CALB (Candida antarctica lipase B) immobilization on APTMS activated carrier via cross-linking method

The Employment of enzymatic catalyss in polymer sytheses have attracted attention, thanks to provides control on stereochemistry, chemoselectivity, and regioselectivity. Unlike organic synthesis, enzymatic polymerization provides yielding non-toxic, biocompatible and biedegradable products. Through these advantages, the products obtained by enzymatic polymerization find widely usage in medical applications such as biomedical engineering, drug release systems and tissue and organ transplantations. As another significant point, some of polymetic products, such as cellulose, chitin, xylan and hyaluronan, are only able to be obtained succesfully by enzymatic polymerization. Poly(ε-caprolactone) (PCL) is a focus of interest because of its advantages over other biopolymers with regards to mainly favorable degradation rate and controllable pore size. Its crystallinity is able to reach up to 69%, depending on the molecular weight in the manner that as the moleculer weight decreases, the crystallinity increases. Thus, its mechanical and thermal properties as well as physical structure are govern mainly by the moleculer weight, varying between 530 and 630 000 g/mol. Other significant propety of PCL is to owning good miscibility with various polymers such as poly (lactic acid), poly(vinyl chloride), poly(bisphenol-A) and poly(styrene–acrylonitrile). PCL is more relatively stable in ambient conditions, highly biocompatible, easy to manufacture, and viable in various type of scaffold. Therefore, inherent properties of PCL reveal themself on the area of tissue engineering. Among various lipases from differient origins, it is observed that Candida antartica lipase B (CALB) showed relatively high catalyhtic activity for the polymerization of ɛ-caprolactone (ɛ-CL) to PCL. Enzymatic synthesis of PCL may be categorized two main pathway, which are polycondensation reaction, and ring opening polymerisation (ROP). As to polycondensation polymerization, beside the necessity of additional process to removal by-products (water), low molecular weight is a drawback on polycondensation reactions. Because of these drawbacks related to polycondensation reaction, ROP of ε-caprolactone came into prominence in PCL synthesis. Nevertheless, enzymes are frigle macromolecules, which need to be immobilized in order to gain stability and to be recovered at the end of the reaction. Besides, it is a desired result of the immobilization process to improve cataltic activity of the enzymes. The reuse capability of immobilized enzymes is an significant determinative factor on immonilization achievement. The commercially derivative of immobilized CALB, Novozyme 435® , is known with its high efficiency on ROP of ɛ-caprolactone (ɛ-CL). However, the high cost of this enzyme, because of the immobilization support material, and enzyme leakage issue are still general concerns. For these reasons, more effective and cheaper immobilization approaches on CALB, which can be alternative to available systems, is a current trend in the literature. In the present study, free CALB, commercially named as Lipozyme® , was immobilized onto rice husk ash (RHA). Rice husk (RH), as agricaltural waste, is an easy accessible and cheap material, which is a possible source for production of amorphous silica when it is treated thermally in controlled temperature and time. The fact that amorphous silica, with very fine particles, has large surface area and porous structure, makes it an attractive choice of carrier for immobilization studies. In this study, cross-linking method was applied by bonding of CALB to the support via a spacer molecule covalently in order to overcome enzyme leakage issue. The performance of the new enzyme preparate, obtained through this process, was evaluated in ROP of ɛ-CL. In order to obtain support material, RH was burned at 600 0C for 6 hours. After thermal treatment, surface modification on the RHA was accomplished in two step. Firstly, the surface was silanized with 3-aminopropyltrimethoxysilane (3-APTMS) to obtain the functional amino groups. In second step, glutaraldehyde was used as cross-linking agent to activate amino-functionalized surface. In order to determinate the best conditions in immobilization parameters, different 3-APTMS and glutaraldehyde concentrations as well as enzyme loading ratios were evaluated during parametric studies. In addition, storage stability, optimum temperature and pH were investigated. Throughout the immobilization study, fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA), and scanning electron microscopy (SEM) were performed in order to characterize RHA, surface-modified RHA, and the new immobilized lipase. In the second phase of this study, performance of immobilized CALB on surface modified RHA by cross-linking metod was evaluated in PCL sythesis. Polymerization reactions of ɛ-CL were conducted in at various periods (6, 24, 48, 72, and 120 hours) and at different temperatures (30, 40, 60, and 80 ֩ C) in order to determinate the best reaction conditions. Besides effects of the enzyme concentration in the polymerization was examinated at different enzyme : monomer ratios. Polymerization reactions were performed with Lipozyme® and Novozyme 435 in optimum reaction conditions conditions in order to compare the results with that of new immobilized enzyme. Molecular weight distributions and structural identification of products were carried out by means of gel permeation chromatography (GPC) and FT-IR spectroscopy. Proton nuclear magnetic resonance spectroscopy (1H-NMR) and X-ray diffraction (XRD) studies were performed in order to reveal crystal structures of the polymer samples. Besides, Thermal analysis of samples was done by TGA and differential scanning calorimetry (DSC) analysis. Structural details of the surface of polymer were characterized by means of SEM. As to the results, obtained throughout the current study, surface modified RHA and enzyme immobilized surface of the support were confirmed by FT-IR and TGA analyses. In comparison with free enzyme, immobilized enzyme remained 73.1 % of relative activity with 98 % protein loading efficiency. The specific activity was recorded as %74 of that of free enzyme. After 3 mounths of storage stability evaluation, the immobilized enzyme retained 67 % of its activity. According to the results, recorded during immobilized enzyme studies, it is indicating enhanced thermal stability that there was not a noticeable decrease in relative activity up to 80 0C for immobilized enzyme. Characterization of polymer chains of product, obtained in enzymatic ROP of ɛ-CL in pre-determinated best reaction conditions, were evaluated and verified by FT-IR and 1H-NMR analyses. According to GPC results of products, yielded by crosslinked lipase, the highest molecular weight was obtained as 13700 g/mol at 60 0C and 24 hours reaction period with 1,5 polydispersity index (PDI) and %92 monomer conversation. These results are promising an energy-saving and eco-friendly enzymatic PCL process with monodispersed products and better stability than that of free enzyme.