Direct Application of Fermented Solid Containing Lipases from Pycnoporus sanguineus in Esterification Reactions and Kinetic Resolution of Sec-alcohols

Abstract

Lipases are widely used as biocatalysts in synthetic applications because of their high chemo-, regio-, and enantioselectivities, which play key roles in the synthesis of esters and the resolution of racemates. These biocatalytic steps are essential for the production of various products, including cosmetic ingredients, building blocks in the pharmaceutical and agrochemical industries. In this study, we produced lipases through solid-state fermentation of agricultural by-products and domestic wastes using the fungus Pycnoporus sanguineus. After fermentation, the dried solids containing lipases from P. sanguineus exhibited high catalytic activity. Lipase production was achieved via solid-state fermentation using a substrate composed of wheat bran and sugarcane bagasse supplemented with either residual frying oil or urea, resulting in an enzymatic activity of 24 U mL⁻¹ after 96 h. The resulting P. sanguineus fermentation solids (PSFS) efficiently catalyzed the esterification of capric acid with ethanol, achieving 95% ester conversion within 28 h. Additionally, PSFS proved to be effective in the kinetic resolution of (RS)-1-phenyl-1-ethanol via transesterification with various acyl donors, selectively forming the (R)-enantiomer. This process yielded a 16% conversion to (R)-1-phenylethyl propionate and an enantiomeric ratio (E) exceeding 200 after 72 h. These results demonstrate the potential of PSFS for applications in ester synthesis and resolution of enantiomerically pure sec-alcohols.

1. Introduction

Pycnoporus sanguineus is recognized for its ability to produce various enzymes of biotechnological interest, particularly ligninolytic enzymes such as laccases [1,2,3], peroxidases, and manganese peroxidases, as well as some hydrolases [4,5,6,7] that can be used in a wide range of industrial processes. However, to the best of our knowledge, there are no reports describing lipases from this fungus.

Lipases (EC 3.1.1.3) catalyze the hydrolysis of triacylglycerols found in oils and fats at the oil-water interface, converting them into free fatty acids, diacylglycerols, monoacylglycerols, and glycerol [8]. These enzymes also catalyze esterification and transesterification reactions in water-restricted environments, making them highly valuable for industrial applications [9,10,11]. Microbial lipases are highly valued for their remarkable stability across broad temperature and pH ranges as well as their sustained catalytic activity in the presence of organic solvents [12]. Additionally, their pronounced regio- and enantioselectivity toward a wide variety of substrates make microbial lipases especially suitable for the synthesis of optically pure compounds, including pharmaceuticals, agrochemicals, and structurally modified lipids [13,14].

Several studies have investigated the use of solid-state fermentation (SSF) for catalytic applications, with particular emphasis on its potential in biodiesel production [15,16] Additionally, SSF is a promising strategy for producing aroma esters that are widely used in the food, pharmaceutical, and cosmetic industries [17]. Another important application of fermented solids is the production of pure enantiomers through kinetic resolution of racemates [18,19,20,21]. Enantioselectivity is crucial in the pharmaceutical industry, and some commercial lipases already exhibit high enantioselectivity [22,23]. However, the use of lipases for industrial-scale kinetic resolution remains limited owing to the high cost of biocatalysts [21,24].

SSF offers an economical alternative for lipase production by utilizing agricultural and domestic waste as substrates, which can significantly reduce the cost of biocatalysts. This approach eliminates the expensive concentration, purification, and immobilization steps required in submerged fermentation processes, allowing the direct use of fermented solids in reaction media. This is particularly advantageous for the production of biotechnologically relevant compounds in the pharmaceutical industry, where reducing lipase costs directly lowers the manufacturing costs of these compounds [18].

To our knowledge, this is the first report describing the use of fermented solids containing lipases from Pycnoporus sanguineus as biocatalysts for esterification and kinetic resolution reactions. The resulting biocatalyst exhibited high catalytic efficiency in both processes, together with remarkable enantioselectivity. These findings highlight the unexplored potential of P. sanguineus-derived lipolytic activity for sustainable ester synthesis and the preparation of enantiomerically pure secondary alcohols.

2. Materials and Methods

2.1. Reagents and Media

The reagents used were ethanol (99.5%) from Êxodo (Sumaré, RJ, Brasil) and n-heptane (99.5%) and n-heptane (99.5%) from Vetec (Rio de Janeiro, Brazil). Decanoic acid (C10:0) (98%), lauric acid (C12:0) (99%), myristic acid (C14:0) (99%), tricaprylin (99%) and triolein (65%) (RS)-1-phenyl-1-ethanol (99%), ethyl acetate (99.5%), isopropenyl acetate (99%), vinyl butyrate (99.5%), and vinyl propionate (99%) were obtained from Sigma-Aldrich (San Louis, MO, USA).

Ground sugarcane bagasse used for the preparation of the SSF medium was kindly provided by a sugar and alcohol company located in Alagoas, Brazil. Wheat bran waste was donated by a local company in Maceió, Alagoas. The chemical composition was determined according to the official methods of the Association of Official Analytical Chemists [25], with results expressed in g 100 g of dry matter for crude protein, neutral detergent fiber, acid detergent fiber, lignin, cellulose, hemicellulose, mineral matter, and ether extracts. The experiments were performed with three replicates, and results were expressed as mean ± standard deviation. Waste frying oil was also donated by a local source in Maceió, Alagoas. All other reagents were of analytical grade and purchased from Brazilian suppliers.

2.2. Microorganism

The filamentous fungus Pycnoporus sanguineus was isolated from the basidiocarp present on dried sugarcane stalks, obtained at the A.C. Simões UFAL campus, and identified through morphological analysis. The P. sanguineus fungal strains were grown for 96 h, respectively, in potato dextrose agar medium with 1% (v v⁻¹) inducer (residual frying oil) at 25 °C and then kept at 4 °C.

2.3. Test Trial for the Production of Lipases in Potato Dextrose Agar with Waste Frying Oil

Filamentous fungi were tested for lipase production according to the methodology described by Kouker and Jaeger [26]. Potato dextrose agar culture medium was prepared with 3% residual frying oil and 4 g L⁻¹ NaCl, sterilized by moist heat in a vertical autoclave at 121 °C and 1 atm for 20 min. Then 0.001% rhodamine B was added (sterilized through filtration with a 0.22 µm membrane). The culture medium (20 mL) was added to the Petri dishes.

The plates were incubated for seven days in a bacteriological oven (Sterilifer—SX1.0DTMC, São Paulo, Brazil) at 25 °C. Subsequently, they were subjected to ultraviolet radiation at 350 nm, and lipase production was evidenced by the formation of an orange fluorescent halo on the surface of the plates [27], which was measured to estimate the apparent lipase activity.

After the fungi had grown on the plates, the growth rate was calculated as the ratio (average colony diameter in cm/growth time in days) of cm day⁻¹. The apparent lipase activity was calculated as the ratio between the average diameter of the halos in centimeters and time in days (cm day⁻¹). The experiment was performed in triplicate, and a negative control was prepared under the same conditions without fungal inoculation.

2.4. Solid State Fermentation (SSF) and Obtaining Solid Fermented by P. sanguineus (SFPS)

Pycnoporus sanguineus, a filamentous basidiomycete, was pre-cultivated on potato dextrose agar (PDA) plates supplemented with 1% used frying oil to induce enzymatic activity. The cultures were incubated at 25 °C for 7 to 9 days to allow for robust mycelial growth. Following incubation, uniform agar plugs approximately 1 ± 0.5 cm in diameter were excised and used as inoculum for SSF. The SSF experiments were conducted in 100 mL Erlenmeyer flasks, each containing 5 g of agricultural by-products (

Table 1. Solid-state fermentation conditions for different agricultural by-products.

Agricultural by-Products	Initial Moisture (%)
Wheat bran	60
Wheat bran and sugarcane bagasse (1:1)	70
Wheat bran, sugarcane bagasse (1:1) and RFO (3%);	70
Wheat bran, sugarcane bagasse (1:1) and urea (3%);	70
Wheat bran, sugarcane bagasse (1:1), RFO (3%) and urea (3%).	70

RFO = residual frying oil.

