Novel cross-linked enzyme aggregates of levanase from Bacillus lehensis G1 for short-chain fructooligosaccharides synthesis: Developmental, physicochemical, kinetic and thermodynamic properties Noor Hidayah Abd Rahman a , Nardiah Rizwana Jaafar a , Abdul Munir Abdul Murad b , Farah Diba Abu Bakar b , Nur Arbainah Shamsul Annuar c , Rosli Md Illias a,c,d, ⁎ a School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81300, Skudai, Johor Bahru, Johor Darul Takzim, Malaysia b School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Malaysia c Health and Wellness Research Alliance, Universiti Teknologi Malaysia, 81310, UTM Skudai, Johor Bahru, Malaysia d Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia abstract article info Article history: Received 2 March 2020 Received in revised form 28 April 2020 Accepted 29 April 2020 Available online 04 May 2020 Keywords: Levanase Cross-linked enzyme aggregates Fructooligosaccharides Short-chain fructooligosaccharides (scFOSs) can be produced from the levan hydrolysis using levanase. Levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to scFOSs. However, the use of free levanase presents a lack of stability and reusability, thus hindering the synthesis of scFOSs for continuous re- actions. Here, CLEAs for rlevblg1 were prepared and characterized. Cross-linked levanase aggregates using glutar- aldehyde (CLLAs-ga) and bovine albumin serum (CLLAs-ga-bsa) showed the best activity recovery of 92.8% and 121.2%, respectively. The optimum temperature of CLLAs-ga and CLLAs-ga-bsa was increased to 35 °C and 40 °C, respectively, from its free rlevblg1 (30 °C). At high temperature (50 °C), the half-life of CLLAs-ga-bsa was higher than that of free rlevblg1 and CLLAs-ga. Both CLLAs exhibited higher stability at pH 9 and pH 10. Hyperactivation of CLLAs-ga-bsa was achieved with an effectiveness factor of more than 1 and with improved catalytic efficiency. After 3 h reaction, CLLAs-ga-bsa produced the highest total scFOSs yield of 35.4% and total sugar of 60.4% per gram levan. Finally, the reusability of CLLAs for 8 cycles with more than 50% activity retained makes them as a potential synthetic catalyst to be explored for scFOSs synthesis. © 2020 Elsevier B.V. All rights reserved. 1. Introduction Enzymes are well known as efficient natural catalysts that exhibit great potential in many applications, ranging from food to pharmaceu- tical industries. Immobilization of enzymes is a preferred approach to increase enzyme activity, stability and thermostability. Compared to free enzymes, immobilized enzymes have several benefits, including high enzyme stability, volumetric and specific activity, and improved reusability and selectivity [1]. Common techniques for enzyme immobi- lization are adsorption, covalent binding, affinity binding, entrapment, and cross-linking [2]. Enzyme immobilization typically involves binding to a support or encapsulation in an inert support, which offer high oper- ational stability. However, some weaknesses of carrier-bound enzymes are low product formation due to a high amount of carrier, loss of en- zyme activity caused by high enzyme loading on the carrier and expen- sive support materials [3,4]. Carrier-free immobilized enzymes offer high productivity and low cost because the support material is not required [5]. Carrier-free forms, such as CLEAs, have several advantages over other immobilized enzymes, including the use of partially purified enzymes and the ability to combine two or more enzymes during immobilization, and the ob- tained CLEAs can also easily be separated by centrifugation [6]. In CLEAs technology, both the purification and immobilization of enzymes are combined into one single operation [4]. Although CLEAs is a versatile method, this technology has some limitations as well. For instance, the compact supramolecular structures cause a mass transfer limitation [7]. Therefore, some improvements are required to minimize the prob- lems associated with CLEAs technology. CLEAs were prepared by phys- ically precipitating the enzyme molecules using either non-ionic polymers, organic solvents or salts. Then, the enzymes were cross- linked using a bifunctional cross-linker, such as glutaraldehyde (GA) [8,9]. However, a low lysine content in some enzymes may reduce the cross-linking efficiency [10,11]. This problem can be solved by adding a polymer containing primary amino acids (polyethyleneimine) or pro- tein feeder (BSA) into the enzyme solution as a source of protein and amino groups to improve the cross-linking [12]. CLEAs preparation can be facilitated by the addition of BSA in the case of low protein International Journal of Biological Macromolecules 159 (2020) 577–589 ⁎ Corresponding author at: Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor Bahru, Johor Darul Takzim, Malaysia. E-mail address: r-rosli@utm.my (R. Md Illias). https://doi.org/10.1016/j.ijbiomac.2020.04.262 0141-8130/© 2020 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac