# Optimizing the second step of the biocatalytic process for green leaf volatiles production: Synthesis by soybean 13-lipoxygenase of 13-hydroperoxides from hydrolyzed hempseed oil

Eva Faillace, Virginie Brunini-Bronzini de Caraffa, Claude Gambotti, Liliane Berti, Jacques Maury, Sophie Vincenti

► To cite this version:

Eva Faillace, Virginie Brunini-Bronzini de Caraffa, Claude Gambotti, Liliane Berti, Jacques Maury, et al.. Optimizing the second step of the biocatalytic process for green leaf volatiles production: Synthesis by soybean 13-lipoxygenase of 13-hydroperoxides from hydrolyzed hempseed oil. Biocatalysis and Agricultural Biotechnology, 2025, 65, pp.103548. 10.1016/j.bcab.2025.103548. hal-05111463

HAL Id: hal-05111463

https://hal.science/hal-05111463v1

Submitted on 6 Feb 2026

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<figure><img src="image_1.png" /></figure>
<table><tr><td>Optimizing the second step of the biocatalytic process for green leaf volatiles production: synthesis by soybean 13-lipoxygenase of 13-hydroperoxides from hydrolyzed hempseed oil</td><td>1</td></tr></table>

<table><tr><td>Authors : Eva Faillace, Virginie Brunini-Bronzini de Caraffa, Claude Gambotti, Liliane Berti, Jacques Maury and Sophie Vincenti*</td><td>6</td></tr></table>

<table><tr><td>Laboratoire de Biochimie et Biologie Moléculaire Végétales, Campus Grimaldi, Université de Corse, CNRS UMR 6134 SPE, BP52, 20250 Corte, France;</td><td>8</td></tr></table>

<table><tr><td>Acknowledgments: This work has been supported by the Collectivité de Corse and the ANR project 2023-2028, PIA UNITI No. ANR-22-EXES-0016.</td><td>10</td></tr><tr><td></td><td>11</td></tr></table>

<table><tr><td>Corresponding author:</td><td>12</td></tr><tr><td>Dr. Sophie Vincenti</td><td>13</td></tr></table>

<table><tr><td>CNRS/Université de Corse UMR 6134, SPE, Laboratoire de Biochimie et de Biologie Moléculaire Végétale, Campus Grimaldi, BP 52, 20250 Corte, France</td><td>14</td></tr><tr><td>Fax: +33 495 450 162</td><td>15</td></tr><tr><td>Email: vincenti_s@univ-corse.fr</td><td>16</td></tr></table>

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**Abstract:** Linoleic and linolenic acid hydroperoxides are intermediates for the biocatalytic synthesis of natural green leaf volatiles (GLVs). GLVs are compounds including short chain volatile aldehydes used in the food and flavor industries for their fresh green aroma. GLVs with high added value can be produced by bioconversion of a vegetable oil, such as hempseed oil, in three steps. Once the oil has been hydrolyzed by a lipase, a lipoxygenase (LOX) acts on free polyunsaturated fatty acids to produce corresponding hydroperoxides, which are then converted into volatile aldehydes and oxoacids by a hydroperoxide lyase. In a previous work, optimization of hempseed oil lipolysis led to a yield of 97.2 ± 3.8 %. In this study, we investigated the second step of biocatalytic GLVs production. We focused on the dioxygenation, by a soybean 13-LOX (Glycine max.), of polyunsaturated fatty acids released after lipolysis of hempseed oil to produce corresponding 13-hydroperoxides. Box-Behnken design and response surface methodology (RSM) were used to determine the optimal reaction conditions to achieve the highest degree of conversion. We tested five factors: temperature, reaction duration, oxygen flow, enzyme load and substrate load. Finally, the defined optimal reaction conditions led to a degree of conversion of polyunsaturated fatty acids into hydroperoxides of 84.15 ± 0.03 %.

**Keywords:** lipoxygenase; biocatalysis; experimental design; response surface methodology (RSM); hempseed oil.

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# 1. Introduction

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Lipoxygenases (LOXs) are non-heme iron-containing dioxygenases that catalyze the regiospecific dioxygenation of polyunsaturated fatty acids (PUFAs) to form corresponding hydroperoxides (HPOs) (Andreou and Feussner, 2009; Lakshmi et al., 2015). In higher plants, they are classified according to their regiospecificity: 9-LOXs (EC 1.13.11.58) dioxygenate PUFAs on C9 to form 9-HPOs and 13-LOXs (EC 1.13.11.12) dioxygenate PUFAs on C13 to form 13-HPOs (Feussner and Wasternack, 2002; Gardner, 1996). LOXs with dual specificity, acting on both C9 and C13 of PUFAs, also exist (Xu et al., 2024). LOXs are involved in a lipid oxidation pathway known as the LOX pathway, which is activated during different stages of plant development or in response to biotic or abiotic stresses (Feussner and Wasternack, 2002; Matsui and Engelberth, 2022; Xu et al., 2024). The LOX pathway leads to the production of phytooxylipins, including green leaf volatiles (GLVs) such as short-chain aldehydes, which are widely used by food and cosmetic industries for their fresh green aroma and their antimicrobial and antifungal properties (Blée, 2002; Gigot et al., 2010; Lanciotti et al., 2004). In plants, GLVs are synthetized through the sequential action of a lipoxygenase (LOX) and a hydroperoxide lyase (HPL). LOX acts on PUFAs, 33 preferably linoleic acid and α-linolenic acid, released from phospholipids and galactolipids by lipolytic enzymes, to produce corresponding fatty acid hydroperoxides (HPO). Then, HPL acts on these HPOs to produce C6 or C9 aldehydes and C12 or C9 oxoacids (Feussner and Wasternack, 2002; Vincenti et al., 2019).