) and functioning as individual bioreactors. Prior to inoculation, the flasks were sterilized in a vertical autoclave (Fanem 415, São Paulo, Brazil) at 121 °C for 20 min under 1 atm pressure. After cooling, three mycelial plugs were aseptically introduced into each flask, which were then incubated at 27 °C in a bacteriological incubator (Sterilifer SX1.0 DTMc, São Paulo, Brazil). All fermentations were performed with three replicates and the results were expressed as mean ± standard deviation.

Following the fermentation process, enzyme extraction was performed by adding sodium phosphate buffer (0.1 mol L⁻¹, pH 7.0) containing 0.6% NaCl at a ratio of 5 mL per gram of fermented substrate. The mixture was subjected to orbital shaking at 165 rpm for 40 min at 27 °C to facilitate enzyme release. Subsequently, the contents were passed through a mesh filter and centrifuged at 15,000× g for 10 min at 4 °C. The resulting supernatant was collected and designated as the crude enzyme extract. Enzymatic activities in the crude extracts were determined according to the procedures described in Section 2.5.

2.5. Determination of Lipolytic and Synthetic Activities

2.5.1. Determination of Hydrolysis Activity in the Crude Enzyme Extract Against Synthetic Substrate P-nitrophenyl Palmitate (p-NPP)

Lipase activity was determined based on the spectrophotometric quantification of p-nitrophenol (p-NP) released during the hydrolysis of p-nitrophenyl palmitate (p-NPP), following the protocol adapted from Ferreira et al. [28]. Two working solutions were prepared: Solution A, consisting of 60 mg of p-NPP dissolved in 20 mL of isopropyl alcohol; and Solution B, composed of 4 g of Triton X-100 and 0.4 g of gum arabic dissolved in 200 mL of sodium phosphate buffer (0.1 M, pH 7.0). The enzymatic assays were carried out in microplate wells by combining 40 μL of Solution A, 120 μL of Solution B, and 40 μL of the crude enzyme extract. Two control blanks were included: a matrix blank (lacking the substrate) and a total blank (lacking the enzyme extract). Reactions were incubated at 40 °C for 1 h, after which p-nitrophenol formation was quantified at 410 nm using a FlexStation 3 microplate reader (Molecular Devices, San Jose, CA, USA).

One unit (U) of lipase activity was defined as the amount of enzyme required to release 1 μmol of p-NP per minute under the assay conditions, using the molar extinction coefficient of p-NP at 410 nm (ε = 9780 M⁻¹ cm⁻¹). Lipase activity was calculated using Equation (1), where Abs is the absorbance at 410 nm, Vt is the total reaction volume, ε is the molar extinction coefficient, Ve is the volume of enzyme extract used, t is the incubation time (min), D is the dilution factor, and 1000 is the conversion factor from mol to μmol.

$$AE\left({\displaystyle \frac{\mathrm{U}}{\mathrm{m}\mathrm{L}}}\right)={\displaystyle \frac{Abs\times Vt}{\epsilon \times Ve\times t}}\times D\times 1000$$

(1)

2.5.2. Determination of the Lipolytic Activities of the Fermented Solid in Aqueous Media Against Natural Substrates

Following fermentation, the triglyceride-hydrolyzing activity of the PSFS was evaluated using a titrimetric approach. Two separate assays were conducted, each employing a specific triacylglycerol substrate—tributyrin or triolein—at a final concentration of 67 mmol L⁻¹. The reaction emulsion was composed of 3% (w/v) gum arabic, 2 mmol L⁻¹ CaCl₂, 2.5 mmol L⁻¹ Tris-HCl, and 150 mmol L⁻¹ NaCl. For each test, 100 mg of the dried PSFS was added to 20 mL of the prepared emulsion and stirred vigorously for 5 min in a temperature-controlled vessel. The enzymatic hydrolysis of triglycerides was monitored by titrating the released free fatty acids with 0.05 mol L⁻¹ NaOH using a pH-Stat system (Metrohm 718 Stat Titrino, Herisau, Switzerland) maintained at pH 7.0. One unit (U) of lipolytic activity was defined as the amount of enzyme required to release 1 μmol of fatty acid per minute under the assay conditions.

