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Enzyme immobilization has several drawbacks that limit to the proper immobilization procedure and its enzymatic reaction. In practice, most of immobilization techniques especially covalent binding will significantly reduce the enzyme activity or retention activity compared to the free enzyme applications, due to inactivation or denaturation of enzyme molecule, which cannot be completely avoided. This reason is reflected to the conformation changes of some amino acid residues during the chemical coupling reaction and immobilization procedure. However, the level of enzyme inactivation or denaturation in immobilization depends on the type of enzyme immobilization techniques, structured support, medium conditions (e.g. buffer solution, pH and temperature) and reaction during preparation of immobilization procedure, due to preserve the enzyme activity as close as to its original performance level by not changing the chemical nature or reactive groups on the enzymes active site (Lalonde and Margolin, 2008).

Covalent immobilization has been proved as the strongest and the most stable interaction technique for enzyme immobilization. However, this technique relatively expensive and it requires a complicated procedure due to additional of chemical coupling agents to perform covalent linkage between enzyme

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molecule and surface support structure, especially for those involving the nano-sized supports (Kim et al., 2006). In normal cases, covalent immobilization of enzyme leads to lower residual enzyme activity, as mentioned previously due to the tendency of some of amino acid residues that are essential for catalytic activity also form covalent linkage on the support (Brena et al., 2006).

Sometimes, the center of active site of enzyme could be modified or altered through chemical coupling reaction, since most chemical coupling agents (e.g.

glutaraldehyde (GA), N-ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and etc.) are toxic and very reactive due to unstable reagents, which it leads to inactivation of the enzyme molecule (Cao, 2006). Thus, appropriate selection of type, method and suitable reaction condition of chemical coupling agents are needs to be concerned for enhancing, stabilizing and optimize the reaction for covalent interaction in enzyme immobilization (Pessela et al., 2007).

Many factors such as moisture absorption, poor compatibility, and poor surface characteristic (hydrophilicity/hydrophobicity) may affect to the behavior of structural properties of CNF support, which make a less attractive toward covalent interaction (Hu et al., 2011). Poor structure properties and incompatible of surface hydrophilicity/hydrophobicity of support may interrupt the interaction mechanism of covalent immobilization of enzyme and reaction of chemical coupling agents, as well as may lead to the weak interaction and enzyme leakage from the surface support (Fang and Szleifer, 2002). Surface modification via chemical-physical treatment could overcome these problems which it able to create a suitable surface interfacial characteristic of CNF for α-CGTase immobilization and interaction via chemical coupling agents (Ferrarotti et al., 2006). The interaction between hydrophobic and hydrophilic characteristic will provide a suitable interface for retaining enzyme activity, as reducing the complimentary unfolding toward hydrophobic-side group in amino acids and increases the reactive functional group of cellulose (Hu et al., 2011).

Packed-bed or fixed bed reactor system is a recent application of EMR in industrial. For the packed bed reactor system, it requires a larger catalyst module to increase the efficiency of enzymatic reaction. Installation of catalyst bead for larger scale system, handling of the catalyst bead such as enzyme immobilization preparation and loading to the reactor column are difficult due to large quantity of bead (Meng et al., 2017). In addition, enzyme has certain life-span and after certain cycles of enzymatic reaction or certain period of operation, the bead must be removed and regenerated, otherwise it is considered as a waste. In facts, a smaller bead is needed to reduce the size of bed of reactor module. Unfortunately, it may result in higher back pressure when operated at high flow rate for achieving high productivity (Wong et al., 2015). Smaller bead always result in higher hydrodynamic resistance. Therefore, feeding pump

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needs to be maintained frequently and involves higher energy consumption for pumping the liquid at high back pressure. Channeling or creaking of bead structure could happen due to high back pressure, and optimum reaction temperature is difficult to maintain due to the temperature gradient (Meng et al., 2017). Thus, the application of immobilized enzyme that fouled on membrane surface as EMR system is one appropriate technique to overcome of these problems, as well as to increase the production yield and its productivity with efficient.

Fouling is a particular major problematic in EMR system, causing dramatic permeate flux declined, decreasing the membrane permeability, reducing the membrane selectivity and increasing the back pressure. Therefore, it reduces the operation performance, restricts to the filtration efficiency, and limit the enzymatic reaction (Nguyen et al., 2012). Another limitation in EMR system is the formation, compression and thickness of cake layer, blocking mechanism and concentration polarization (CP) between foulant layer and feed flow lead to the major permeate flux declined. Controlling the fouling distribution and thickness of cake layer could excel the enzymatic reaction performance and increase the permeate flux of membrane operation (Pulido, 2016).

1.3 Objectives

The specific objectives of this research are:

1. To characterize the properties of cellulose nanofiber (CNF) support derived from kenaf bast fiber.

2. To evaluate the performance of immobilized α-CGTase via covalent binding on CNF support by chemical coupling agents.

3. To apply the immobilized α-CGTase on CNF using UF membrane reactor system based on the membrane fouling concept.

4. To develop the dynamic mathematical modelling for the mass transfer and reaction kinetic of fouled α-CGTase-CNF layer in UF system as enzymatic membrane reactor.

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13 1.4 Scope of study

This study emphasized on covalent immobilization of α-CGTase on CNF support via interaction of ligand-spacer arm and its application in ultrafiltration (UF) membrane system using membrane fouling concept. This research approach has been organized based on four objectives, which every objectives represent as a major research works. First objective is focused on the preparation, isolation and characterization of CNF support derived from kenaf bast fiber via chemical-physical treatment as support structure for α-CGTase immobilization.

Second objective is involved the covalent immobilization of α-CGTase on CNF support via ligand-spacer arm interaction as a chemical coupling agents [α-CGTase(GA1,12-diaminododecane)CNF]. Then, the immobilized α-CGTase on CNF was assayed using soluble starch to evaluate the enzyme activity and its properties during enzymatic reaction. Third objective is subjected to the application of immobilized α-CGTase using stirred-cell UF membrane system.

This system was prepared by loading the α-CGTase-CNF into stirred-cell reactor module by introducing the pneumatic pressure, to form a thin fouled α-CGTase-CNF layer on the surface of membrane. This work was undertaken to study the performance of membrane operation and its enzymatic reaction. Forth objective is developed the dynamic mathematical modelling based on the fouled α-CGTase-CNF layer in membrane system. Dynamic mathematical modelling was develop due to study the performance of component balance, mass transfer and reaction kinetic that occurred on the fouled α-CGTase-CNF layer during membrane operation, as well as elucidated the possible significance variables that effect to the performance of enzymatic reaction in the system. The overview of this research work and its graphical abstract were described as in Figure 1.4.

and Figure 1.5.

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Figure 1.4: Overview of the research work.

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Figure 1.5: Graphical abstract of the research work.

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