To meet the growing demand for natural flavoring compounds, the use of LOX pathway enzymes as biocatalysts in biocatalytic processes offers significant potential, enabling the production of natural
GLVs with high value-added (Gigot et al., 2010; Vincenti et al., 2019). In these processes, a vegetable oil is bioconverted into GLVs in three steps through the action of lipase, LOX and HPL. To enable the use of such a process on a larger scale, each biocatalytic reaction must first be optimized.

In a previous work, we were interested in the optimization of the first step of the biocatalytic process: the lipolysis of several vegetable oils. In particular, we optimized the hydrolysis reaction of hempseed oil by Candida rugosa lipase (CRL). The defined optimal conditions allowed to reach a hydrolysis rate of 97.2 ± 3.8%.

In the present study, we focused on the optimization of the second step of the biocatalytic process: the dioxygenation by soybean 13-LOX of PUFAs released after lipolysis of hempseed oil to produce 13-HPOs. The 13-LOX (LOX-1) from soybean (Glycine max) is the most extensively studied LOX and the most widely used enzyme both in the form of flours and as a commercial enzyme (Gardner, 1996). It is the most referenced enzyme for the conversion of PUFAs into corresponding HPOs in biocatalytic processes using the LOX pathway enzymes to produce C6 flavoring molecules (Coenen et al., 2022; Crouvisier Urion et al., 2022; Elshof et al., 1996; Fauconnier and Marlier, 1996a; Gala Marti et al., 2021; Kermasha et al., 2001).

We proposed to test the influence of different factors (temperature, reaction duration, oxygen flow, enzyme load and substrate load) on the dioxygenation reaction of PUFAs by soybean 13-LOX. Response surface methodology (RSM) and experimental Box-Behnken design were used to identify the best factor levels in order to determine the optimal reaction conditions and maximize the degree of
conversion of PUFAs from hydrolyzed hempseed oil into HPOs by soybean 13-LOX. Finally, the best 77
conditions of reaction were determined for the degree of conversion. 78

**2. Materials and Methods** 79
80

**2.1. Hempseed oil composition and hydrolyzed oil conditioning** 81
82

The hempseed oil is a vegetable oil with high contents of PUFAs and low contents of oleic acid and 83
saturated fatty acids (Callaway and Pate, 2009). Indeed, the selected hempseed oil contains 75.6% 84
PUFAs (56.6% linoleic acid and 19.0% linolenic acid), 12.7% saturated fatty acids and only 4.4% oleic 85
acid (Faillace et al., 2023). 86

This low content in oleic acid should be considered carefully, as preliminary tests showed that an 87
increasing concentration of oleic acid can lead to a decrease in LOX activity (Supplementary Materials 88
– Fig. S1). 89

After lipolysis of hempseed oil with CRL under the optimal conditions (Faillace et al., 2023), the reaction 90
medium is poured into a separating funnel and left overnight at +4°C in order to separate the aqueous 91
phase (containing the reaction medium, lipase and glycerol) and the lipid phase (containing free fatty 92
acids and remaining of mono-, di- and triglycerides). The lipid phase is then recovered, nitrogen-flashed 93
and stored at -20°C until use. 94
## 2.2. Enzymatic assays and determination of the optimal temperature and pH 95

### 2.2.1. Enzyme and Substrate preparation for enzymatic assays 96

Soybean 13-LOX (*Glycine max*. ) was purchased from Sigma-Aldrich (Darmstadt, Germany), and it is an 98
enriched preparation of LOX-1. Several other isoforms of the enzyme can be present in these 99
preparations, including LOX-2 and LOX-3, which exhibit dual specificity (Fauconnier and Marlier, 100
1996b). 101

The enzyme was prepared at a concentration of 1 mg of commercial powder.mL⁻¹ into phosphate 102
buffer saline (PBS) 1X. 103

The substrate, i.e. linoleic acid for enzymatic assays was prepared following the method described by 104
Anthon and Barrett (Anthon and Barrett, 2003) at a concentration of 25 mM in the presence of 105
Tween20 at a concentration of 11 mM. 106

### 2.2.2. Enzymatic assays 107

The LOX activity was carried out at 30°C, on Specord205 Analytik Jena (lena, Germany), by continuously 108
monitoring the increase in absorbance at 234 nm for 140 seconds corresponding to the formation of 109
the conjugated diene system of the hydroperoxide by the LOX (Axelrod et al., 1981). The 1 mL reaction 110
mixture was composed of 50 mM borate buffer pH 9 and 10 µL of enzyme preparation. The reaction 111
was started by adding the substrate i.e. linoleic acid to have an initial substrate concentration of 250 112
µM at the start of the measurement. The LOX activity was calculated using the extinction coefficient 113
ε=25,000 L.mol⁻¹.cm⁻¹, corresponding to conjugated dienes (Pérez et al., 1999), and was expressed in 114
enzyme units (U). One unit of LOX activity was defined as the amount of enzyme that catalyzes the
formation of 1 µmol of HPOs per minute.
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2.2.3. Optimum temperature and pH
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The optimal pH was determined by varying the pH from 6 to 10 in 0.5 increments. The optimal

temperature was determined over a temperature range of 5 to 45 °C in 5 °C increments. These
determinations were carried out with linoleic acid as substrate.