2.5.3. Determination of the Lipolytic Activities of the Fermented Solid in Organic Media

To evaluate hydrolytic activity in an organic medium, 10 mL of the reaction mixture were transferred into 12 mL hermetically sealed glass vials equipped with Teflon-lined caps, each containing 600 mg of lyophilized PSFS. The reaction system comprised 9.8 mL of n-heptane, 70 mmol L⁻¹ triolein, and 0.2 mL of distilled water (2% v/v). The vials were incubated at 40 °C under continuous agitation at 180 rpm. At predetermined time intervals, 100 μL aliquots were withdrawn from the reaction mixture and analyzed for residual free fatty acid content using the Lowry–Tinsley colorimetric method [29]. One unit (U) of lipase activity was defined as the amount of enzyme that catalyzes the release of 1 μmol of fatty acids per minute under the specified experimental conditions.

2.5.4. Determination of the Esterification Activities of the Fermented Solid in Organic Media

Esterification reactions were carried out in n-heptane to evaluate the catalytic activity of the PSFS in ethyl ester synthesis [30]. To investigate the influence of fatty acid chain length on enzymatic performance, three saturated fatty acids were tested: decanoic acid (C10:0), lauric acid (C12:0), and myristic acid (C14:0).

Reactions were conducted in 12 mL screw-capped glass vials containing 600 mg of dry PSFS and 9.8 mL of n-heptane supplemented with 210 mmol L⁻¹ ethanol and 70 mmol L⁻¹ of the respective carboxylic acid. The mixtures were incubated at 40 °C under agitation at 200 rpm. At regular intervals, 100 μL aliquots were collected and analyzed for residual free fatty acids using the Lowry–Tinsley colorimetric method [29].

One unit (U) of esterification activity was defined as the amount of enzyme capable of converting 1 μmol of fatty acid per minute under the specified conditions. In this study, both hydrolytic and esterification activities are expressed as units per mL (U mL⁻¹).

2.6. Kinetic Resolution of (RS)-1-phenyl-1-ethanol Catalyzed by the Lipases Present in the Dry Fermented Solid Produced by P. sanguineus (SFPS)

Kinetic resolution reactions were performed in hermetically sealed 12 mL glass vials, each containing 600 mg of PSFS, corresponding to 0.6 U of triolein-hydrolyzing activity (based on a specific activity of 1.0 U g⁻¹). The reaction medium consisted of 8 mL of n-heptane containing racemic 1-phenyl-1-ethanol (12.5 mmol L⁻¹) and vinyl acetate (50 mmol L⁻¹) as the acyl donor. Reactions were carried out in an orbital shaker at 200 rpm and 40 °C.

To assess the impact of acyl donor type on enzymatic resolution, alternative esters—ethyl acetate, isopropenyl acetate, vinyl propionate, and vinyl butyrate—were also tested under the same conditions. At designated time points, 40 μL aliquots were collected and analyzed by gas chromatography using a chiral column (as described in Section 2.7).

The absolute configurations of the resulting alcohol and ester enantiomers were determined by comparing retention times with those obtained from parallel reactions catalyzed by Candida antarctica lipase B (CALB), a biocatalyst with well-established (R)-enantiopreference for these substrates [31]. Enantiomeric excess values for both substrate (ee_s, %) and product (ee_p, %) were calculated as follows:

$${ee}_s(\mathrm{\%})={\displaystyle \frac{[S-R]}{[R+S]}}\times 100$$

(2)

$${ee}_p(\mathrm{\%})={\displaystyle \frac{[R-S]}{[R+S]}}\times 100$$

(3)

where R is the concentration of the (R)-enantiomer (substrate or product), and S is the concentration of the (S)-enantiomer (substrate or product).

