ABSTRACT
This chapters analyzes the degradation of poly(e-caprolactone) (PCL) by three fungal lipases from Mucor miehei, Candida rugosa, and Rhizhopus arrizhus in a solvent-free system. To evaluate the activity of the three lipases, we utilized olive oil as the substrate. Mucor miehei lipase proved to be the best biocatalyst for both substrates: The data presented suggest it has a better capability to hydrolyze triglycerides found in olive oil. This property is further enhanced when the enzyme is immobilized on a glass surface in monolayers ranging between 1 and 5. We analyzed the degradation of PCL by mass spectrometry discovering that only Mucor miehei lipase was able to completely degrade a PCL film. Moreover, mass spectrometry results evidenced the enzyme action of separating
plastic oligomers of PCL and of further hydrolyzing oligomers previously detached. 8.1 Introduction
8.1.1 BiodieselBiodiesel is an alternative fuel for diesel engines. Its primary advantages are that it is one of the renewable fuels currently available and is also non-toxic and biodegradable. It can also be used directly in most diesel engines without requiring extensive engine modifications.Rudolf Diesel tested vegetable oil as fuel for his engine (Shay, 1993). With the advent of cheap petroleum, appropriate crude oil fractions were refined to serve as fuel and diesel fuels and diesel engines evolved together. In the 1930s and 1940s, vegetable oils were used as diesel fuels from time to time, but usually only in emergency situations. Recently, because of increases in crude oil prices, limited resources of fossil oil and environmental concerns there has been a renewed focus on vegetable oils and animal fats to make biodiesel fuels. Continued and increasing use of petroleum will intensify local air pollution and magnify the global warming problems caused by CO2 (Shay, 1993). In a particular case, such as the emission of pollutants in he closed environments of underground mines, biodiesel fuel has the potential to reduce the level of pollutants and the level of potential or probable carcinogens (Krawczyk, 1996).Fats and oils are primarily water-insoluble, hydrophobic substances in the plant and animal kingdom that are made up of one mole of glycerol and three moles of fatty acids and are commonly referred to as triglycerides (Sonntag, 1979a). Fatty acids vary in carbon chain length and in the number of unsaturated bonds (double bonds). 8.1.2 LipasesLipases (EC 3.1.1.3) are a class of enzymes that catalyze hydrolysis of triglycerides with the formation of fatty acids and glycerol (Gutiérrez-Ayesta et al., 2007) at lipid/water interface. The biochemical characteristics of several microbial lipases, fungal and
mammalian were extensively evaluated (Dominguez et al., 2006; Kovac et al., 1996; Sharma and Chisti, 2001). Their active site is composed of a “catalytic triad” of three amino acids (Ser-His-Asp/Glu) (Joseph et al., 1997; Ninia and Sardab, 2001; Varfolomeev et al., 2005) and when they are placed at lipid/water interface, an hydrophobic patch around the catalytic triad is creates (Kovac et al., 1996) allowing the enzyme’s activation (Joseph et al., 1997; Ninia and Sardab, 2001; Varfolomeev et al., 2005). Lipases have an excellent catalytic activity and can also catalyze the reverse reaction, the esterification. These features make the use of lipases suitable in many biotechnological applications (Babu et al., 2007) as the synthesis of biopolymers and biodiesel (Bernardes et al., 2007; Salis et al., 2005; Zhao et al., 2007), and the production of pharmaceuticals, chemicals and soaps (Jaeger and Eggert, 2002).In a previous study (Pastorino et al., 2004), we described the lipase-catalyzed degradation of PCL in organic solvent (toluene). Because of the high toxicity of many organic solvents used for the dissolution of plastic, this chapter compares the hydrolytic activity of three lipases from different fungal origin-Mucor miehei, Candida rugosa, and Rhizhopus arrizhus-in a solvent-free system with the aim of developing a new technological application based on innovative principles. In Sivozhelezov et al. (2009), we exploited the possibility of employing Langmuir-Blodgett (LB)-based protein structures to use in biocatalysis, exemplified by lipase with olive oil as substrate; we demonstrated that it was possible to form even more active lipase nanostructures by the LB technique at the airwater interface, proving that the Langmuir film provides a better catalytic effect in lipase than a mere oil-water boundary.The goal of this study was to analyze the degradation in a solvent free system of PCL, which is a biodegradable aliphatic polyester, well known for its high mechanical compatibility with many polymers and good adhesion to a broad spectrum of substrates (Tsuji et al., 1996). 8.2 Materials and MethodsLipases from Mucor miehei and Candida rugosa were purchased from Sigma-Aldrich (St. Louis, MO), lipase from Rhizopus arrizhus was obtained from Fluka (Milan, Italy). MilliQ (Millipore) water was used for preparation of enzyme solution. Lipase was utilized
without any further purification. Poly(e-caprolactone) MW 80 kDa was from Sigma-Aldrich (St. Louis, MO). Olive oil and all the chemicals for enzymatic activity assay (sodium taurocholate, NaCl, and CaCL2) were produced by Fluka (Milan, Italy). 2,5 Dihydroxybenzoic acid (DHB) (Bruker Daltonic, Leipzig, Germany) was used as matrix for matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. All the others chemicals (potassium phosphate, NaI, NaOH, TFA, and acetonitrile) were purchased from Sigma. 8.2.1 Measurement of the Enzymatic Activity of LipaseEnzymatic activity of lipase was determined by modifying the standard test Sigma (N° 62300A2) (Worthington, 1988) using as substrate olive oil; 10 µL of enzyme from a stock solution (10 mg/mL) were added at the reaction mixture composed by potassium phosphate buffer 10 mM, Na taurocholate 1.5% NaCl 3M and CaCl2 75 mM. Subsequently, the pH was adjusted to 7.8 using NaOH 100 mM. The reaction mixture was magnetically stirred at 37°C for 30 min. The amount of fatty acids released was determined by titration with NaOH 100 mM.