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2.2.4. Temperature-dependent inactivation of soybean 13-LOX
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The effect of temperature on enzyme inactivation was investigated by measuring LOX activity on
linoleic acid after incubation at different temperatures (4, 10, 20 and 30°C) in 50 mM borate buffer pH
9, over periods ranging from 10 to 90 minutes with a 10-minute increment.
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The inactivation rates were estimated by calculating the first-order inactivation constant (k) on semi-
logarithm plots, using the following equation: ln (A/A0) = - k/t(min), where A0 and A represent specific
activity of the soybean 13-LOX (µmol.min⁻¹.mg⁻¹of protein) respectively at t0 and at 10, 20, 30, 40, 50,
60, 70, 80 or 90 minutes.
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The half-life time (t₁/₂), defined as the time after which the initial LOX activity is halved, was calculated
as follows: t₁/₂ = ln(2)/k.
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2.2.5. Protein assay
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Protein content was determined using the Lowry method with bovine serum albumin as standard
(Lowry et al., 1951).
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## 2.3 Optimization of the degree of conversion using a Box-Behnken design and statistical analysis

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In order to optimize the dioxygenation reaction of PUFAs from hydrolyzed-hempseed oil by soybean 137
13-LOX, a Box-Behnken design has been set up. A RSM was used to (i) evaluate the influence of five 138
factors (temperature, reaction duration, O₂ flow, enzyme load and substrate load) on the conversion 139
of PUFAs into HPO by the 13-LOX, and (ii) determine the optimum levels of these factors that maximize 140
the degree of conversion. Preliminary experiments helped to define the limit values of the design. 141
Table 1 provides information on the factor levels coded -1, 0, and +1 for low, middle, and high values, 142
respectively. 143

The experimental design was generated using Minitab Software (Release 18) and consisted of 138 144
experimental runs, including 3 repetitions. All experiments were led in a randomized order. The 145
experimental design with response values is shown in the Supplementary Materials, Table S1. 146

All reactions were monitored by a bioreactor (Lambda Minifor Laboratory fermenter) to control the 147
reaction parameters, and a cryostat (Julabo CD-1001F) was used to control the system temperature. 148
The reaction mixture (total volume of 50 mL) was prepared as follows: the mass of fatty acids predicted 149
by the experimental design, was emulsified with a fraction of 50 mM Borate buffer at pH 9 before 150
being clarified with NaOH 1N, then added to the rest of the 50 mM borate buffer solution at pH 9. The 151
mixture was then placed in a 0.3L bioreactor vessel (Lambda Minifor Laboratory fermenter) and the 152
pH was adjusted to 9. The reaction was started by simultaneous addition of enzyme (amount defined 153
by the design) and O₂ (flow rate defined by the design) and lasted for the duration defined by the 154
design. The reaction was carried out at the design-defined temperature and agitation was set at 5Hz 155
to avoid excessive foaming. At the end of the reaction, the action of the LOX was stopped by addition 156
of HCl 1N until the pH dropped to 3. 157

The response surface analysis module of the Minitab Software was used to analyze the design. In order 158
to fit the mathematical model, to the experimental data, regression analyses were carried out using 159
the following generalized second-order polynomial equation: 160

$$ \hat{Y} = \beta_0 + \sum_{i=1}^{I} \beta_i X_i + \sum_{i=1}^{I} \beta_{ii} X_i^2 + \sum_{i} \sum_{j} \beta_{ij} X_i X_j \quad 161 $$

where $\hat{Y}$ is the predicted response (degree of conversion), $\beta_0$ is the model constant, $I$ is the number of 162
factors (five in this study), $\beta_i$ is the linear coefficient associated with factor $X_i$, $\beta_{ii}$ is the quadratic 163
coefficient associated with factor $X_i$, and $\beta_{ij}$ is the interaction coefficient between factors $X_i$ and $X_j$. To 164
achieve clarity, factors $X_i$ will be coded as follows: temperature (T), reaction duration (D), $O_2$ flow (F), 165
enzyme load (E) and substrate load (S). 166

This equation describes the contribution of the various factors on the response i.e. the degree of 167
conversion and was used to predict the optimal values of the factors. 168

An analysis of variance (ANOVA) was performed to estimate the level of significance of each 169
coefficient. Model fit to data is verified by the $R^2$ coefficient. The p-value was checked to find out the 170
significance of all the fitted equation terms at 5% of significance. 171

The response surface plots of the predicted model were obtained by depicting two significant factors 172
within the experimental range and keeping the other factors at their central level. 173
Finally, the model allowed to determine the factor values leading to a maximum degree of conversion 174
of PUFAs from hydrolyzed hempseed oil into corresponding HPOs. 175

