Journal of Molecular Catalysis B: Enzymatic 134 (2016) 9–15 Contents lists available at ScienceDirect Journal of Molecular Catalysis B: Enzymatic j ourna l ho me pa ge: www.elsevier.com/locate/molcatb Biocatalytic epoxidation of -pinene to oxy-derivatives over cross-linked lipase aggregates Madalina Tudorache a , Andreea Gheorghe a , Ana S. Viana b , Vasile I. Parvulescu a,∗ a University of Bucharest, Department of Organic Chemistry, Biochemistry and Catalysis, Bd. Regina Elisabeta 4-12, Bucharest 030016, Romania b Centro de Química e Bioquímica, Fcauldade de Ciências, Universidade de Lisboa, Campo Grande 1749-016 Lisboa, Portugal a r t i c l e i n f o Article history: Received 10 May 2016 Received in revised form 31 July 2016 Accepted 11 September 2016 Available online 12 September 2016 Keywords: Cross-linked lipase aggregates -Pinene Epoxidation Green process Magnetic particles Enzyme secondary-structure a b s t r a c t Lipase-based cross-linked aggregates were investigated for a non-specific reaction, i.e. the epoxidation of -pinene to its oxygenated derivatives. The activity of the biocatalysts has been evaluated in a green context, i.e. ethyl acetate as both acetate-supplier and organic solvent with H 2 O 2 /UHP/TBHP as oxidant. Screening of the lipase sources indicated Aspergillus niger lipase as the most efficient biocatalyst for this reaction. Different immobilization protocols ((i) cross-linked enzyme aggregates (CLEA), (ii) cross- linked enzyme aggregates onto magnetic particles (CLEMPA) and (iii) covalent immobilized enzyme (CIE) onto magnetic particles (MP)) were evaluated considering the activity as main parameter. Thus, CLEA and CLEMPA afforded better epoxidation yields of -pinene towards CIE. The investigated biocatalytic systems allowed to transform -pinene into oxigenated derivatives with industrial and commercial appli- cations (e.g. -pinene oxide, camphene, pinanediol and camphonelic aldehyde). FTIR investigations on the biocatalysts revealed the effects of the immobilization protocol on the enzyme secondary-structure. Additionally, textural characterizations were performed by Scanning Electron Microscopy (SEM), Trans- mission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) analysis. © 2016 Elsevier B.V. All rights reserved. 1. Introduction -Pinene is the main component of the monoterpene fraction in some essential oils (e.g. mastic oil) and turpentine, which is a paper and pulp industry residue available in bulk quantities at a low price. Approximately 350,000 tons of turpentine are produced annually worldwide [1]. Commonly, its applicability is limited to fuels for the recovery boilers, although it could be effectively utilized in other processes on site. On the other side, -pinene is considered as a renewable raw resource with a great potential for the production of pharmaceuticals, agrochemicals and other fine chemicals [2]. Epoxidation may provide an efficient route to achieve the sus- tainability in the valorization of -pinene and its derivatives (e.g. -pinene oxide, verbenone, verbenol, campholenal, pinanediol, camphene, etc). It affords the introduction of an oxygen atom into the olefinic terpene skeletons [3] leading to oxidation prod- ucts that are usually found in the plant kingdom [4,5], but at low concentration. Molecules incorporating these structures exhibit important flavor/fragrance (sandalore (Givaudan) and polysantol ∗ Corresponding author. E-mail addresses: v parvulescu@yahoo.com, vasile.parvulescu@g.unibuc.ro (V.I. Parvulescu). (Firmenich)) and anti-cancinogenic behavior [6]. Also, verbenol is a well-known aggregation pheromone of the bark beetle that is uti- lized in the forestall pest control. However, to achieve them in an efficient way new challenging synthetic routes are necessary [7]. Biocatalytic methods offer a fair balance between efficiency, environmental friendly and cost aspects [8]. -Pinene treated with an oxidation agent (e.g. hydrogen peroxide – H 2 O 2 or ureea- hydrogen peroxide – UHP) and carboxylic acids in an organic medium using lipases (e.g. Candida antarctica B), chloroperoxidases (e.g. Caldariomyces fumago) or laccases (e.g. Trametes versicolor, Trametes hirsuta or Botrytis cinerea) as biocatalysts led to the desired epo-oxidation products [9–12]. Epoxidation is achieved under mild conditions by employing peroxy-carboxylic acids pro- duced continuously in situ by lipase-catalyzed perhydrolysis of the corresponding carboxylic acid [10]. Additionally, derivatives of the carboxylic acids, such as ethyl acetate, phenyl acetate and dimethyl carbonate were also used as a perhydrolysis substrate and solvent [13–15]. Noteworthy, for lipase, the biocatalyst performances have even been improved by the immobilization of the enzyme either in organic-modified clay or in smectite nanoclay [16,17]. Thus, the immobilization of the lipase onto the hydrophobic surfaces resulted as a promising strategy for the preparation of an efficient biocata- lyst for the epoxidation of -pinene. http://dx.doi.org/10.1016/j.molcatb.2016.09.009 1381-1177/© 2016 Elsevier B.V. All rights reserved.