Studies on the Catalytic Properties of Crude Freeze-Dried Preparations of Yarrowia lipolytica Extracellular Lipases for Geranyl Ester Derivative Synthesis

Abstract

The study aimed to evaluate the impact of selected factors of the freeze-drying process on the hydrolytic and synthetic activity of the extracellular lipases of Y. lipolytica KKP 379 and to attempt the use of the crude enzyme preparation as a biocatalyst in the synthesis of geranyl 4-hydroxyphenylpropanoate. Antioxidant and antibacterial properties of the geranyl ester derivative were also investigated in order to evaluate their usefulness as a novel food additive. The studies confirmed that freeze-drying was an effective method of dehydrating yeast supernatant and allowed for obtaining lyophilizates with low water activity from 0.055 to 0.160. The type and concentration of the additive (2-6% whey protein hydrolyzate, 0.5% and 1% ammonium sulphate) had a significant effect on the hydrolytic activity of enzyme preparations, while the selected variants of drying temperature during the freeze-drying process were not significant (10 °C and 50 °C). Low yield of geranyl 4-hydroxyphenylopropionate was shown when the lyophilized supernatant was used (5.3%), but the yield of ester synthesis increased when the freeze-dried Y. lipolytica yeast biomass was applied (47.9%). The study confirmed the antioxidant properties of the synthesized ester by the DPPH• and CUPRAC methods, as well as higher antibacterial activity against tested bacteria than its precursor with 0.125 mM MIC (minimal inhibitory concentration) against L. monocytogenes.

Keywords: Yarrowia lipolytica; antibacterial; antioxidant; biocatalyst; cryoprotectant; esterification; freeze-drying; lipase.

Figure 1

Scheme of geranyl 4-hydroxyphenylpropanoate synthesis.

Figure 2

Influence of water activity (a_w) on the hydrolytic activity of freeze-dried enzyme preparations with the addition of 1% ammonium sulphate. Experiments were performed in triplicate.

Figure 3

Effect of the used additive on the hydrolytic activity of freeze-dried preparations. Significant differences are marked with letters of the alphabet. Experiments were performed in triplicate.

Figure 4

Effect of the secondary drying temperature on the hydrolytic activity of freeze-dried preparations. Significant differences are marked with letters of the alphabet. Experiments were performed in triplicate.

Figure 5

The effect of the applied cryoprotectant and the secondary drying temperature during the freeze-drying process on the hydrolytic activity of the lyophilized enzyme preparation (calculated per 1 g of the final preparation taking into account the presence of both the lyophilized supernatant and the addition of cryoprotectant). Experiments were performed in triplicate.

Figure 6

The effect of the applied cryoprotectant and the secondary drying temperature during the freeze-drying process on the hydrolytic activity of the lyophilized enzyme preparation (calculated per 1 g of the preparation, referring only to the lyophilized supernatant, excluding the presence of the cryoprotectant). Experiments were performed in triplicate.

Figure 7

Hydrolytic activity of selected freeze-dried enzyme preparations after storage in a desiccator for 4 months (calculated per 1 g of the final preparation taking into account the presence of both the lyophilized supernatant and the addition of cryoprotectant). Significant differences are marked with letters and a number given in a subscript indicate the compared values. Experiments were performed in triplicate.

Figure 8

Hydrolytic activity of selected freeze-dried enzyme preparations after storage in a desiccator for 4 months (calculated per 1 g of the preparation, referring only to the lyophilized supernatant, excluding the presence of the cryoprotectant). Significant differences are marked with letters and a number given in a subscript indicate the compared values. Experiments were performed in triplicate.