## 2.4. Hydroperoxides quantification and degree of conversion calculation 176

When a dioxygenation reaction is completed, 2mL of the reaction medium are taken directly from the bioreactor, in order to determine the amount of HPO formed by spectrophotometry at a wavelength of 234 nm, using the molar extinction coefficient ε=25,000 L.mol⁻¹.cm⁻¹. 178
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The degree of conversion was calculated from the amount of PUFAs released from the hydrolysis of hempseed oil, using the following equation: 181
182

$$ \text{Degree of conversion (\%)} = \frac{n_{\text{HPO}}}{(n_{\text{FAR}} \times 75.6\%)} \times 100 \qquad 183 $$

where n<sub>HPO</sub> corresponds to the number of moles of HPO formed (mmol), n<sub>FAR</sub> corresponds to the number of moles of total fatty acids released after the lipolysis of hempseed oil and present in the medium when the reaction starts, and 75.6% corresponds to the content of PUFAs in the hempseed oil. 184
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# 3. Results and discussion 188

## 3.1 Initial materials 189

### 3.1.1 Hydrolysed Hempseed oil analysis 190

In a previous work (Faillace et al., 2023), we optimized the lipolysis of hempseed oil by CRL and quantified the fatty acids released by FTIR analysis. The measured hydrolysis rate was 97.2 ± 3.8%. 191
192
Preliminary tests showed that free glycerol present in the medium has an inhibitory effect on 13-LOX 193
activity (Supplementary Material - Fig. S2). In order to remove glycerol from the hempseed oil lipolysis 194
reaction medium, the lipid phase was separated from the aqueous phase by decantation at 4 °C 195
overnight. An FTIR analysis was then carried out to verify the absence of glycerol in this lipid phase 196
(Figure 1.B). Commercial linoleic acid (Figure 1.A) and commercial glycerol (Figure 1.C) were used as a 197
control. 198

The FTIR profiles (Figure 1) show the absence of glycerol in the recovered lipid phase, indicating the 199
efficient removal of this compound during decantation. 200

201

Additional preliminary tests have also shown that a high proportion of oleic acid has an inhibitory effect 202
on LOX activity, with a 50% loss of activity in the presence of 40% oleic acid (Supplementary Materials 203
- Fig. S1). The high PUFAs content (75.6%) and the low oleic acid content (4.4%) of the hempseed oil 204
make it a relevant candidate for use in a biocatalytic reaction involving LOX. 205

### 3.1.2 Protein assays of the enzyme preparation 206

Lowry method (Lowry et al., 1951) used to determine protein content shows that 1 mg of commercial 207
powder corresponds to 1 mg of protein. 208

Moreover, 1 mg of commercial powder, and thus 1 mg of protein, correspond to 40 ± 3 units of 209
enzyme. 210

## 3.2 Optimal pH and optimal temperature, and temperature-dependent inactivation 211

The influence of the pH and temperature on the soybean 13-LOX activity is presented in the Figure 2. 212
The optimal pH is 9 and more than 80% of the specific activity is preserved over a pH range of 8,5 to 213
10. At pH 8, only 55% of the specific activity is preserved (Figure 2.A). The optimal temperature is 30°C 214
but more than 75% of the specific activity is preserved between 25 and 45°C (Figure 2.B). 215

The optimal pH of 9 was selected as the reaction pH and not as a tested parameter in the design due 216
to a better solubility of fatty acids at pH 9 than at lower pH values. Furthermore, even though the 217
commercial powder used contains predominantly the LOX-1 isoform which is a 13-LOX and is most 218
effective at pH 9.0-9.5, other LOX isoforms have been found, such as LOX-2 and LOX-3, which have a 219
dual specificity and are most active at pH 6-7 (Axelrod et al., 1981). Thus, maintaining a basic pH 220
particularly favors LOX-1 activity, which specifically catalyzes the dioxygenation of PUFAs on C13 221
(Elshof et al., 1996; Fauconnier and Marlier, 1996b). 222

Nevertheless, the optimal temperature was not selected, and the limit values chosen for the 223
experimental design are between 4 and 16°C. Oxygen is more soluble at temperatures lower than at 224
30°C, and acts as a co-substrate in the dioxygenation reaction of PUFAs by LOX (Zheng and Brash, 225
2010). In addition, soybean 13-LOX (LOX-1) is an enzyme known for its ability to produce secondary 226
products (Crouvisier Urion et al., 2022; Vliegenthart and Veldink, 1991). Operating at a temperature 227
below +20°C, especially when coupled with variations in oxygen flow, may further decrease the 228
enzyme's ability to generate secondary compounds, due to reduced enzyme efficiency (Crouvisier 229
Urion et al., 2022). The enzyme inactivation over time at different temperatures (4, 10, 20, 30°C) has 230
been studied, and the results shown in Figure 3 and Table 2 have confirmed the stability of the LOX 231
over time at temperature chosen for the design. 232
The semi-logarithmic plots of the inactivation kinetics showed straight lines, with R² ranging from 0.91 to 0.99 (Figure 3). These results suggest that LOX temperature-dependent inactivation followed first-order kinetics.

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This study on temperature-dependent inactivation shows that the enzyme remains stable for several hours at temperatures between 4°C and 10°C (Figure 3).

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The first order inactivation constant (k) and the half-life time (t₁/₂) of LOX were calculated (Table 2). At 4°C, k is 9.3 times lower than at 10°C, and 58.3 times lower than at 30°C. In contrast, k at 30°C is 6.3 times higher than at 10°C. Incubation at 20°C results in a 3.8-fold decrease in the inactivation constant compared to 30°C (Table 2). The t₁/₂ of soybean 13-LOX at 4°C is 83.24 hours, which progressively decreases to 11.96 hours at 10°C, 7.16 hours at 20°C, and 1.84 hours at 30°C (Table 2).

