*2.1. Materials*

Sunflower oil for food use was obtained from a local market. Its standard fatty acids profile is: 63.5% linoleic acid, 24% oleic acid, 6.5% palmitic acid, 5% stearic acid and 2% of palmitoleic, with minor amounts of linolenic, behenic, and cetoleic acids. It exhibits a kinematic viscosity value of 32 mm2/s. The water content as determined by the Karl Fisher method was <0.08% and acidity degree 0.2%, expressed as oleic acid content. The palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid methyl esters used as standards were obtained from AccuStandard, Inc. 125 Market Street, New Haven, CT 06513, USA and methyl heptadecanoate was purchase from Sigma–Aldrich, San Luis, Misuri, Estados Unidos. All of them were chromatographically pure. Absolute ethanol and sodium hydroxide pure analytical compounds (99%) used were purchased from Panreac, Carrer del Garraf, 2, 08211 Castellar del Vallès, Barcelona, Spain. The Lipozyme RM IM, a *Rhizomucor miehei* lipase immobilized in beads from macroporous anion exchange resins was kindly provided by Novozymes A/S, Krogshøjvej 36, 2880 Bagsværd, Denmark.

#### *2.2. Immobilization of Rhizomucor Miehei Lipase on Inorganic Supports*

#### 2.2.1. Immobilization of Lipozyme RM IM by Physical Adsorption

The physical adsorption of Lipozyme RM IM has been studied on two different inorganic supports, a natural sepiolite (Tolsa S.A., Zaragoza, Spain) and a commercial silica gel. The sepiolite is a cheap natural silicate with high surface area (226 m2/g) and a fibrous structure. The theoretical formula of the unit cell is Si12O30Mg8(OH)6(H2O)4·8H2O, where the Si4+ and the Mg2+ can be partially substituted by Al3+, Fe2+ and alkaline ions. Each Mg atom completes its coordination with two molecules of water (Figure 1). The physical adsorption of lipases on sepiolite requires a previous acid demineralization step, in which the different metal hydroxides, i.e., Al, Fe, alkaline ions and mostly Mg, are extracted [28]. In this case, 40 g of the sepiolite was stirred at RT with a 1M solution of hydrochloric acid (HCl), until no presence of magnesium is detected (24 h).

Afterwards, the channels of the sepiolite can be filled with the lipase, producing its immobilization by physical adsorption. For its part, the immobilization on a silica gel does not require any activation treatment of the silica. Thus, the physical immobilization was carried out according to the following procedure. In a 25 mL round bottom flask, 0.2 g support, 0.01 g of Lipozyme RM IM lipase and 3.5 mL of absolute ethanol are mixed and stirred for 30 min at 700 rpm and 35 ◦C. As a matter of density of the solids, the corresponding amount of demineralized sepiolite employed as inorganic support was 1.0 g. The biocatalysts obtained by immobilization will be denoted as Lipo-Sep and Lipo-silica, either if Lipozyme was adsorbed on demineralized sepiolite or on silica-gel.

**Figure 1.** Structure of hydrated natural sepiolite.

2.2.2. Covalent Immobilization of Lipozyme RM IM on Sepiolite

Analogously to the physical adsorption, sepiolite cannot be directly employed as support for covalent immobilization of lipases. First of all, it has to be subjected to a surface activation process by sol-gel precipitation of AlPO4 on powdered solid sepiolite, in a proportion Sepiolite/AlPO4 80/20 [24]. Then, two different linkers, *p*-hydroxybenzaldehyde or 4-aminobenzylamine, were employed to interact with the Bronsted acid sites of the support, following a reported procedure [28], as it is shown in Schemes 2a and 3a,b. If 4-aminobenzylamine is employed to modify the functional groups in the surface, tereftaldicarboxaldehyde is also added to react with 4-aminobenzylamine and form the imines bond. Briefly, the immobilization of Lipozyme was carried out at room temperature, by introducing the functionalized inorganic solid (0.2 g) with the Lipozyme RM IM (0.01 g) in a reaction flask with 6 mL of ethanol, stirring in a refrigerator for 24 h. Finally, prior to its use, ethanol (6 mL) was added to the mixture and the solid, with the immobilized Lipozyme was separated by centrifugation. These supports will be denoted as 1-Sep/AlPO4 for the support modified with *p*-hydroxybenzaldehyde and 2-Sep/AlPO4 for the support modified with 4-aminobenzylamine and tereftaldicarboxaldehyde. Finally, the covalent immobilization of the lipase on the modified amorphous Sepiolite/AlPO4 can be achieved by chemical interaction of the organic groups available in the lipase either with the amino group or the aldehyde group, Schemes 2b and 3c. The final biocatalysts will be denoted as Lipo-1Sep/AlPO4 and Lipo-2Sep/AlPO4.

$$\begin{array}{c} \text{(a)} \end{array} \tag{1} \quad \text{@)} \text{H}\_{\text{H}} + \text{ (b)} \text{H} \xrightarrow{\text{O}^{\text{-}}} \bigvee \quad \text{---} \quad \text{@-} \bigvee \quad \text{-} \quad \text{@-} + \text{ (a)}$$

(b) 

**Scheme 2.** Solid phase synthesis, step by step, for the immobilization of the enzyme through the ε-amino groups of the lysine residues. In Step 1, surface OH groups of the supported Sepiolite/AlPO4 are activated by microwave heating with p-hydroxybenzaldehyde; before covalent immobilization of the enzyme through the ε-amino group of lysine residues (step 2).

**Scheme 3.** Solid phase synthesis, step by step, for the immobilization of the enzyme through the ε-amino groups of the lysine residues. In Step 1, surface OH groups of the supported AlPO4/sepiolite are activated by microwave heating with 4-aminobenzylamine; In Step 2, tereftaldicarboxaldehyde is reacted through imines bonds also obtained by microwave heating; Finally, in Step 3 covalent immobilization of the enzyme is obtained through the ε-amino group of lysine residues. It must be clarified that water is formed in steps (**a**)–(**c**).

Figure 2 shows the inorganic supports employed to immobilize the Lipozyme RM IM. As can be seen, despite the fact 1 g of sepiolite was employed instead of 0.2 g of the rest of the solids, the final volume was similar for all the supports.

**Figure 2.** Inorganic supports used to immobilize Lipozyme RM IM: (**a**) demineralized sepiolite, (**b**) silica gel, (**c**) AlPO4/sepiolite activated with 4-aminobenzylamine and terephthalato dicarboxaldehyde (**d**) AlPO4/sepiolite activated with *p*-hydroxybenzaldehyde.
