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An industrial enzyme, Cyclodextrin glucanotransferase (CGTase), was immobilised in polyvinyl alcohol (PVA) nanofiber (average diameter around 200 nm) membrane via co-electrospinning of the CGTase/ PVA mixture followed with glutaraldehyde vapour phase cross-linking. Addition of enzyme with concentration ranging from 1.5 to 7.5 % to the PVA solution (8 wt%) caused significant changes to the liquid jet behaviours which consequently affected the nanofiber structures and sizes. Incorporation of CGTase in the PVA membrane was confirmed by Raman spectroscopic analysis. The Raman spectra also showed no structural changes occured to the enzyme after subjected to the electrostatic spinning and cross-linking reaction. The immobilised enzyme showed excellent catalytic efficiency with up to 3.6 times higher enzyme loading, 25 % higher activity and good reusability in comparison with CGTase/PVA film made up from the same starting solution (control). (Received 25 May, 2017; Accepted 3 August, 2017) # corresponding author: Tel: +81 42 3887987; E-mail: wuled@cc.tuat.ac.jp Preparation and Characterisation of Cyclodextrin Glucanotransferase Enzyme Immobilised in Electrospun Nanofibrous Membrane Suryani Saallah, M. Nazli Naim, Mohd Noriznan Mokhtar, Noor Fitrah Abu Bakar, Masao Gen, and I. Wuled Lenggoro *1Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan *2Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia *3Faculty of Chemical Engineering, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia *4School of Energy and Environment, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China *5Department of Chemical Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan *6Biotechnology Research Institute, Universiti Malaysia Sabah, 88400, Kota Kinabalu, Sabah, Malaysia J. Fiber Sci. Technol., 73(10), 251-260 (2017) doi 10.2115/fiberst.2017-0035 ©2017 The Society of Fiber Science and Technology, Japan Journal of Fiber Science and Technology (JFST), Vol.73, No. 10 (2017) 251 continuous process (Hwang and Gu, 2013). Despite these advantages, extending the use of immobilised enzymes to various fields and applications requires further improvement and a better understanding of the current techniques to enhance their catalytic efficiency. To date, many types of materials have been designed for enzyme support or carrier. Properties of these support materials, particularly the size and structure, play an important role in determining the immobilised enzyme performance. Reduction of the support geometric size can improve the catalytic efficiency by providing high surface area for enzyme attachment (Sardar and Ahmad, 2015). In this sense, growing interest is being shown in integrating enzymes with nanostructured materials, particularly nanofibers. Electrospinning is a process in which electrostatic force is applied to a polymer solution to generate fibers with diameters ranging from several microns down to smaller than 100 nm (Sill and von Recum, 2008). Nanofibers produced through electrospinning offers several outstanding features for enzyme immobilisation; a) size, morphology and composition can be control to meet the requirements of enzyme support, b) ease of functionalisation for various purposes, c) high porosity and interconnectivity to reduce mass transfer limitation throughout the meshes and d) the fibers can be collected in the form of membrane to facilitate the biocatalyst recovery and reuse, which would allow them to be applied in an enzymatic membrane bioreactor (Agarwal et al., 2008; Herricks et al., 2005; Kim & Kim, 2017; Tran & Balkus, 2012). The utilisations of electrospun nanofibers for enzyme immobilisation have been achieved mainly through physical adsorption and covalent binding. Both of these methods allow the enzyme to “only” attach on the outer surface of the support, creating monolayer coverage of enzyme which might limit the loading capacity. To encounter this issue, a simple method which involves the encapsulation of an enzyme by co-electrospinning of enzyme and polymer solution mixture has been introduced (Dai et al., 2013; Fazel et al., 2016; Sakai et al., 2008). Enzymes such as lipase (Sakai et al., 2008; Sawada, et al., 2012), laccase (Dai et al., 2013), horseradish peroxidase (HRP) (Fazel et al., 2016) and acetylcholinesterase (Moradzadegan et al., 2010) have been successfully immobilised in polymer membranes with a remarkable improvement in enzyme loading efficiency. Their catalytic performances were also remained upon immobilisation, confirming that either the electric charge applied during the electrospinning process or the interference of polymer materials had minimal or no negative effects on the enzyme structure and catalytic function. In spite of the simplicity of the co-electrospinning strategy and the excellent features possessed by the