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Thus, these results show that the enzyme is stable for temperatures ranging from 4 to 16°C and reaction times of up to 150 minutes which are the limit values defined in the design.

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### 3.3 Optimization of the reaction using a Box-Behnken design

245

The optimization of the dioxygenation of PUFAs from hydrolyzed hempseed oil by soybean 13-LOX was performed using RSM. A Box-Behnken design was used to evaluate the influence of temperature (T), reaction duration (D), dioxygen flow (F), enzyme load (E) and substrate load (S) on the degree of conversion.

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The results obtained from the 138 experimental runs, carried out according to the Box-Behnken design, are summarized in Supplementary Materials, Table S1. The degrees of conversion obtained fluctuate

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from 3.06 % to 84.72%. Experimental data were subject to a regression analysis. A second-order polynomial was fitted to the experimental values of degree of conversion. The equation shown in the Table 3 models the relationship between the five factors and the degree of conversion.

R² coefficient of the model is 87.79% and the adjusted R² is 85.70% meaning that more than 85% of the variability of the response can be explained by the model (Table 3). These values confirm a satisfactory adjustment of the model to the experimental data. So, the model can be used to analyze the influence of the five factors, determine the optimal values of these factors and finally predict the degree of conversion that can be obtained under optimal conditions.

The significance of the fit of the second-order polynomial for degree of conversion was assessed by performing an analysis of variance (ANOVA). A *p*-value lower than 0.05 was considered significant. The model was simplified, by keeping terms showing significant *p*-value (Table 4). The complete model and the pareto plot can be found in Supplementary data, Table S2 and Figure S3 respectively.

The RSM analysis showed a *p*-value lower than 0.001 for each linear terms T, D, F, E and S, indicating the five factors have a significant influence on the degree of conversion. Statistically, only the quadratic terms T², E² and S² are significant (*p*-value lower than 0.01). Regarding the interaction terms, T×F, T×E, T×S, D×F, D×E, D×S and F×E are significant (*p*-value <0.05).

The effects of cross term product factors on the degree of conversion can be examined using three-dimensional (3D) response surface plots. Only significant interactions are retained in Figure 4. The other 3D response surface plots and all the contour plots can be found in the Supplementary Data,
Figure S4 and Figure S5 respectively. On each plot, the three other factors are maintained at their middle level. The holding values are 10°C for T, 85 min for D, 0.275 L.min⁻¹ for F, 105 U for E, and 550 mg for S (Table 1).

Concerning the interaction terms T x F, T x S and T x E (Figure 4 a, b, c), high degrees of conversion are achieved at lowest reaction temperature. The degree of conversion decreases as the reaction temperature is increased. Increasing the reaction temperature promotes the further oxidation process and degradation of HPO into undesirable by-products that absorb at a wavelength of 278 nm instead of 234 nm (Blasi et al., 2021; Crouvisier Urion et al., 2022; Pérez et al., 1999). These are not taken into account for the degree of conversion calculation, therefore increasing the reaction temperature decreases the calculated degree of conversion.

In Figure 4.a, the highest degree of conversion (65%) is obtained at 4°C with 0.5 L.min⁻¹ oxygen flow rate. At 4°C, increasing the O₂ flow leads to a rise of the degree of conversion (from 40 to 65%). According to the literature, lower temperatures enhance oxygen solubility, which results in a higher concentration of dissolved oxygen which promotes greater enzyme efficiency and thus leads to a higher degree of conversion (Elshof et al., 1996; Martini et al., 1994; Tromans, 1998; Zheng and Brash, 2010).

In Figure 4.b, the substrate load has minimal influence on the degree of conversion compared to temperature.

In Figure 4.c, the degree of conversion increases as the temperature decreases and the enzyme load increases.
Considering the interaction term D x F (Figure 4.d), for reaction times exceeding 130 minutes, high degrees of conversion (above 50%) are achieved regardless of the oxygen flow. In contrast, at higher oxygen flow (exceeding 0.3 L.min⁻¹), the degrees of conversion raise from 35% to 50%, between 20 to 150 minutes of reaction respectively.

Concerning D x S interaction (Figure 4.e), for low substrate quantities (100 mg), high degrees of conversion (above 60%) are achieved regardless of the reaction duration, as even for the shortest duration tested (20 minutes), the degree of conversion exceeds 60%. On the other hand, when a large amount of substrate is supplied, the duration reaction has a strong influence, since the degree of conversion increases with increasing reaction duration. Thus, for 1,000 mg of substrate, the degree of conversion is less than 10% after 20 min, but reaches 50% after 150 min.

Regarding D x E interaction (Figure 4.f), for enzyme quantities below 80 U, the degrees of conversion remain inferior to 30% regardless of the reaction duration. In contrast, higher enzyme amounts result in increased degrees of conversion as the reaction duration is increased, with yields rising from around 30% at 20 minutes to over 60% at 150 minutes. Moreover, for reaction times exceeding 100 minutes, increasing the enzyme quantity leads to a boost in degrees of conversion, reaching up to 60% at 150 minutes with 200 U of enzyme.