The conversion (c, %) and enantiomeric ratio (E) were calculated from the ee_s and ee_p values according to [32].

$$c\left(\mathrm{\%}\right)={\displaystyle \frac{\left[{ee}_s\right]}{{[ee}_s+{ee}_p]}}\times 100$$

(4)

$$E={\displaystyle \frac{\mathrm{l}\mathrm{n}\left[\left(1-c\right)\left(1-{ee}_s\right)\right]}{\mathrm{l}\mathrm{n}\left[\left(1-c\right)\left(1+{ee}_s\right)\right]}}$$

(5)

2.7. Gas Chromatography

Samples obtained from the kinetic resolution experiments were analyzed using gas chromatography on a GC-17A system (Shimadzu Co., Kyoto, Japan) equipped with a hydrogen flame ionization detector (FID) and a chiral capillary column (CP Chirasil-DEX CB; 25 m × 0.25 mm × 0.25 μm). Injections were performed in split mode, with an injection volume of 0.5 μL. Nitrogen was used as the carrier gas at a constant flow rate of 1 mL min⁻¹. The injector and detector temperatures were set at 245 °C. The oven temperature was initially held at 110 °C and then ramped at 1 °C min⁻¹ until reaching 120 °C, where it was maintained for 10 min.

3. Results and Discussion

3.1. Chemical Composition of Wheat Bran

The chemical composition of raw wheat bran was analyzed to quantify the nutrients present and evaluate its use in SSF as a support for the growth of P. sanguineus.

Table 2. Chemical composition of wheat bran in 100 g of dry solid.

Components (%)	% (g 100 g−1)
Crude protein	20.1 ± 0.9
Ether extract	88.9 ± 0.2
Mineral material	6 ± 0.5
NDF	53.6 ± 2.8
ADF	13.7 ± 1.1
Ash	3.9 ± 1.9
Lignin	3.5 ± 0.5
Cellulose	10.3 ± 1.3
Hemicellulose	39.9 ± 1.9

Abbreviations: NDF, neutral detergent fiber; ADF, acid detergent fiber. (n = 3, mean ± standard deviation).

shows the percentages of the components analyzed based on the dry matter of the wheat bran. Similar results were obtained by Ghodrat et al. [33], with contents of crude protein (15.7% ± 0.8), neutral detergent fiber (45% ± 2), acid detergent fiber (13.8% ± 0.7), lignin (4% ± 0.2), cellulose (9.6% ± 0.5) and hemicellulose (31.6% ± 1.6). However, the ether extract content was 2% ± 0.1, considerably lower than that found in this study. According to Apprich et al. [34], the chemical composition of wheat bran can vary due to intrinsic factors, such as the variety of wheat used, and extrinsic factors, such as the soil, growing conditions, and storage, which explain the observed differences.

Wheat bran is composed of lignocellulose, the most abundant biomass in nature, and is primarily composed of lignin, cellulose, and hemicellulose. Wheat bran is primarily composed of lignocellulose, the most abundant biomass in nature, and consists largely of lignin, cellulose, and hemicellulose. However, its relatively high protein content and low lignin concentration distinguish it from other lignocellulosic biomasses [34]. These unique characteristics make wheat bran particularly suitable for biotransformation processes, as it offers not only lignocellulosic material but also a rich source of proteins and fats. In such processes, microorganisms utilize organic carbon from lignocellulose along with the proteins and lipids present as essential nutrients for enzyme production [35].

3.2. Preliminary Test of Lipase Production by P. sanguineus in Potato Dextrose Agar with Residual Frying Oil

A lipase production test using the filamentous fungus P. sanguineus grown on potato dextrose agar with used frying oil and rhodamine B yielded positive results. An orange halo formed around the colonies, indicating lipase activity. This color change was caused by the reaction of fatty acids produced during enzymatic hydrolysis with rhodamine B. Based on this observation, we confirmed that P. sanguineus is a lipase producer. The calculated growth rate was 1.32 cm day ⁻¹, while the apparent lipase activity rate was 0.34 cm day ⁻¹. The results were obtained by calculating the mean colony diameter and halo size during the growth period. The experiment was performed in triplicate, and a negative control without fungal inoculum was included under identical conditions. The observed growth rate of 1.32 cm day⁻¹ indicates that P. sanguineus demonstrates steady and moderate growth on a medium supplemented with residual frying oil. The corresponding apparent lipase activity suggests a proportional relationship between fungal biomass development and enzyme production. The use of waste frying oil likely enhances lipase synthesis by serving as a rich source of triglycerides, thereby stimulating enzymatic hydrolysis. These findings highlight P. sanguineus as a promising candidate for sustainable lipase production, particularly in industrial processes that utilize low-cost renewable substrates.

