Effect of silanization agent on the immobilization of lipase on a silica-based support material

Enzymatic syntheses for industrial applications have attracted attention recently due to their advantages over conventional chemical syntheses. Utilization of enzymes provides clean and environmental friendly processes in addition to high selectivity and specificity. However, in order to be adapted for industrial applications, enzyme should be engineered through several processes. Immobilization of enzyme is one of these engineering process and has become an attractive technique to improve enzyme stability and activity. Immobilization is a technique which is applied in order to improve enzyme activity and stability. In general, interactions between the enzyme and a solid support material are desired so that the enzyme is localized in a specific region. The catalytic activity is conserved, even improved. Additionally, immobilization also permits repeated use of enzymes by recovery. This allows continuous and economical processes for industrial applications. It also prevents protein contamination of the product. There are several methods of immobilization, mainly physical adsorption, entrapment, covalent attachment and cross-linking. Physical adsorption is the simplest immobilization technique. It depends on weak interactions between the enzyme and a solid support material. Though it is an economic method, enzyme leakages due to weak interactions may cause difficulties. Entrapment is achieved by occlusion of the enzyme in a polymer matrix. The enzyme is protected from the effects of the environment. However, mass transfer limitations occur. The difference of this method is that the enzyme is not bounded to the polymer matrix. During covalent attachment, functional groups of the enzyme create strong covalent bonds. It is important to ensure that catalytic sites of the enzyme are not involved in binding. Cross-linking is achieved by the intermolecular linkages between the proteins or between the proteins and the solid support material. A solid support material is required for the immobilization procedure on which the enzyme will be bound. A suitable support material should be considering several criteria. It should have resistance to physical forces, inertness towards enzymes. It should also be accessed easily and cheap, biocompatible and resistant to microbial contamination. Support material can be divided into two groups: organic and inorganic support materials. Support materials can be used after surface modification for improved results. One of the most commonly used surface modification technique is silanization. In case of materials having hydroxylated surfaces, modification occurs through these hydroxyl groups and generally organosilanes are used as silanization agent. Hydroxyl groups on the surface react with active groups of the silanization agent, generally alkoxy groups. Trifunctional silanes have higher reactivity and monolayers of trifunctional silanes are believed to yield well-ordered and compact films. Most widely used trifunctional organosilanes are 3-aminopropyl triethoxysilane (3-APTES), 3-aminopropyl trimethoxysilane (3-APTMS) and 3-glycidyloxypropyl trimethoxysilane (3-GPTMS). Rice husk is high silica containing material which is obtained from rice production process as by-product. Silica is considered to be a suitable support material for enzyme immobilization. Additionally, it is a cheap and easily accessible material since it is a by-product. Utilization of this by-product may be considered as a waste management strategy. Lipases may find various applications in different industries. For example they can be used as additives in detergents and responsible for removal of oil stains since they catalyze hydrolysis of fats. In pulp and paper industry, lipases are used for removal of pitch whose deposition reduces the quality of pulp. Lipases are also used to replace the conventionally used emulsifiers in food industry and for flavor development. Lipases can be mainly used for biodiesel production in energy industry in order to replace fossil fuels with renewable energy sources. Biopolymers are generally preferred for medical application since they are biodegradable and biocompatible with human body thus nontoxic. Poly (ε-caprolactone) (PCL) is a biodegradable and biocompatible polymer which has been extensively studied for medical applications. PCL is synthesized by ring opening polymerization (ROP) reaction of ε-caprolactone and the reaction can be catalyzed by lipase eznyme. In this study, free Candida antarctica lipase B (CALB L) was immobilized on modified rice husk ash (RHA). By this procedure, it is aimed to increase catalytic activity and improve stability of CALB. RHA was chosen since it is an easily accessible and cheap material. Additionally, its high silica content makes RHA a suitable choice since it is known that silica is successfully used as a support material for enzyme immobilization. As a first step, RHA was obtained by burning rice husks at 600 °C for 6 h. Surface modification was achieved by using two different silanization agents, 3-glycidyloxypropyl trimethoxysilane (3-GPTMS) and 3-aminopropyl trimethoxysilane (3-APTMS). The modified RHA were used as support materials for immobilization of CALB by physical adsorption. Enzyme loading ratio and concentration of silanization agent were evaluated as part of the optimization studies. Furthermore, pH, thermal, storage and operational stabilities were studied; optimum pH and temperature of the immobilized lipase samples were determined. Characterization of RHA, surface modified RHA and immobilized lipase samples were done by Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA), scanning electron microscopy (SEM) and Brunauer-Emmett-Teller (BET) surface area analysis. FT-IR and TGA results showed that both 3-GPTMS and 3-APTMS modifications were successfully achieved. Surface area analysis also showed the textural structures of the support material and modified support materials. Immobilization of lipase on these modified samples was also successful; reaching immobilization efficiencies of 88.5 % and 90.8 % for 3-GPTMS and 3-APTMS, respectively. Catalytic activity of CALB was retained and specific activities were increased by approximately 1.8 folds for both types of immobilized enzyme samples. This value is close to the specific activity of a commercially available immobilized CALB, Novozyme 435® (2.2 times higher than free CALB). Stabilization experiments showed that the immobilize CALB samples have remarkable pH and temperature stability, and storage and operational stabilities. Based on these results, it can be suggested that CALB immobilized onto 3-GPTMS and 3-APTMS silanized RHA may become an attractive alternative to other commercially available CALB. Catalyzing polymerization reactions, especially lactone monomers, is one of the applications of immobilized lipases that attracted attention. Polycaprolactone (PCL) synthesis via enzymatic ring opening polymerization (ROP) reaction has been widely studied in recent years. There is a commercially available immobilized CALB, Novozyme 435®, which is known to be efficiently utilized for PCL polymerization. In further studies, these immobilized CALB onto 3-GPTMS and 3-APTMS silanized RHA may be adapted for PCL polymerization. These immobilized lipases may also be used in co-polymerization reactions with other monomers which may allow faster kinetics and yield polymer chains with higher molar masses. Such an application exists in literature.