Finally, for the plot showing F x E interaction (Figure 4.g), for enzyme load below 100 U, the degree of conversion remains beneath 30% regardless of the oxygen flow. In contrast, for enzyme load above 100 U, a higher degree of conversion is observed with an increased oxygen flow rate, reaching 55% with 200 U of enzyme and an oxygen flow rate of 0.5 L.min⁻¹.
The RSM approach determines the relationships between the factors studied (temperature, reaction duration, oxygen flow, enzyme load, substrate load) and the response in order to provide optimal factor values to optimize the reaction while maximizing the degree of conversion. This optimization mainly consists of reducing the reaction duration and the amount of enzyme loaded while maintaining a high degree of conversion. The RSM also predicts the degree of conversion that could be achieved under these optimal conditions. This prediction is then subject to experimental validation. The results are presented in Table 5.

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The optimal conditions consist of carrying out the reaction at 4°C for 51.5 minutes with 96.88 U of soybean 13-LOX per 100 mg of substrate and a dioxygen flow rate of 0.49 L.min⁻¹. Under these conditions, the degree of conversion obtained experimentally is 84.15 ± 0.03%. This result is consistent with the predicted value, considering a standard deviation of 0.18% which confirms the adequacy between the model and the experimental results (Table 5).

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We have shown that 1 mg of commercial powder enriched with LOX-1 corresponds to 1 mg of protein, and to an enzymatic activity of 40 ± 3 units. In our optimized reaction, we use 96.88 U of enzyme, i.e. 2.5 mg of protein, to convert 100 mg of substrate. Thus, the substrate-to-catalyst ratio, which reflects the mass of the substrate required relative to the enzyme mass supplied, is 40.

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The defined conditions use 380 enzyme units per mmol of substrate to convert an initial concentration of 5.21 mM PUFAs in a 50 mL volume.

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A large-scale reaction of dioxygenation of PUFAs from hydrolyzed hempseed oil with soybean 13-LOX in a reaction volume of 500 mL was performed in a one-liter bioreactor, under optimal conditions defined (96.88 U of soybean 13-LOX per 100 mg of substrate, dioxygen flow rate of 0.49 L.min⁻¹, 4°C, 51.5 minutes). A degree of conversion of 86.15 ± 0.08% was obtained, indicating a successful 10-fold scale-up.

Several studies have led to the development of processes for producing 13-HPO from hydrolyzed oils (Aziz et al., 2014; Drönner et al., 2023; Elshof et al., 1996; Fauconnier and Marlier, 1996a; Gala Marti et al., 2021; Márczy et al., 2002; Németh et al., 2004). The reaction conditions used and the degrees of conversion achieved in each study are detailed in Table S3.

Most studies use crude extracts or enzyme preparations from soybeans as source of 13-LOX, to convert PUFAs of hydrolysed vegetable oils. Crude extracts from soybean flour (Elshof et al., 1996) and soybean flour suspensions (Drönner et al., 2023) led respectively to degree of conversions of 80% and 60% on hydrolyzed safflower oil. Márczy et al. (2002) and Németh et al. (2004) used LOX-1 from defatted soybean meal, achieving 72% and 62% degree of conversion on hydrolyzed sunflower and linseed oils. Degrees of conversion of 94% were obtained using an extract from soybean seeds (Fauconnier and Marlier, 1996a).

Fewer studies employed commercial soybean 13-LOX, with yields of 74% (Aziz et al., 2014) and 83% (Gala Marti et al., 2021) on hydrolyzed safflower oil.

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  <tbody>
    <tr>
      <td>Our study uses 18.6 units of commercial soybean 13-LOX per mmol of PUFAs, which is the lowest enzymatic load compared to the works cited in the table S3, such as those of Aziz et al. (2014) and Gala Marti et al. (2021), which respectively use 337.5 and 20,000 units of commercial soybean 13-LOX per mmol of PUFAs, or those of Márczy et al. (2002) and Németh et al. (2004) which respectively use 60 and 35 units of LOX from soybean meal per mmol of PUFAs. However, some studies worked at much higher substrate concentrations (up to 100–142.8 mM) (Elshof et al., 1996; Fauconnier and Marlier, 1996a; Gala Marti et al., 2021; Márczy et al., 2002; Németh et al., 2004). Notably, Elshof et al. (1996) achieved 80% degree of conversion with 28 U per mmol of PUFAs, but under continuous substrate addition, a strategy differing from batch reactions.</td>
      <td>350</td>
    </tr>
    <tr>
      <td>Gala Marti et al. (2021) reported yields of over 80% using 2,000,000 U of LOX to convert 100 mM of linoleic acid from hydrolyzed safflower oil (equivalent to 20,000 units per mmol of linoleic acid) in a 50 mL volume. Later, Drönner et al. (2023) scaled up this process to a 2.5 L bioreactor and achieved a degree of conversion of 60%, using 1,500,000 U of LOX to convert 30 mM of linoleic acid (equivalent to 35,000 units per mmol of linoleic acid) released from hydrolyzed safflower oil. Although this remains smaller than the volumes used in some studies (e.g., 2 L, Elshof et al., 1996; 10 L, Fauconnier and Marlier, 1996), it demonstrates good scalability potential.</td>
      <td>359</td>
    </tr>
    <tr>
      <td>The optimized dioxygenation reaction catalyzed by soybean 13-LOX can be integrated to biocatalytic processes to produce GLVs with industrial interest. We previously optimized the lipolysis of hempseed oil using CRL, achieving the reaction in 4.5 hours with an hydrolysis rate of 97±3.8% (Faillace et al., 1996).</td>
      <td>366</td>
    </tr>
    <tr>
      <td></td>
      <td>367</td>
    </tr>
    <tr>
      <td></td>
      <td>368</td>
    </tr>
    <tr>
      <td></td>
      <td>369</td>
    </tr>
  </tbody>
</table>
2023). The sequential combination of reactions catalyzed by CRL and soybean 13-LOX now allows for an efficient bioconversion of hempseed oil into HPO, with yields of 97.2±3.8 % and 84.15±0.03% respectively for the first and second steps completed in 5.5 hours of reaction time.

