Immobilization of a Cutinase from Fusarium oxysporum and Application in Pineapple Flavor Synthesis

Abstract

In the present study, the immobilization of a cutinase from Fusarium oxysporum was carried out as cross-linked enzyme aggregates. Under optimal immobilization conditions, acetonitrile was selected as precipitant, utilizing 9.4 mg protein/mL and 10 mM glutaraldehyde as cross-linker. The immobilized cutinase (imFocut5a) was tested in isooctane for the synthesis of short-chain butyrate esters, displaying enhanced thermostability compared to the free enzyme. Pineapple flavor (butyl butyrate) synthesis was optimized, leading to a conversion yield of >99% after 6 h, with an initial reaction rate of 18.2 mmol/L/h. Optimal reaction conditions were found to be 50 °C, a vinyl butyrate/butanol molar ratio of 3:1, vinyl butyrate concentration of 100 mM, and enzyme loading of 11 U. Reusability studies of imFocut5a showed that after four consecutive runs, the reaction yield reaches 54% of the maximum. The efficient bioconversion offers a sustainable and environmentally friendly process for the production of "natural" aroma compounds essential for the food industry.

A Middle-Aged Enzyme Still in Its Prime: Recent Advances in the Field of Cutinases

“…Cut-CLEA presented a higher T opt by 10 • C, and retained its activity at 60 • C after 1 h, while the free enzyme was stable up to 40 • C. Furthermore, the immobilized cutinase increased its stability in organic solvents, and especially in polar ones. A recombinant F. oxysporum cutinase was also immobilized following CLEA methodology using the crude intracellular fraction of the E.coli host [90]. Its thermostability increased 3-fold at 35 • C, and it could be reused for the synthesis of butyl butyrate up to four times, with less than 50% reduction of yield.…”

“…The yields for both types of reactions in non-conventional media, such as isooctane or heptane, are high in most cases (>95%) [25,162]. The highest yields for butyl butyrate synthesis have been achieved in isooctane by esterification using cutinase from M. cinnamonea (McCut) (97%) [51], and by transesterification with cutinase from F. oxysporum (imFocut5a) (>99%) [90]. In both cases the reactions reached equilibrium in less than 6 h, but the amount of imFocut5a utilized was almost 20 times lower than the one of McCut.…”

Effect of the Immobilization Strategy on the Efficiency and Recyclability of the Versatile Lipase from Ophiostoma piceae

“…Apart from lipases, other enzymes from the α/β hydrolase family have demonstrated their ability to produce these aliphatic esters. For example, esterifications catalyzed by cutinases have shown to be very efficient with substrates with 4–7 carbon atoms, and several reports deal with their use to produce of short-chain esters [31,32,33,34,35].…”

Immobilization of Active Substances in Food Using Self‐Organized Patterned Porous Film via Breath Figure Approach

“…The immobilization can be realized by three main types of methods: encapsulation, covalent binding to support, and crosslinking method. Relevant technical strategies have been broadly adopted for the immobilization of protein and enzymes such as top‐down techniques, [14] photoetching technique, [15] colloidal crystal, [16] microphase separation self‐assembly method and so forth [11b,17] . For instance, Lynne Regan reported an engineered surface‐active biofilm proteins building blocks method for immobilization of protein to a surface [18] .…”

“…Christian J. Doonan constructed a porous hydrogen‐bonded organic framework (HOF) coating enzymes including catalase, alcohol oxidase and urea, and the encapsulation retains enzymatic activity [19] . Evangelos Topakas reported the immobilization of a cutinase from Fusarium oxysporum by cross‐linking methods and their application in pineapple flavour synthesis [20] . Marwa I.Wahba fabricated the porous chitosan beads to acquire higher activities of immobilized β‐d‐galactosidase for winemaking applications [21] .…”