Immobilization of lipases from Rhizomucor miehei and Thermomyces lanuginosus by adsorption on variously grafted silica gels

Twenty variously surface-modified mesoporous silica gels were studied as carriers for immobilization by hydrophobic adsorption of the lipases from Rhizomucor miehei (RmL) and Thermomyces lanuginosus (TlL). Several of the surfacemodified silica gels studied proved to be advantageous supports for RmL and TlL resulting in novel biocatalysts of high activity and enantioselectivity in the kinetic resolution of racemic 1-phenylethanol rac-1. The fact that it were different grafting methods which led to the most efficient supports for RmL and TlL indicated that the selection of optimal support for the immobilization of a particular lipase cannot be predicted.


Introduction
Biocatalysis with isolated enzymes can simplify and improve organic syntheses even on an industrial scale [1].Lipases (EC 3.1.1.3)are essential in the digestion, transport and processing of lipids (e.g.triglycerides, fats, oils) in most, if not all, living organisms.Lipases are versatile biocatalysts which can provide regio-and enantioselectivity in a wide range of reactions [2,3].Consequently, they are one of the most extensively utilized biocatalysts in organic synthesis [4,5].
Immobilization can enhance activity, thermal and operational stability, and also reusability of enzymes which are essential advantages in industrial applications [6,7].Among the many available immobilization methods, including adsorption, covalent attachment to solid supports and entrapment within polymers [8][9][10][11], hydrophobic adsorption onto suitable carriers was found to be an efficient way not only for immobilization but also for the separation of lipases [12].Lipases immobilized by various procedures proved to be useful both in batch mode and in continuous-flow biotransformations [13][14][15][16].
Since we have found that surface-modified silica gels proved to be efficient supports for adsorptive immobilization of lipases A and B from Candida antarctica (CaLA and CaLB) and those from Pseudozyma aphidis (PaL) [12], Pseudomonas fluorescens (Lipase AK) and Burkholderia cepacia (Lipase PS) [17], we were prompted to extend our studies to lipases from the thermophilic filamentous fungi Rhizomucor miehei (RmL) and Thermomyces lanuginosus (TlL) supported by variously grafted silica gels as well.
2 Experimental section 2.1 Chemicals and enzymes Racemic 1-phenylethanol rac-1 and vinyl acetate were obtained from Sigma-Aldrich.All solvents of analytical grade or higher were products of Merck.Solutions of lipases from Rhizomucor miehei (>20000 U g 1 , Cat. no: L4277) and Thermomyces lanuginosus (>100000 U g 1 , Cat. no: L0777) were purchased from Sigma-Aldrich.Davisil R 250 [40-63 µm] was the product of W. R. Grace & Co. Etched silica gel was prepared from Davisil R 250 by shaking in ethanol containing cc.NH 4 OH (0.5 v/v%) for 4 days.Surface functionalized silica gels were the products of SynBiocat Ltd.

Adsorption of enzymes on surface modified silica gels
Lipase solution (RmL or TlL; 1.25 mL) was dissolved in Tris buffer (11.25 mL, 100 mM, pH=7.5, ionic strength controlled with NaCl) then surface functionalized silica gel (250 mg, as indicated in Tab. 1 and Tab. 2) was added to the solution.The resulting suspension was shaken at 400 rpm and 4˚C for 18 h.The supported lipase was filtered off with a glass filter (G4), washed with 2-propanol (5 mL, twice), hexane (5 mL), dried at room temperature (2 h) and stored at 4˚C.