Even if the commercial powder used contains several isoforms of soybean LOX, LOX-1 which specifically catalyzes the dioxygenation on C13 of PUFAs is predominant and the basic pH (9) of the reaction favors this LOX-1 activity which therefore allows the majority formation of 13-HPO. For the last step of the biocatalytic process, the use of an HPL acting specifically on 13-HPO such as the recombinant olive 13-HPL (Jacopini et al. 2016) will allow the selective production of C6 aldehydes and C12 oxoacids.

## 4. Conclusions

In this study, the second-step of the biocatalytic production process of GLVs was investigated.

We focused on the dioxygenation, by soybean 13-LOX, of PUFAs released after lipolysis of hempseed oil to produce corresponding 13-HPO.

Experimental design and RSM were used to evaluate the influence of five factors (temperature, reaction duration, dioxygen flow, enzyme load and substrate load) on the degree of conversion.

According to the experimental design, 138 conditions were tested and the data analysis allowed to determine optimal reaction conditions while maintaining high degree of conversion. Thus, a degree of conversion of 84.15 ± 0.03% was obtained in just over 50 minutes with a substrate-to-catalyst mass ratio of 40.
The sequential process involving the lipolysis of hempseed oil by CRL and the conversion of the released PUFAs into HPOs by soybean 13-LOX enables the synthesis of 13-HPOs. These compounds can then serve as a substrate for 13-HPL (Jacopini et al., 2016), leading to the production of C6 GLVs, which are valuable in industrial applications such as natural flavors and fragrances.

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      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
      <td>445</td>
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      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
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      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
      <td>447</td>
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      <td>448</td>
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      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
      <td>449</td>
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      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
      <td>450</td>
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    <tr>
      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
      <td>451</td>
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    <tr>
      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
      <td>452</td>
    </tr>
    <tr>
      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
      <td>453</td>
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    <tr>
      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
      <td>454</td>
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      <td>455</td>
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      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
      <td>456</td>
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    <tr>
      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
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      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
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      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
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      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
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      <td>Tromans, D., 1998. Temperature and pressure dependent solubility of oxygen in water: a thermodynamic analysis. Hydrometallurgy 48, 327–342. https://doi.org/10.1016/S0304-386X(98)00007-3</td>
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    <td>473</td>
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    <td>474</td>
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    <td>475</td>
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    <td>476</td>
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    <td>477</td>
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</table>
**Tables Caption**

**Table 1:** Experimental level of factors used in the trials

**Table 2:** Inactivation constant (k) and half-life time ($t_{1/2}$) of the LOX at the different temperatures.

**Table 3:** Response surface model analysis of the degree of conversion; second-order polynomial equation and determination coefficients.

**Table 4:** Response surface quadratic model analysis of degree of conversion level obtained, after model simplification—analysis of variance (ANOVA).

**Table 5:** Predicted and measured degrees of conversion with optimal conditions of temperature, reaction duration, O₂ flow, enzyme load and substrate load for the action of soybean 13-LOX on hydrolyzed hempseed oil.
<table><caption>TABLE 1</caption><thead><tr><th rowspan="2">Factors</th><th colspan="3">Coded factors levels</th></tr><tr><th>-1 (Low)</th><th>0 (Middle)</th><th>+1 (High)</th></tr></thead><tbody><tr><td>Temperature (°C)</td><td>4</td><td>10</td><td>16</td></tr><tr><td>Reaction duration (min)</td><td>20</td><td>85</td><td>150</td></tr><tr><td>O<sub>2</sub> flow (L.min<sup>-1</sup>)</td><td>0.05</td><td>0.275</td><td>0.5</td></tr><tr><td>Enzyme load (U)</td><td>10</td><td>105</td><td>200</td></tr><tr><td>Substrate load (mg)</td><td>100</td><td>550</td><td>1,000</td></tr></tbody></table>
<table><caption>TABLE 2</caption><thead><tr><th></th><th><i>k</i><sup>a</sup></th><th><i>t</i><sub>1/2</sub> (<i>h</i>)<sup>b</sup></th></tr></thead><tbody><tr><td>4°C</td><td>0.00011 ± 0.00013</td><td>83.24</td></tr><tr><td>10°C</td><td>0.00102 ± 0.00050</td><td>11.96</td></tr><tr><td>20°C</td><td>0.00165 ± 0.00027</td><td>7.16</td></tr><tr><td>30°C</td><td>0.00641 ± 0.00109</td><td>1.84</td></tr></tbody></table>

