Lipazın inorganik taşıyıcıda immobilizasyonu ve polikaprolakton sentezi

In recent years, aliphatic polyester synthesis via enzymatic ring opening polymerization (ROP) of lactones has become an attractive research area. There is a considerable interest on utilization of enzymatically synthesized aliphatic polyesters for biomedical applications due to their biodegradability, biocompatibility, and outstanding mechanical properties. In addition, catalysis with enzymes provides achievement of non-toxic materials which is essential for biomedical implementations such as drug delivery systems, tissue engineering, and medical devices. Polycaprolactone (PCL) is an aliphatic polyester that can be synthesized via ROP of ɛ-caprolactone (ɛ-CL). Among other aliphatic polyesters, PCL has a relatively low melting temperature (Tm), between 59-64 ̊ C, and low glass transition temperature (Tg), between -60-10 ̊ C. Moreover its decomposition temperature is about 350 ̊ C which provides high thermal stability. Besides good viscoelastic and rheological features, these thermal properties make PCL to be easily manufactured. In addition, PCL is highly soluble in variety of solvents such as tetrahydrofuran (THF), toluene, chloroform, benzene and dichloromethane and it is compatible with other polymers which makes possible to obtain blends. Furthermore, PCL is a suitable biopolymer for biomedical purposes due to its biocompatibility and biodegradability. When compared with the other biodegradable polymers, PCL degradation process is slower in vivo. This long-term biodegradation behavior provides advantage for drug delivery application of PCL. As a result of these advantageous properties, PCL has become an important subject for polymer science and achievement of enzymatic PCL synthesis has received attention for biotechnological applications. ROP of ɛ-CL can be catalyzed by both biocatalysts (enzymes) and organometallic initiators. Organometallic initiators such as Zn, Al, Sn, and Ge, cannot be removed completely from polymer matrix at the end of the polymerization which may lead toxic effects when used as a biomaterial. On the other hand by using biocatalysts, this problem can be overcome since they can be removed easily at the end of the reaction and they are non-toxic and eco-friendly catalysts. Moreover, biocatalysts can catalyze polymerization at mild reaction conditions (relatively low temperatures and pressures). However, biocatalysts may lose their activities after long reaction periods and may have stability troubles. Thus, enzyme immobilization is the most widely used strategy to overcome utilization problems of enzymes such as low stability and lack of ability to be reused. By the immobilization of enzymes, enzyme activity and stability can be improved, enzyme can be recovered at the end of the reaction and reused which may provide a potential to enzyme to be used in a continuous process. Candida antarctica lipase B (CALB) is one of the most efficient and selective lipase that can catalyze esterification and transesterification reactions. Its immobilized form is widely used as a biocatalyst and commercially available as Novozyme 435®. It is known with its effectivity on ROP of ɛ-CL. However, Novozyme 435® is immobilized on acrylic resin which is mechanically and thermally weak support. Therefore, development of new carriers became an attractive research area to develop such an effective lipase that can catalyze polymerization reactions as efficiently as Novozyme 435®. In this work, free CALB (CALB L, Lipozyme®) was immobilized on an inorganic support material, rice husk ash (RHA), since it is a cheap and plentiful (by-product of rice production) material. Furthermore, silica is a widely used support material for enzyme immobilization. Since RHA is rich with SiO2 content (up to 95%), it is a promising support material. Therefore in this study it was aimed to show the efficiency of lipase immobilized on RHA with an application; catalysis of PCL synthesis. For this purpose, firstly support material was prepared and lipase was immobilized via two methods; physical adsorption and cross-linking. Then, PCL was synthesized with these new immobilized enzymes. At support preparation and immobilization part, first of all RHA was obtained from the burning of rice husks at 600-650 ֩ C for 6 hours. Then, by the use of a silanization agent 3-aminopropyl triethoxysilane (3-APTES), surface modification of RHA was achieved and functional amine (-NH2) groups were added to the surface. Immobilization of lipase was performed by physical adsorption and cross-linking with glutaraldehyde. For the optimization of new immobilized lipases; different 3-APTES and glutaraldehyde concentrations and enzyme loading ratios were also tried. In addition, storage stability, operational stability, optimum temperature and pH were investigated. Moreover, fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA), and scanning electron microscopy (SEM) were used for the characterizations of RHA, surface-modified RHA, and immobilized lipases. The second part of this study, which is another novel work, is PCL synthesis via immobilized lipases on surface-modified RHA with physical adsorption (AD) and cross-linking (CR) methods. Polymerization reactions were carried out in toluene which was shown before as an efficient solvent for ROP of ε-CL. For the determination of optimum polymerization conditions of immobilized lipases, polymerizations were performed for various reaction times (6, 24, 48, 72, and 120 hours) at different temperatures (30, 40, 60, and 80 ֩ C). After obtaining the optimum polymerization conditions of immobilized lipases, effect of different enzyme concentrations were investigated and enzyme recycling studies were done. In addition, polymerizations via Lipozyme® and Novozyme 435® were also carried out at these conditions for the comparison of the performances of home-made and commercial enzymes. Molecular weight distributions and chain structures of the polymer samples were compared by gel permeation chromatography (GPC) and hydrogen nuclear magnetic resonance spectroscopy (1H-NMR), respectively. In addition, for the characterization of chain structures, FT-IR was also used. Thermal properties of the polymer samples were obtained by TGA and differential scanning calorimetry (DSC) analysis. To record the crystal structure of polymer samples, X-Ray diffraction (XRD) analysis was carried out. Moreover, for determination of surface structures, SEM was used. At the end of this study, surface-modified RHA was successfully obtained and addition of –NH2 groups to the surface was showed by FT-IR and TGA analyses. Both AD and CR lipases were achieved with an immobilization efficiency of about 90%. In addition, the activities of AD and CR lipases were measured as 92.3% and 78.8% of the activity of free lipase, respectively. Furthermore, it was found that, specific activities became 1.8 and 2.0 folds greater than the specific activity of free lipase after immobilization via cross-linking and physical adsorption methods, respectively. Moreover, their specific activities were close to the specific activity of Novozyme 435® which has a specific activity of 2.2 folds greater than free lipase. By using AD lipase, PCL (its structure was verified by FT-IR and 1H-NMR) was synthesized with a molecular weight of 14000 g/mol at the end of 48 hours reaction period at 60 ֩ C. Similarly by the use of CR lipase, 11580 g/mol molecular weight was reached at the end of 24 hours reaction period at 40 ֩ C. Finally, immobilized lipases were recycled six times at their best reaction conditions. At the end of six reaction cycles, 34.1% and 35.7% of their initial activities were remained for AD and CR lipases, respectively. These results indicated that lipase immobilized by cross-linking and adsorption not only had good activity recovery, but also remarkable stability, better reusability and application adaptability than free lipase. Moreover, in further studies, development of nonohybrids may be possible by grafting CALB immobilized onto inorganic support RHA into PCL matrix. There exists such an successful application with montmorillonite and sepiolite as inorganic support materials in literature.