Cellulase and Xylanase Immobilized on Chitosan Magnetic Particles for Application in Coconut Husk Hydrolysis

Cellulase and xylanase were covalently immobilized on chitosan magnetic particles. They were employed as catalysts to produce reducing sugar from coconut husk individually and simultaneously. Fourier Transform Infrared Spectroscopy (FT-IR) and Bradford’s protein analysis confirmed that the enzymes were covalently immobilized on the support. Cellulase from Aspergillus niger and Trichoderma reesei immobilized on chitosan magnetic microparticles yielded 0.352 g/L and 0.316 g/L of reducing sugar respectively, while immobilized xylanase from Trichoderma longibachterium yielded 0.432 g/L. The simultaneous use of cellulase and xylanase produced 0.8034 g/L of sugar, and the addition of glutaraldehyde as a cross-linking agent increased the amount of reducing sugar. Enzyme could maintain its activity at 91% for up to five cycles. Using nanosized particles resulted in a sugar yield of 0.49 g/l and 54.7 % of its activity maintained after five cycles. 

Cellulase; Chitosan magnetic particles; Coconut husk; Immobilization; Xylanase

Lignocellulose can be converted to reducing sugar for biofuels since it consists of cellulose, hemicellulose and lignin (Alftrén & Hobley, 2014). Coconut husk, which is an abundant lignocellulosic substrate in Indonesia, is a valuable substance as a future source of energy. However, the complex structure of lignocellulosic substrate is a major obstacle in its degradation process. The most widely known method to degrade lignocellulose is by chemical or physical pre-treatment, followed by hydrolysis. Enzymatic hydrolysis has been proven to improve the yields of sugar produced, has high selectivity, low energy costs and mild operating conditions (Zang et al., 2014). However, the expense of the enzyme and its specific activity for particular substrates are drawbacks in the application on a complex substrate such as lignocellulose (Han et al., 2018; Song et al., 2016).

The combination of xylanase and cellulase has numerous advantages; for example, xylanase can degrade the xylan which is attached to the cellulose surface and block the access of cellulase to cellulose (Mardawati et al., 2018). Since xylose is generated from xylan hydrolysis, the addition of xylanase not only makes the cellulose accessible to cellulase but at the same time also generates more sugar (Jia et al., 2015). The use of multiple enzymes simultaneously can also be achieved by utilizing crude enzyme. Crude enzyme from A. niger contains numerous enzymes, including cellulases and xylanases. Furthermore, utilizing crude enzyme will make the overall process more economical. The other strategy for employing the enzymes more economically is by reusing them in the hydrolysis reaction through immobilization. This also improves their stability and adaptability in various conditions, such as temperature and pH (Han et al., 2018).

Covalent binding is commonly used as an immobilization technique, since it restricts leaching of the enzyme because of the stable covalent bond with the support (Cheng-Kang & Au-Duong, 2018). Cellulase has been shown to be able to covalently immobilize on the surface of carrageenan (Yuan et al., 2016); polystyrene, polypropylene and polyethylene (Ahirwar et al., 2017); and chitosan (El-Ghaffar & Hashem, 2010; Manrich et al., 2010). In addition, xylanase has been shown to covalently immobilize on chitosan (Manrich et al., 2010); alginate beads (Jampala et al., 2017); and magnetic nanoparticles (Soozanipour et al., 2015; Shahrestani et al., 2016). As support for enzyme immobilization, chitosan, a natural polymeric support, has previously been reported to have the ability to support cellulase and xylanase since it has various functional group, and is inexpensive, inert, hydrophilic and biocompatible (Osuna et al., 2012). Micro-sized and nanosized chitosan magnetic particles were used since their larger surface area will resolve the mass transfer resistance between the immobilized enzyme and the substrate. Chitosan will act as a coating on the magnetic particles as it delivers a functional group for covalent binding. To enhance the binding, a cross-linking agent, glutaral dialdehyde (GDA), was also supplemented

Cellulase and xylanase were successfully immobilized covalently on chitosan magnetic particles. The FT-IR spectra convinced the covalent bond and the existence of GDA. The use of cellulase and xylanase collectively in the optimum ratio significantly enhanced the yield of sugar, and the immobilized cellulase and xylanase could be utilized several times without loss of activity. This simultaneous use of three enzymes immobilized on chitosan magnetic particles has economic potential for use as a biocatalyst in lignocellulose hydrolysis.

The authors are grateful to the Directorate General of Resources for Science, Technology and Higher Education, Ministry of Research, Technology and Higher Education of the Republic Indonesia (128/SP2H/PTNBH/DRPM/2018) for the funding provided for this study.

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