<footnote><sup>a</sup> Inactivation constant (k) was calculated with the following equation: ln (A/A0) = - k/t(min) ; <sup>b</sup> Half-life time (t<sub>1/2</sub>) was calculated as follows: t<sub>1/2</sub> = ln(2)/k.</footnote>
<table><caption>TABLE 3</caption><thead><tr><th>Equation</th><th>R<sup>2</sup></th><th>Adjusted R<sup>2</sup></th></tr></thead><tbody><tr><td>Degree of conversion (%) =<br>45.4 + 0.31 x T + 0.056 x D + 84.5 x F + 0.2154 x E - 0.0997 x S + 0.1187<br>x T<sup>2</sup> - 0.000103 x D<sup>2</sup> - 26.6 x F<sup>2</sup> - 0.000977 x E<sup>2</sup> + 0.000040 x S<sup>2</sup> -<br>0.01171 x T x D - 5.25 x T x F - 0.01101 x T x E - 0.002704 x T x S -<br>0.357 x D x F + 0.001409 x D x E + 0.000306 D x S + 0.278 x F x E +<br>0.0089 x F x S + 0.000076 x E x S</td><td>0.8779</td><td>0.8570</td></tr></tbody></table>
<table><caption>TABLE 4</caption><thead><tr><th>Source<sup>a</sup></th><th>Sum of Squares</th><th>Degree of Freedom</th><th>F value</th><th>p-value<sup>b</sup></th></tr></thead><tbody><tr><td>T</td><td>9,976.7</td><td>1</td><td>153.74</td><td>0.000</td></tr><tr><td>D</td><td>3,919.7</td><td>1</td><td>60.4</td><td>0.000</td></tr><tr><td>F</td><td>1,079.2</td><td>1</td><td>16.63</td><td>0.000</td></tr><tr><td>E</td><td>8,252.3</td><td>1</td><td>127.16</td><td>0.000</td></tr><tr><td>S</td><td>20,747.8</td><td>1</td><td>319.71</td><td>0.000</td></tr><tr><td>T<sup>2</sup></td><td>478.2</td><td>1</td><td>7.37</td><td>0.008</td></tr><tr><td>E<sup>2</sup></td><td>2,034.3</td><td>1</td><td>31.35</td><td>0.000</td></tr><tr><td>S<sup>2</sup></td><td>1,736.8</td><td>1</td><td>26.76</td><td>0.000</td></tr><tr><td>T x F</td><td>603.4</td><td>1</td><td>9.3</td><td>0.003</td></tr><tr><td>T x E</td><td>472.6</td><td>1</td><td>7.28</td><td>0.008</td></tr><tr><td>T x S</td><td>639.8</td><td>1</td><td>9.86</td><td>0.002</td></tr><tr><td>D x F</td><td>326.9</td><td>1</td><td>5.04</td><td>0.027</td></tr><tr><td>D x E</td><td>907.9</td><td>1</td><td>13.99</td><td>0.000</td></tr><tr><td>D x S</td><td>959</td><td>1</td><td>14.78</td><td>0.000</td></tr><tr><td>F x E</td><td>424.3</td><td>1</td><td>6.54</td><td>0.012</td></tr><tr><td>Lack of fit</td><td>2,991.2</td><td>20</td><td>3.15</td><td>0.000</td></tr><tr><td>Pure error</td><td>4,601.4</td><td>97</td><td>-</td><td>-</td></tr><tr><td>Total</td><td>62,186.4</td><td>137</td><td>-</td><td>-</td></tr></tbody></table>

<footnote><sup>a</sup> Factors coded as follows: temperature (T), duration reaction (D), O₂ flow (F), enzyme load (E) and substrate load (S); <sup>b</sup> p-value < 0.05 indicates a statistical significance of the factor.</footnote>
<table><caption>TABLE 5</caption><thead><tr><th>Optimum conditions of reaction</th><th>Predicted degree of conversion (%)</th><th>Measured degree of conversion (%)</th></tr></thead><tbody><tr><td>Temperature (T)</td><td>4°C</td><td></td></tr><tr><td>Reaction duration (D)</td><td>51.5 min</td><td></td></tr><tr><td>O<sub>2</sub> flow (F)</td><td>0.49 L.min<sup>-1</sup></td><td>84.3</td><td>84.15 ± 0.03</td></tr><tr><td>Enzyme load (E)</td><td>96.88 U</td><td></td></tr><tr><td>Substrate load (S)</td><td>100 mg</td><td></td></tr></tbody></table>
**Figures Caption**

**Figure 1:** FTIR Analysis of commercial linoleic acid (A), lipid phase recovered after lipolysis of hempseed hydrolysis (B) and commercial glycerol (C). The red arrow indicates the ester group corresponding to the remaining mono-, di-, and triglycerides in the lipid phase.

**Figure 2:** Influence of pH (A) and temperature (B) on the specific activity of soybean 13-LOX.

**Figure 3:** Kinetics of LOX inactivation over time at different temperatures, where $A_0$ and A represent LOX specific activity (µmol.min⁻¹.mg⁻¹of protein) respectively at t0 and 10, 20, 30, 40, 50, 60, 70, 80 or 90 minutes for 4°C (●-), 10°C (►-), 20°C (*-) and 30°C (---).

**Figure 4:** Three-dimensional (3D) response surface plots of the degree of conversion (%) of PUFAs from hydrolyzed hempseed oil into 13-HPOs, as function of: T and F (a), T and S (b), T and E (c); D and F (d), D and S (e), D and E (f); F and E (g).
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>