Carbohydrate Polymers 82 (2010) 600–604 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Immobilized cellulase by polyvinyl alcohol/Fe 2 O 3 magnetic nanoparticle to degrade microcrystalline cellulose Hongdong Liao a , Ding Chen b , Li Yuan b , Mang Zheng a , Yonghua Zhu a,∗∗ , Xuanming Liu a,∗ a State Key Laboratory of Chemo/Biosensing and Chemometrics, Department of Life Science and Technology, Hunan University, Changsha, 410082, PR China b College of Materials Science and Engineering, Hunan University, Changsha, 410082, PR China article info Article history: Received 21 April 2010 Accepted 12 May 2010 Available online 21 May 2010 Keywords: Cellulase immobilization Polyvinyl alcohol/Fe2O3 nanoparticle Microcrystalline cellulose Degradation Ball milling abstract In an attempt to improve the enzymatic efficiency in ball mill, a novel immobilized cellulase on polyvinyl alcohol/Fe 2 O 3 magnetic nanoparticle with high activity was synthesized and characterized by transmis- sion electron microscopy, Zetasizer, Fourier transform infrared (FTIR) spectroscopy and vibrating sample magnetometry. It was found that the immobilized cellulase was a kind of spherical complex with approx- imate 270 nm, 4.87 A m 2 kg -1 , and mainly constructed by the accumulation of 10 nm Fe 2 O 3 nanoparticles with the diffused polymer. When the immobilized cellulase was applied to degrade microcrystalline cel- lulose by combining with wet ball milling, the yielded glucose was 1.89 mg mL -1 , at least three times than the sum of individual yield. The immobilized cellulose maintained 40% activity even after four cycles of reuse. From these results, it can be concluded that the immobilization of cellulase with wet ball milling is a novel method to significantly improve the efficiency of cellulose conversion. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Cellulose can be specifically hydrolyzed into oligomeric or sol- uble sugar by cellulase and is thus expected as a renewable source of fuels and chemicals. But the susceptibility of cellulose to enzyme degradation was limited for the structural complexity, particularly for hydrogen-bonded and ordered microcrystalline cellulose (MCC) (Mansfield & Meder, 2003). As a result, the complete conversion usually required large cellulase loadings, which increase cost and accordingly limited its economic feasibility. Several methods have been developed to improve the efficiency of enzyme degradation. Among of them, ball milling has been rec- ognized as one of the most effective treatments for its contribution to reducing particle size and crystallinity of cellulose (Hendriks & Zeeman, 2009). The rate and extent of saccharification of cellu- lose could be improved by simultaneous ball milling and enzyme hydrolysis (Mais, Esteghlalian, Saddler, & Mansfield, 2002). How- ever, our research showed that severe wet ball milling greatly destroyed the activity of cellulose (Zhou et al., 2010). Many doc- uments have reported that the immobilization on nanoparticles can efficiently maintain enzyme activity and get some applica- ble performance such as low cost and high stability (Dincer & Telefoncu, 2007; Ho, Mao, Gu, & Li, 2008; Li, Yoshimoto, Fukunaga, & ∗ Corresponding author. Tel.: +86 731 88821721; fax: +86 731 88821721. ∗∗ Corresponding author. Tel.: +86 731 88821565; fax: +86 731 88822606. E-mail addresses: zyh20@hotmail.com (Y. Zhu), xml05@126.com (X. Liu). Nakao, 2007; Liao et al. 2008; Wu, Yuan, & Sheng, 2005), however, there was rare report about the technology of nano-immobilized cellulase combining with mechanical pretreatments for effective hydrolysis of cellulose, not to mention MCC with high-crystallinity property. In this work, a novel magnetic nanoparticle immobilized with cellulase under microemulsion system (ICM) was synthesized and applied to improve the hydrolysis efficiency of MCC combing with ball milling. 2. Experimental 2.1. Materials MCC and cellulase (R-10) were purchased from Dingguo Biologic Technique Company (Beijing, China). PVA with 1750 ± 50 degree of polymerization and 98% of degree of hydrolysis was purchased by Yufeng Chemical Reagent Glass Instrument Co. Ltd. (Changsha, China). All other agents used were of analytic grade. 2.2. Immobilized cellulase by polyvinyl alcohol/Fe 2 O 3 magnetic nanoparticle The magnetic Fe 2 O 3 nanoparticle was prepared as described in the literature (Chastellain, Petri, & Hofmann, 2004). PVA/Fe 2 O 3 nanoparticle-bound immobilized cellulase was prepared by cycli- cally freezing and thawing procedure under microemulsion system. PVA (20 mg) was dissolved in 4 mL sodium citric acid buffer (0.05 M, 0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbpol.2010.05.021