Immobilization of the cross-linked para-nitrobenzyl esterase of Bacillus subtilis aggregates onto magnetic beads Jae-Min Park a,1 , Mina Kim a,1 , Ju-Yong Park b , Dong-Hun Lee a , Kyu-Ho Lee c , Jiho Min b, **, Yang-Hoon Kim a, * a Department of Microbiology, Chungbuk National University, 410 Sungbong-Ro, Heungduk-Gu, Cheongju 361-763, South Korea b Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University, 664-14 1-Ga, Duckjin-Dong, Duckjin-Gu, Jeonju 561-156, South Korea c Department of Environmental Science, Hankuk University of Foreign Studies, Kyunggi-Do 449-791, South Korea 1. Introduction Para-nitrobenzyl esters serve as protecting groups of the intermediates in the manufacture of clinically important oral b- lactam antibiotics, in which de-esterification of the intermediates is required for a final product synthesis [1,2]. As some cases of PNB ester removal in organic solvent generate large amounts of zinc- containing waste material, an environmentally benign process, such as an enzymatic process that creates no metal intermediates, is highly desirable for large scale industrial applications [3,4]. Bacillus subtilis para-nitrobenzyl esterase (PNBE) has been known to catalyze the hydrolysis of the p-nitrobenzyl esters of various cephalosporin derived antibiotics to the corresponding free acid and p-nitrobenzyl alcohol [5,6]. Recently, enzyme immobilization techniques using a variety of materials as carrier solid supports have attracted increasing interest with improvements in activity and or stability of the enzyme without adverse affects on its catalytic activity [7]. While the covalent attachment of enzymes onto carriers results in high loading of enzymes and also showed that the enzyme activity and stability can be improved, since the maximal loading capacity with covalent attachment is limited by a monolayer coverage of covalently immobilized enzyme molecules it is anticipated that coatings of cross-linked enzyme aggregates (CLEA) could further improve the enzyme loading, thus increasing overall enzyme quality [8,9]. CLEA of various enzymes have been studied and reported including, but not limited to, CLEA of tyrosinase and penicillin acylase [9,10]. The use of magnetic particles for the immobilization may also improve overall enzyme quality with the following advantages: ease of recovery from a reaction mixture using a magnetic field, improved stability for repeated usage in continuous bioseparation processes, and a greater control over the catalytic process [11]. In this study, the enzyme PNBE was immobilized onto the amino groups of magnetic beads activated with glutaraldehyde (GA). The resulting covalently attached enzyme was also cross-linked with glutaraldehyde forming PNBE-CLEA. The immobilized PNBE-CLEA was character- ized and compared to the corresponding free enzyme with respect to pH and temperature. Thermal and storage stabilities of prepared enzymes under various experimental conditions were determined for further characterization. Lastly, the recycling activity of the PNBE-CLEA immobilized on magnetic beads was also measured. Process Biochemistry 45 (2010) 259–263 ARTICLE INFO Article history: Received 10 June 2009 Received in revised form 31 August 2009 Accepted 17 September 2009 Keywords: Bacillus subtilis Cross-linked enzyme aggregates Immobilization Magnetic beads Para-nitrobenzyl esterase ABSTRACT The para-nitrobenzyl esterase (PNBE), which was encoded by pnbA gene from Bacillus subtilis, was immobilized on amino-functionalized magnetic supports as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE-CLEA immobilized on the magnetic beads using glutaraldehyde as a coupling agent was 31.4 mg/g of beads with a 78% activity recovery after the immobilization. The performance of immobilized PNBE-CLEA was evaluated under various conditions. As compared to its free form, the optimal pH and temperature of PNBE-CLEA were 1 unit (pH 8.0) and 5 8C higher (45 8C), respectively. Under different temperature settings, the residual enzyme activity was highest for the PNBE-CLEA, followed by covalently fixed PNBE without further cross-linking and the free PNBE. During 40 days of storage pried, the PNBE-CLEA maintained more than 90% of its initial activity while the free PNBE maintained about 60% under the same condition. PNBE-CLEA also retained more than 80% activity after 30 reuses with 30 min of each reaction time, indicating stable reusability under aqueous medium. ß 2009 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +82 43 261 3575; fax: +82 43 264 9600. ** Co-corresponding author. Tel.: +82 63 270 2436; fax: +82 63 270 2306. E-mail addresses: jihomin@chonbuk.ac.kr (J. Min), kyh@chungbuk.ac.kr (Y.-H. Kim). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.09.015