Immobilization of pectinase from Leucoagaricus gongylophorus on magnetic particles Paulo Roberto Adalberto, a Francisco Jos e dos Santos, b Camilla Calemi Golfeto, a M^ onica Rosas Costa Iemma, a Dulce Helena Ferreira de Souza a and Quezia Bezerra Cass * a Received 23rd May 2012, Accepted 17th August 2012 DOI: 10.1039/c2an35682a Polygalacturonases (EC 3.2.1.15) hydrolyze the a-1,4-glycosidic linkages in polygalacturonic acid chains. The interest on specific inhibitors of pectinase and the versatility of magnetic support for enzyme immobilization endorsed the preparation of an immobilized enzyme reactor (IMER). This work presents the synthesis of CoFe 2 O 4 amino-derivatives, which was employed as the support for the immobilization of pectinases from Leucoagaricus gongylophorus. Amino-functionalized CoFe 2 O 4 was obtained from glyceryl-derivatized CoFe 2 O 4 and was characterized by infrared spectroscopy and electronic microscopy. The immobilized enzyme maintained the same thermal, chemical and kinetic behaviour of the free enzyme (T opt 60  C; pH opt 5.0; K app M ¼ 0.5 mg min 1 ; V app max z 5.0 mmol min 1 mL 1 ). The straightforward synthesis of CoFe 2 O 4 derivatives and the efficiency of immobilization offer wide perspectives for the use of the developed new IMER. Introduction The symbiotic relationship of Atta sexdens with basidiomycete Leucoagaricus gongylophorus is an important neotropical herbivorous system. 1,2 Ants manure a fungal crop in subterra- nean gardens within their colonies. The grown fungus provides nutrients in the form of specialized hyphal swellings called gongylidia, which contain a tasty mixture of carbohydrates, amino acids, proteins, lipids, and vitamins. 2 Such fungus is thought to be the sole source of nutrient for the queens, larvae and alates, whilst supplementing the diet of adult workers. 3 The colonies of leaf-cutting ants have been likened to a super- organism whose ecological efficiency has resulted in a major human agricultural pest, liable for significant economic losses in South American agriculture. 1,4 L. gongylophorus efficiently hydrolyzes pectin. Pectinases are produced by symbiotic fungus cultures higher than amylase (7 times), xylanase (24 times), CMCase (38 times), and cellulase (200 times). 5 The pectolytic enzymes are broadly known as pec- tinases, and depending on their mode of action are classified as hydrolases, 6,7 esterases 8,9 and lyases. 10 These enzymes, recently described in animals, 11,12 are commonly produced by bacteria 13,14 and fungi 15,16 such as L. gongylophorus. 4,5 Pectin, rather than other polysaccharides, seems to be the main leaf material to support the fungus garden. 3 Recent reports have indicated a central role of pectinase in mold nutrition. 4,5 Therefore, a suit- able route for a biorational colony control would be disruption of pectin degradation. Pectinases have widespread application 17–21 and the techno- logical relevance of this enzyme complex has created the motif for the development of pectinase bioreactors in a variety of supports. 22–24 Because of their advantages over soluble enzymes, immobilized enzyme reactors (IMERs) 25,26 have been helpful in practical and theoretical enzymatic studies. 27,28 However, the task of continuous digestion of pectic substances by immobilized pectinase suffers constraints due either to losses of activity, which take place during immobilization, 22,23 or to rheological limita- tions in the operation of the bioreactor. Super paramagnetic iron oxide nanoparticles (SPIONs) present wide applicability. 29–31 Technology focused on the synthesis of functionalized nanostructured magnetic materials has undergone great development, and new materials have been described as supports for enzyme immobilization. 32,33 In this context, magnetic IMERs with pectolytic activity emerge as a promising alternative. Throughout this work, a cobalt ferrite amino-derivative is described as the support for the immobili- zation of L. gongylophorus polygalacturonase. The results herein reported present cobalt ferrite as a feasible material for this purpose. Experimental Chemicals Yeast Nitrogen Base (YNB), 3,5-dinitrosalicylic acid (DNS), and polygalacturonic acid were purchased from Sigma (St Louis, a Departamento de Qu ımica, Universidade Federal de S~ ao Carlos, Cx. Postal 676, S~ ao Carlos, 13565-905, S~ ao Paulo, Brazil. E-mail: quezia@ pq.cnpq.br; Fax: +55-16-3351-8350; +55-16-3351-8087 b Procell Biologics, Avenida Comendador Alfredo Maffei, 4001, 13561-270, S~ ao Carlos, SP, Brazil This journal is ª The Royal Society of Chemistry 2012 Analyst, 2012, 137, 4855–4859 | 4855 Dynamic Article Links C < Analyst Cite this: Analyst, 2012, 137, 4855 www.rsc.org/analyst PAPER Downloaded by UNIVERSIDADE FEDERAL SAO CARLOS on 05 February 2013 Published on 17 August 2012 on http://pubs.rsc.org | doi:10.1039/C2AN35682A View Article Online / Journal Homepage / Table of Contents for this issue