Lipases (triacylglycerol acyl hydrolases, EC 3.1.1.3) are widely used enzymes in biocatalysts that have essential roles in organic chemistry since they have a broad specificity and can react with various substrates [1] . The solubility of lipases in water is very poor, that is why the reaction usually occurs at an aqueous-organic interface at which they perform better catalytic activity than at aqueous solution [2, 3] . Lipases can hydrolyze triglycerides at lipid-water interfaces since the hydrolytic reaction is reversible in a nonaqueous medium; they can also catalyze the formation of acylglycerols from glycerol and free fatty acids [4]. Applications of lipases in different industries have been steadily increasing, especially during the last few years. They have been used particularly in the food industry, in the pharmaceutical industry, in the pulp and paper industries, and in the chemical industries [5].
Enantioselectivity of the lipases has significant enzyme property for many applications, such as preparation of optically pure alcohols and esters, with a special interest in the pharmaceutical industry [6]. In order to produce bioactive compounds such as natural products, agrochemicals, and pharmaceuticals, enantiopure alcohols are significant constituents [7]. In a biological system, two enantiomers of a chiral compound may exhibit very distinct behaviors. Indeed, when a racemic mixture is used as a chiral drug, whose enantiomers display different biological effects, generally only one of the enantiomers is biologically active, whereas the other is either inactive or produces side-effects, including toxicity. For this reason, there has been a huge demand to produce optically pure drugs in the pharmaceutical industry [8–12]. Lipases generally exhibit better enantioselectivity in the kinetic resolution of secondary alcohols than in the kinetic resolution of primary or tertiary ones [13]. Candida antarctica lipase B (Cal-B) is a serine hydrolase and the most commonly used lipase compared with others [14]. Both its free and immobilized forms show very good activity and stability towards kinetic resolution of secondary alcohols [15]. Various studies have been published about the lipase-catalyzed kinetic resolution of 1-phenyl ethanol. In some of these studies, Cal-B was immobilized on different support materials or other lipases that have been used as biocatalysts. For example, in a recent study by Abahazi et al. [16], Cal-B was covalently immobilized onto bisepoxide-activated amino alkyl polymer resins, and then it was used to catalyze the kinetic resolution of 1-phenylethanol. They achieved high enantiomeric excess (ee > 99%) and enantioselectivity (E > 200) values. Also, the results represented that the immobilized enzyme can be used repeatedly and show long-term stability. In another work by Souza et al. [17], commercially available Cal-B (immobilized on a macroporous polyacrylate resin) was chosen as a biocatalyst for the kinetic resolution of 1-phenylethanol and high enantioselectivity (ee = 98, E= 200) was obtained.
In order to enhance the enantioselectivity and stability of an enzyme, there are different biochemical techniques. The most commonly used of these techniques is the immobilization of enzymes. A proper immobilization method can alter the selectivity of lipases and improve catalytic activity and biochemical properties [5, 18]. The use of immobilized enzymes as biocatalysts has drawn attention in recent years. They provide several advantages over their soluble forms; for example, they can be separated easily from the mixture, and they are generally more stable, enable production of pure products, have the ability to reuse the enzyme [19], increase storage stability, protect tertiary structure of enzyme [20], and minimize the inhibition by any environmental conditions [21].
Enzymes can be immobilized onto supports covalently via chemical interactions between active groups of the supports and amino acid groups of the enzyme molecules [13]. The immobilization of enzymes with covalent binding generally cause denaturation of enzyme and the loss of catalytic activity due to the limitation of conformational changes and generation of rigid structures along immobilization. On the other hand, the stability of enzymes will be increased considerably, and the leakage of the enzyme could be decreased [22]. Alternatively, it may be beneficial to utilize some additives such as substrates, ligands, and substrate analogues in order to protect the active center of the enzyme during the immobilization process. Therefore, when the active center of the enzyme captured by a substrate molecule, the conformational change of the enzyme can be reduced, and its catalytic activity can be protected during covalent binding [23]. During the covalent attachment procedure, the most critical point is the preference of a suitable solid support. Support materials should have high surface area and low cost; in addition, they should be environmentally friendly and nontoxic [24]. The interaction between enzyme and support material is a crucial factor since the properties of the support can modify the activity of the biocatalyst and enzyme loading [25].
There are many supports used for lipase immobilization, for example, a variety of nanostructures that provide low mass-transfer resistance and a large surface area, which upgrade binding efficiency, increase interaction with the enzyme, and develop the longterm storage and reusing of the enzyme [26]. The utilization of magnetite ( Fe3O4) as a nanocarrier for the covalent immobilization of enzymes is notably promising since it has high surface area [27–29]; it allows recyclability and repeatability of biocatalysts having long-term stability [30], allows satisfactory separation under external magnetic fields [31], and supplies high enzyme loading per unit mass of support [32]. Another support material that can be used as an immobilization matrix is GO, which has attracted great attention on account of its definite chemical and surface properties [33]. Various functional groups such as C=O, -OH, -O- and -COOH in the structure of GO permit it to be hydrophilic and disperse easily in water and organic solvents. Thus, a suitable interaction occurs between graphene oxide and enzyme molecules [34, 35]. The combination of Fe3O4 nanoparticles with GOnanosheets offers another promising support material. Due to the very high surface area and strong interaction of GO nanosheets with Fe2+/Fe3+ ions, Fe3O4 nanoparticles with small particle size and large surface area can easily prepared, separately. In addition, the Fe3O4 nanoparticles on GO nanosheets prevent the aggregation of the GO nanosheets and maintain the large surface area of the support material. Moreover, because of the excellent magnetic properties of Fe3O4nanoparticles, the GO/Fe3O4 nanocomposite can separated easily with an external magnetic field. In the literature, there are some studies about the immobilization of different kind of lipases onto GO/Fe3O4 nanocomposite [36–38]. Xie and Huang [37] immobilized Candida rugosa lipase, and they obtained activity recovery as 64.9%. The immobilized enzyme was used to catalyze the transesterification of soybean oil with methanol.
In this study, it is aimed to increase the enzymatic activity and enantioselectivity of Cal-B by immobilizing it onto Fe3O4 nanoparticles, GO nanosheets and GO/ Fe3O4 nanocomposite. For this purpose, GO, Fe3O4, and GO/ Fe3O4 nanostructures were firstly prepared; subsequently, they were immobilized by covalent attachment of Cal-B and finally, the different immobilized samples were used as biocatalysts in the enantioselective transesterification reaction of (R, S)-1-phenylethanol for the first time in the literature. In addition, the reusability of the immobilized enzymes was examined; high catalytic activities of the enzymes were verified.