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 O.; 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 theproduction 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 inthe food and pharmaceutical industries. Most are extracted from natural sources, so they aredifficult to isolate from their matrix. On the other hand, the procedures based on the productionof 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 greatdisadvantage for the industry. To overcome such drawbacks, in recent years the use of enzymesimmobilized in inorganic supports has emerged. These heterogeneous enzymatic catalysts havethe following advantages: it increases the useful life of the enzyme and allows the biocatalyst tobe reused, facilitates its separation from the reaction mixture, and allows less aggressive reactionconditions 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 ofPseudomonas fluorescens lipase (LPF) to produce amyl acetate.Methods: A solid enzymaticcatalyst called LOBE (Low Ordered Biosilicified Enzyme) was synthesized from LPF, docecylamineand tetraethyl orthosilicate (TEOS) by biosilicification, it is an enzymatic mineralization with anorganic silicon precursor.On the other hand, SBA-15 or M-GM mesoporous silicas weresynthesized using the sol-gel method, using TEOS as silicon source, pluronic acid P123 or glycerolmonostearate as structure directing agent, respectively. The resulting solids were filtered, washed,dried, and subsequently calcined. Part of the material obtained was modified post-synthesis bywet 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 mMphosphate buffer, pH 7 to immobilize the lipase on the synthesized materials and obtain thebiocatalysts LPF/SBA-15, LPF/Ca/SBA-15, LPF/M-GM or LPF/Ca/M-GM. The reaction was performedin 10 mL glass batch reactors with an orbital shaker (180 oscillations per minute) at 37°C. Thereactors 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 gaschromatography.Results: The presence of the immobilized enzyme in the synthesized solids wasevidenced by Infrared Spectroscopy (IR). Subsequently, was evaluated the activity of theheterogeneous enzymatic catalysts in the transesterification reaction between vinyl acetate andisoamyl alcohol to obtain isoamyl acetate. The biocatalysts LPF/Ca/SBA-15, LPF/M-GM, andLPF/Ca/M-GM presented a low specific activity (0.05 U/mg), even less than the free enzyme (0.19U/mg). In the case of SBA-15 modified with Ca, it can block pores, reducing the surface to fix theenzyme and in addition, the basic properties acquired when the support was modified with analkaline-earth metal, such as Ca, could inhibit the enzyme. In the case of the M-GM material, itdoes not offer an ordered pore system of the SBA-15 type for the enzyme to fix. These could bethe causes of the lower activity detected. On the other hand, the LPF/SBA-15 (0.19 U/mg) andLOBE (0.20 U/mg), biocatalysts showed a specific activity like the free enzyme, with the advantagethat they can be reused. Conclusion: It was possible to immobilize the LPF on the different solidsupports and to detect transesterification activity in all the biocatalysts. Being the biocatalystsLPF/SBA-15 and LOBE the most active, with a specific activity of 0.19 U/mg (40% conversion) and0.20 U/mg (15,75% conversion), respectively. The biocatalysts offer the possibility of reusing inbach or in continuous flow reactions to obtain a Fine Chemical product used as flavoring andflavoring from a more sustainable process.