Improving the esterification activity of Pseudomonas fluorescens and Burkholderia cepacia lipases via cross-linked cyclodextrin immobilization† Ivaldo I. Junior, a Emanuela Calcio Gaudino, b Katia Martina, b Giancarlo Cravotto, b Rafael Luque c and Rodrigo O. M. A. de Souza * a The search for a new, efficient and sustainable matrix for biocatalyst immobilization is a growing area in biotechnology. Our proposed approach deals with the utilization of solid cross-linked b-cyclodextrin as supports for enzyme immobilization. Results obtained in terms of enzyme activity and thermal stability of novel immobilised materials have been found to remarkably improve those obtained using commercial immobilized enzymes in esterification reactions (e.g., monostearin synthesis). Introduction Lipases are triacylglycerol acyl hydrolytic enzymes that have found applications as hydrolytic, esterication and trans- esterication biocatalysts. 1 Upon immobilization, supported enzymes can further provide an easily separable and reusable system (together with enhanced product recovery) which boasts of enhanced resistance to deactivation as compared to free enzymes. 2–4 Immobilization has several implications when generating increasingly stable biocatalysts compatible with continuous processing technologies. 5 Various strategies to immobilize enzymes on a number of supports have been reported. These range from the more extended and widely employed physical methods (e.g., adsorption, entrapping and/ or electrostatic immobilization) to chemical protocols including covalent immobilization. 4 Cyclodextrins (CDs) are a class of macrocyclic structures comprising (a-1,4)-linked b-D-glucopyranose units that possess a relatively non-polar cavity. The internal hydrophobic cavity and the external hydrophilic rim of CDs render them as ideal candidates for modelling host–guest interactions, 6–8 drug delivery, 9 catalysis, 10,11 chiral separation 12 and molecular recognition in self-assembled monolayers. 12 b-CD has proven to be a good enzyme support, with a number of contributions reporting signicant efficiencies in promoting catalytic processes both in water and organic solvents. The addition of b- CD to solutions containing lipases has been reported to enhance reaction rates as well as enantioselectivity and lipase stability. 13,14 Furthermore, CD immobilized Candida rugosa lipase offered important advantages (e.g., thermal stability) with respect to its free enzyme counterparts. 14–17 Biodiesel comprising alkyl esters of long chain fatty acids has been proposed as a suitable bio-derived replacement for petroleum diesel as a means to reduce gaseous pollutant emissions including CO, SO x , and organic compounds. 18 The properties of biodiesel are similar to those of petroleum-based diesel, allowing its use either as a substitute for diesel fuel or more commonly in fuel blends. Several strategies for biodiesel production have been reported in recent years and include homogeneous/heterogeneous 19 and biocatalytic triacylglyceride transesterication protocols, with most commonly extended low molecular weight alcohols. 20 Mono- and diacylglycerols (MAG and DAG) are well-known biodegradable, biocompatible, nontoxic and nonionic surfac- tants widely used in food, pharmaceutical and industrial applications. 21 Constituted of a hydrophobic and hydrophilic part, the hydrophobic part consists of fatty acids (i.e., lauric, myristic, palmitic, oleic and stearic acid), whereas the hydro- philic part can be formed of glycerol or one of its ester deriva- tives of organic acids including lactic, citric, acetic or tartaric acid. MAG and DAGs are commonly produced on the basis of the batch alkaline catalyzed chemical glycerolysis of natural oil and fats at high temperatures (220–250  C) and elevated pres- sures under nitrogen atmosphere. Besides the high energy consumption of their preparation, high temperatures are responsible for low yields (<50%) and poor product quality which leads to dark-coloured and burned-tasting product a Departamento de Qu´ ımica Orgˆ anica, Federal University of Rio de Janeiro, Brazil. E-mail: souzarod21@gmail.com b Dipartimento di Scienza e Tecnologia del Farmaco, University of Turin, Via P. Giuria 9, 10125 Torino, Italy c Departamento de Qu´ ımica Org´ anica Universidad de C´ ordoba, Edicio Marie Curie (C- 3), Campus de Rabanales, Ctra Nnal IV-A, Km 396, E14014, Cordoba, Spain. E-mail: q62alsor@uco.es † Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra03797a Cite this: RSC Adv. , 2014, 4, 45772 Received 2nd June 2014 Accepted 1st July 2014 DOI: 10.1039/c4ra03797a www.rsc.org/advances 45772 | RSC Adv., 2014, 4, 45772–45777 This journal is © The Royal Society of Chemistry 2014 RSC Advances PAPER Published on 01 July 2014. Downloaded by UNIV FEDERAL DO RIO DE JANEIRO on 08/01/2015 01:47:23. View Article Online View Journal | View Issue