In other studies, the same procedure was used to estimate lipase production by the fungi Trichothecium roseum [36] and Rhizomucor sp. [37]. According to Singh and Mukhopadhyay [38], preliminary tests for detecting lipase activity in filamentous fungi on plates are highly useful. However, it is important to consider the resulting variability, which can depend on factors such as the composition of the culture medium and cultivation conditions, including carbon and nitrogen sources, pH, and temperature. Additional challenges may arise from rapid colony growth in certain species, low lipolytic activity in others, and the possibility of dye reactions with nonenzymatic metabolites, which could lead to misleading results. The filamentous fungus studied here was inoculated into SSF using different agricultural by-products for lipase production.

3.3. Production of Lipases by P. sanguineus by SSF

P. sanguineus produced lipases in SSF using wheat bran and sugarcane bagasse, as well as wheat bran and sugarcane bagasse, in addition to frying oil and/or urea (Figure 1).

The highest lipase production by P. sanguineus in SSF was achieved with wheat bran, which showed an enzymatic activity of 24 U mL⁻¹ against p-NPP (Figure 1). Sugarcane bagasse was added to wheat bran as a spacer to facilitate fungal hyphal growth between substrate particles; however, this did not result in increased production. Similarly, the lipase inducer residual frying oil was added to the solid medium, yet lipolytic activity was lower under these conditions (Figure 1). The addition of urea as a nitrogen source also led to a marked decrease in lipase production, with ~79% reduction in activity compared to the control. This inhibitory effect may be related to the rapid hydrolysis of urea and the subsequent accumulation of ammonia, which can repress lipase secretion. Although only one concentration was tested in this study, it is possible that varying urea concentrations could modulate the production profile, which merits investigation in future studies.

The addition of urea as a nitrogen source also led to a significant decrease in lipase production, with more than 70% reduction in activity compared to the control medium containing wheat bran as the nitrogen source.

In a study on lipase production by the filamentous fungi Aspergillus sp., Fusarium sp., and Penicillium sp. using SSF with various substrate combinations (sugarcane bagasse, wheat bran, and soybean meal), different formulations were tested to enhance the enzyme yield [39]. The highest lipase activity for Aspergillus sp. was observed with a substrate mixture of 7.5 g wheat bran, 2.5 g sugarcane bagasse, and 7.5 g soybean meal, reaching approximately 11 U mL⁻¹. A similar level of activity was recorded for Fusarium sp. under the same conditions, and for Penicillium sp., optimal lipase production (10 U mL⁻¹) occurred using 7.5 g of soybean meal and 2.5 g of sugarcane bagasse [39]. In contrast, the addition of sugarcane bagasse negatively impacted lipase production by Fusarium sp., which showed its highest activity (2.5 U mL⁻¹) when cultivated with 5.0 g each of soybean meal and sugarcane bagasse [39].

Hermansyah et al. [40] investigated lipase production by the filamentous fungus Rhizopus oryzae during SSF and submerged fermentation (SF). To optimize enzyme production, they varied the substrate concentration (wheat bran) from 0.5% to 2% and the inducer concentration (coconut oil) from 2% to 8%. The highest lipase production was observed in SF, with an activity of 63 U mL⁻¹, using 6.9% inducer and 1.9% substrate. The authors suggested that increasing the lipid concentration enhanced both intracellular and extracellular lipase activities. However, high lipid concentrations can cause cytotoxicity. A low substrate concentration is preferred because higher carbon sources can inhibit extracellular lipase production.

In this study, lipase production by P. sanguineus was not enhanced by substrate combinations or the addition of inducers. The highest enzyme production was achieved using wheat bran waste without the addition of additional nutrients. The chemical composition of the waste was sufficient to induce fungal lipase production by the fungus (Figure 1). It is likely that the residual frying oil was toxic to microorganisms, as temperature-induced structural changes in the oils during food preparation could have occurred.

