Heterogeneous enzymatic catalysts: role of the support in the activity of Pseudomonas fluorescens lipase used for amyl acetate synthesis.

CARRILLO, GERMÁN; VASCHETTO, ELIANA G.; FERRERO, GABRIEL ORLANDO; EIMER, GRISELDA A.

Rosario

Congreso; V Encuentro & Workshop de la Red Argentina de Tecnología Enzimática; 2023

Red Argentina de Tecnología Enzimática

Background: Among the reactions of economic importance in the fine chemicals industry is the production of esters, compounds with high added value that make up fragrances and flavors. These substances are produced by esterification or transesterification reactions and are used in the food and pharmaceutical industries. Most are extracted from natural sources, so they are difficult to isolate from their matrix. On the other hand, the procedures based on the production of esters by traditional routes require the use of inorganic catalysts at temperatures between 200-250°C and the generation of by-products that contaminate the desired product, this being a great disadvantage for the industry. To overcome such drawbacks, in recent years the use of enzymes immobilized in inorganic supports has emerged. These heterogeneous enzymatic catalysts have the following advantages: it increases the useful life of the enzyme and allows the biocatalyst to be reused, facilitates its separation from the reaction mixture, and allows less aggressive reaction conditions to be used. This translates into simpler processes with greater efficiency and selectivity, promoting Green Chemistry. In this work it is shown how the support affects the activity of Pseudomonas fluorescens lipase (LPF) to produce amyl acetate.Methods: A solid enzymatic catalyst called LOBE (Low Ordered Biosilicified Enzyme) was synthesized from LPF, docecylamine and tetraethyl orthosilicate (TEOS) by biosilicification, it is an enzymatic mineralization with an organic silicon precursor.On the other hand, SBA-15 or M-GM mesoporous silicas were synthesized using the sol-gel method, using TEOS as silicon source, pluronic acid P123 or glycerol monostearate as structure directing agent, respectively. The resulting solids were filtered, washed, dried, and subsequently calcined. Part of the material obtained was modified post-synthesis by wet impregnation to incorporate Ca (2.5%), obtaining the materials Ca/SBA-15 or Ca/M-GM. Then, the mesoporous supports were suspended in a 5 mg/mL enzyme solution of LPF in 25 mM phosphate buffer, pH 7 to immobilize the lipase on the synthesized materials and obtain the biocatalysts LPF/SBA-15, LPF/Ca/SBA-15, LPF/M-GM or LPF/Ca/M-GM. The reaction was performed in 10 mL glass batch reactors with an orbital shaker (180 oscillations per minute) at 37°C. The reactors had 87.5 mg of biocatalysts per reaction or 25 mg of free enzyme. The vinyl acetate: isoamyl alcohol ratio was 1:1.5, the product was determined at 2 and 24h by gas chromatography.Results: The presence of the immobilized enzyme in the synthesized solids was evidenced by Infrared Spectroscopy (IR). Subsequently, was evaluated the activity of the heterogeneous enzymatic catalysts in the transesterification reaction between vinyl acetate and isoamyl alcohol to obtain isoamyl acetate. The biocatalysts LPF/Ca/SBA-15, LPF/M-GM, and LPF/Ca/M-GM presented a low specific activity (0.05 U/mg), even less than the free enzyme (0.19 U/mg). In the case of SBA-15 modified with Ca, it can block pores, reducing the surface to fix the enzyme and in addition, the basic properties acquired when the support was modified with an alkaline-earth metal, such as Ca, could inhibit the enzyme. In the case of the M-GM material, it does not offer an ordered pore system of the SBA-15 type for the enzyme to fix. These could be the causes of the lower activity detected. On the other hand, the LPF/SBA-15 (0.19 U/mg) and LOBE (0.20 U/mg), biocatalysts showed a specific activity like the free enzyme, with the advantage that they can be reused. Conclusion: It was possible to immobilize the LPF on the different solid supports and to detect transesterification activity in all the biocatalysts. Being the biocatalysts LPF/SBA-15 and LOBE the most active, with a specific activity of 0.19 U/mg (40% conversion) and 0.20 U/mg (15,75% conversion), respectively. The biocatalysts offer the possibility of reusing in bach or in continuous flow reactions to obtain a Fine Chemical product used as flavoring and flavoring from a more sustainable process.