Results
Modification of the surface of a mesoporous silica gel (Davisil R 250; particle size: 40-63 µm, pore diameter: 25 nm) with a selection of mono-and disubstituted alkoxysilanes as reported earlier [12] enabled us to prepare nineteen mechanically stable silica based supports of varying hydrophobicity.
Although lipases from Rhizomucor miehei and Thermomyces lanuginosus are commercially available in immobilized forms [e.g. the Cross-Linked Enzyme Aggregate (CLEA) or Immobead 150 variants of TlL or RmL from Sigma-Aldrich], studies with variously immobilized forms of CaLB [13,17] indicated that hydrophobic adsorption on a modified surface might result in useful biocatalysts.Therefore, the adsorptive immobilization of lipases from Rhizomucor miehei and Thermomyces lanuginosus (RmL and TlL) was performed as described earlier for CaLA and CaLB [12] as well as for Lipase AK and Lipase PS [17].Soluble RmL or TlL was diluted in TRIS buffer (pH=7.5)then various grafted silica supports were added to the lipase solution and the resulting suspensions were shaken at 4˚C for 18 h.Activity and selectivity of the immobilized biocatalysts were tested in the enantiomer selective acylation of racemic 1phenylethanol rac-1 (Fig. 1) and characterized by the specific biocatalyst activity (U B ), conversion (c) of the substrate, and enantiomeric excess (ee) and enantiomeric ratio (E) of the product.Conversion (c) and enantiomeric excess (ee) in the kinetic resolutions of rac-1 were determined by GC.Enantiomeric ratio (E) for the reactions was calculated from c and ee (R)−2 [18].Specific biocatalyst activity (U B ) of the various biocatalysts in the acylation of rac-1 was calculated using the equation U B = n P /(t × m B ) (where n P [µmol] is the amount of the product (R)-2, t [min] is the reaction time and m B [g] is the mass of the applied biocatalyst).

Studies with immobilized Rhizomucor miehei lipase (RmL)
First, biocatalysts prepared by hydrophobic adsorption of RmL on mesoporous silica gel, etched silica gel and nineteen variously grafted silica gel supports were investigated (Tab.1).Because productivity and selectivity differences were most pronounced at low conversions, values after 1 h acylation of racemic 1-phenylethanol rac-1 were used for comparison (Tab.1).

Studies with immobilized Thermomyces lanuginosus lipase (TlL)
Next, the hydrophobic adsorption of TlL on mesoporous silica gel, etched silica gel and further nineteen variously grafted silica gel supports was studied (Tab.2).Similarly to the RmL biocatalysts the catalytic properties of our TlL preparations were compared at low conversions.Thus, the bioconversions of racemic 1-phenylethanol rac-1 after 1 h reaction time were evaluated (Tab.2).

Discussion
We have found that both RmL (Tab. 1) and TlL (Tab.2) can be efficiently immobilized on surface-modified mesoporous silica gel supports.

Grafting function on silica gel
The catalytic properties of the TlL biocatalysts immobilized on the modified silica-gels depended also significantly on the surface properties of the silica support (Tab.2).The various TlL biocatalysts catalyzed the conversions of the rac-1 to acetate (R)-2 after 1 h reaction time between 1.2%-20.0%.

Grafting function on silica gel
Our results have shown that the optimal method of enzyme immobilization depended both on the nature of the substrate and the reaction conditions [13], [14]].This study indicated that using a broad selection of variously grafted silica gels for the immobilization of RmL and TlL provided a selection of biocatalysts with a wide range of activity and selectivity when applied for the kinetic resolution of racemic 1-phenylethanol rac-1.

Conclusions
The various mesoporous surface grafted silica gels proved to be efficient supports for the adsorptive immobilization of lipases from Rhizomucor miehei (RmL) and Thermomyces lanuginosus (TlL) influencing significantly the activity and enantiomer selectivity of the resulting biocatalysts in the kinetic resolution of rac-1.The fact that in both cases the highest activity or the highest selectivity was achieved with different supports indicated that there is no golden rule for support selection.Although different versions of a particular lipase might be optimal for different substrates, it is expected that the large assortment of the surface modified silica gel supports investigated in this study can provide us with immobilized RmL and TlL biocatalysts useful in selective biotransformations of other valuable compounds as well.