Sujatha and Dhandayuthapani [41], in their study on the production conditions of lipases by Bacillus licheniformis KDP in SF, observed that lipase activity decreased as the urea concentration in the medium increased. At 0.1 mg L⁻¹ of urea, the activity was 12 U mL⁻¹, but it dropped to less than 1.0 U mL⁻¹ when the urea concentration was raised to 1.0 mg L⁻¹. In contrast, lipase production increased when the ammonium nitrate concentration was elevated. The authors have suggested that urea may be toxic to microorganisms by inhibiting lipase activity.

Tan et al. [42], in their study on lipase production by Penicillium camembertii Thom PG-3 under SF conditions using a medium containing 4% soybean meal, 75% olive oil, and other components, have reported that inorganic nitrogen sources such as ammonium (NH₄⁺) and nitrate (NO₃⁻) enhanced lipase production, whereas organic sources like urea had an inhibitory effect. Fermentation supplemented with 0.1% (v/v) ammonium sulfate, ammonium nitrate, and urea yielded enzyme activities of 126, 70, and 25 U mL⁻¹, respectively. Similarly, Kumar et al. [43] investigated lipase production by Bacillus coagulans BTS-3 and found that the enzyme yield varied with the nitrogen source used. Fermentations with 0.1% (v/v) ammonium sulfate, yeast extract, and urea resulted in activities of 0.42, 0.36, and 0.12 U mL⁻¹, respectively.

In this study, lipase production by P. sanguineus was negatively affected by the presence of urea in the culture medium (Figure 1). It is possible that urea is toxic to the fungus or interferes with the metabolic regulation of enzyme production. The fermented and freeze-dried solids containing P. sanguineus lipases derived from the same SSF were then applied as biocatalysts in esterification reactions and kinetic resolution via transesterification.

3.4. Application of the Fermented Solids That Contained Lipases from P. sanguineus

Hydrolysis Activity and Esterification Reactions Using the P. sanguineus Lipases Present in the Dry Fermented Solids

The hydrolytic activity of the dry SFPS against olive oil and tributyrin was 10.2 ± 0.6 and 100.2 ± 1.2 U mL⁻¹, respectively, indicating a preference for a triglyceride with shorter-chain fatty acids. Tributyrin offers advantages as a lipase substrate, such as ease of dispersion without the use of detergents and water-soluble hydrolysis products [44]. Additionally, its small size may facilitate access to the catalytic sites of the enzyme. The performance of PSFS in esterification reactions using fatty acids with varying chain lengths was also evaluated. The results presented in

Table 3. Synthesis of ethyl esters catalyzed by lipases present in the dry fermented solid by P. sanguineus (PSFS).

Acyl Donor	Conversion (%)
	1.5 h	24 h	28 h
Decanoic acid (C10:0)	17 ± 0	86 ± 2	95 ± 2
Lauric acid (C12:0)	15 ± 3	78 ± 3	89 ± 3
Myristic acid (C14:0)	8 ± 0	72 ± 4	74 ± 1

Reaction conditions: 600 mg of SFPS in 10 mL of the reaction medium. Ethanol 210 mmol L-1, fatty acid 70 mmol L ⁻¹ in n-heptane. For the tests, the molar ratio (ethanol)/(fatty acid) was 3:1, at 40 °C and 200 rpm. Residual fatty acid content was analyzed using the Lowry–Tinsley method. The values presented are the mean of triplicate tests ± the standard error of the mean.

demonstrate the preference of SFPS for short-chain fatty acids (C10).

The highest conversion was 95% when decanoic acid was used in the synthesis of ethyl decanoate. This was followed by 89% conversion when lauric acid was used in the synthesis of ethyl laurate and 74% conversion to myristate when myristic acid was used as the acyl donor. SFPS showed potential for catalyzing ester synthesis reactions with high conversion values. Thus, SFPS can be an alternative biocatalyst in industrial applications because several industrialized products are made up of esters, such as biofuels and emollients for cosmetics and aromas [16,45]. Soares et al. [16] have reported the production of an SF composed of sugarcane bagasse, 23% residual palm oil, and a nutrient solution by Penicillium roqueforti ATCC 10110 after 72 h of fermentation. The fermented solid exhibited a hydrolytic activity of 31.8 ± 3 U mL⁻¹ S against olive oil. The same preparation could also catalyze the synthesis of ethyl oleate in n-heptane at 40 °C, achieving a 36% yield after 96 h. The fermented solid catalyzed the esterification of oleic acid with ethanol in a solvent-free system, achieving a 69% conversion in 48 h [46].