electrospun polymeric nanofibrous membranes, to this extent, very few reports concerning nanofibers as a support for enzyme immobilisation have been published. Previously, the authors have shown that CGTase could be successfully immobilised into a nanofibrous polyvinyl alcohol (PVA) membrane (Saallah et al., 2016) by co-electrospinning of CGTase and PVA mixture, which allow the enzyme to attach and encapsulated in the nanofibers. The PVA solution is rich with hydroxyl groups that enable the formation of secondary bonding with the enzyme molecules. Therefore, by mixing the enzyme with PVA, the complex 3-dimensional structure and strong molecular interactions between the enzyme molecules could be interrupted (Wu, Yuan, & Sheng, 2005), which allow the formation of CGTase/PVA nanofibers by electrospinning. However, the previous study only focused on the feasibility of immobilising the CGTase enzyme in the PVA nanofiber at a low enzyme concentration (1 % v/v) and the structural changes that might occur during the immobilisation process were not comprehensively considered. Morevover, although several studies related to enzyme immobilisation using co-electrospinning approach have been done in the past, to our knowledge, no studies have been conducted to investigate the effect of addition of enzyme to the polymer solution on the behaviour of the electrified jet. This information is of interest to gain a thorough understanding and better control of the electrospinning process which affect the properties and performance of the immobilised enzyme/ nanofiber. In the present study, the effect of varying the CGTase enzyme concentration from 1.5 to 7.5% v/v on the electrospinning process and the nanofibrous membrane morphology and size were investigated. Efficiency of the immobilised CGTase/PVA nanofiber was investigated by analysing the enzyme loading, activity and reusability. The CGTase/PVA film made up from the same electrospinning solution was used as a control. 252 Journal of Fiber Science and Technology (JFST), Vol.73, No. 10 (2017) Electrospinning parameters Ambient conditions Voltage -9 kV Relative humidity 50 – 57 % Flow rate 0.2 ml/hr Temperature 23.6 – 25.4 C Tip to collector distance 10 cm Duration 3 h Materials and Methods Materials Polyvinyl alcohol (PVA) (85000-124000 Da, 99 % degree of hydrolysis), Glutaraldehyde (GA) solution (25 % in water), Bradford reagent, and bovine serum albumin (BSA, 67000 Da) were supplied by SigmaAldrich, USA. CGTase (EC 2.4.1.19) from Bacillus macerans was purchased from Amano Enzyme, Inc., Japan. α-cyclodextrin (α-CD) was purchased from Acros Organics, USA. Soluble starch and hydrochloric acid (32 %w/v) were provided by Merck, Germany. All other chemicals used were of analytical reagent grade. Preparation and characterisation of CGTase/PVA solution A PVA solution with a concentration of 8 % w/v was prepared by dissolving PVA powder in acetate buffer (100 mM, pH 6) under mild stirring at 90 ̊C. The solution was allowed to cool to room temperature before mixing with CGTase to obtain a CGTase/PVA mixture with 1.5 to 7.5 % v/v CGTase concentration. Prior to electrospinning, properties of the CGTase/ PVA solutions which include the viscosity and conductivity were analysed using a Cannon-Fenske viscometer (Cannon Instrument Company, USA) and conductivity meter (Mettler Toledo, Switzerland), respectively, to study its effect on the electrospinning and fiber formation. Properties of the samples were checked regularly to ensure that the prepared solutions had constant properties prior to electrospinning. Formation ofCGTase/PVAmembrane by electrospinning The CGTase/PVA membrane was fabricated using the similar electrospinning system described in Saallah et al. (2016) with slight modifications on the experimental conditions. The system consisted of a high voltage supply, a stainless steel capillary (16 G, length × OD × ID = 80 × 1.61 × 1.25 mm) connected to a syringe pump and a grounded rotating collector covered with aluminium foil (Fig. 1(A)). The electrospinning modes were observed using a digital camera (Nikon, D 90) equipped with a macro lens (Nikon, AF Micro-Nikkor 105 mm f/2.8 D). Table 1 summarises the parameters used for the fabrication of the CGTase/PVA membrane. The membrane obtained was then dried overnight in a desiccator before being detached and subjected to glutaraldehyde vapour phase crosslinking vapour for 2.5 hours at an ambient temperature in the presence of hydrochloric acid (HCl) as a catalyst (3:1 ratio of GA: HCl). Scanning electron microscopy (SEM) analysis A Scanning Electron Microscope (JSM 6510, JEOL, Japan) was used to observe the morphology of the immobilised enzymes. The analysis was performed using an accelerating voltage of 10 kV. All samples were sputter-coated with gold (JFC 1200, JEOL, Japan) under vacuum before the SEM observations. The images obtained from t