic methods are very sensitive and require very small amounts of paper (Attanasio et al. 1995; Capitani et al. 1996). To check the obtained results, X-Ray powder diffractometry was also used in conjunction with standard chemical met- hods for the analysis of hemicelluloses. Experimental procedures Preparation of handsheets Bleached cotton linters obtained from Cartiere Miliani Fabriano SPA, Italy, was soaked in water and beaten in a Valley beater to 40°SR. Handsheets of 60 gIm2 were prepared by a dynamic sheet former (Techpap, France) with acidic sizing. Nordic Pulp and Paper Research Journal Vol 1 3 no. 2,1998 Brought to you by | Miami University Libraries (Ohio) Authenticated Download Date | 10/28/18 6:59 PM