Long-chain ethyl esters (C10-C14), including ethyl oleate, are used in many cosmetic components, such as emollients in sunscreens and emulsion-forming agents [47,48]. Some of these esters are found in fine perfumes, shampoos, soaps, and other personal care products, as well as in non-cosmetic products such as household cleaners and detergents. Their use worldwide is in the order of millions of tons per year [49]. Given their industrial relevance, several studies have reported the synthesis of the ethyl esters of capric, lauric, and myristic acids using lipases in various reaction media (

Table 4. Examples of esterification reactions of different carboxylic acids with ethanol catalyzed by lipases.

Ester	Source of lipase	Conditions	Conversion (%)	Reference
Ethyl caprate	A. niger	Ethanol (5 g L−1), capric acid 10 mM, 8 h, 28 °C	85	[50]
Ethyl laurate	C. antarctica B	Ethanol (5.8 mol L−1), lauric acid (2.9 mol L−1), microwave (850 W), 10 min, 45 °C	98.5	[51]
Ethyl laurate	C. antarctica B	Ethanol (17.12 mol L−1), lauric acid (5.02 mol L −1), ultrasonic bath (100 W), 40 min, 50 °C	96.87	[52]
Ethyl laurate	C. antarctica B	Ethanol (50 mmol), lauric acid (50 mmol), 10 min, 60 °C	92.5	[53]
Ethyl laurate	A. flavus	Ethanol (0.0625 mmol), lauric acid (0.125 mmol), 24 h, 40 °C	95	[54]
Ethyl myristate	B. cepacia	Ethanol, lauric acid molar ratio (8:1), microwave (50 W), 25 min, 45 °C	Productivity of 0.12 mol h−1 mg−1	[55]

).

In the study of Xu et al. [50], the enzymatic synthesis of ethyl caprate has been reported for the first time using a strain of A. niger–designated CGMCC–which was characterized to efficiently synthesize ethyl esters of fatty acids in an aqueous phase using whole cells. Optimal culture conditions for lipase production included a culture temperature of 28 °C, with the culture medium consisting of lactose as the carbon source and (NH₄)₂SO₄ as the nitrogen source. The ester conversion value reached 85% in 8 h. Jaiswal and Rathod [51] used microwave-assisted synthesis of ethyl laurate using lauric acid and ethanol catalyzed by CALB. The effects of operational parameters such as the molar ratio (lauric acid/ethanol), enzyme loading, temperature, and molecular sieves were systematically studied. A maximum conversion of 98.2% was obtained in 10 min compared with the conventional method, where the reaction required 4 h to reach 92.4% conversion. The optimum parameters for microwave-assisted synthesis were a 1:2 molar ratio of lauric acid to ethanol, temperature of 45 °C, enzyme amount of 1.8% (w/w), and molecular sieves of 1.5% (w/w).

Gawas and Rathod [52] focused on the intensification of CALB-catalyzed ethyl laurate synthesis from ethanol and lauric acid under ultrasound-assisted solvent-free conditions but with the stepwise addition of ethanol. The optimal conditions were found to be 1:2 lauric acid/ethanol molar ratio, 2% enzyme loading, 50 °C temperature, and 40 min reaction time. The highest conversion of 96.87% was obtained with the stepwise addition of ethanol in the presence of ultrasound and mechanical stirring. Kanwar et al. [56] have reported 66% conversion of ethyl laurate in n-nonane at 65 °C after 15 h by hydrogel immobilized lipase of Bacillus coagulans MTCC-6375. Solarte et al. [54] enhanced the synthesis of ethyl laurate to 95% yield in 24 h by using the whole cell lipase from A. flavus. This result was similar to that described by Fernandes et al. [57], who reported a 92% yield of ethyl laurate using Lipolase, a commercial preparation containing the lipase from Thermomyces lanuginosus. Recently, Carvalho et al. [55] applied bioimprinting techniques to B. cepacia lipase with myristic acid. In the reaction with the best esterification results with ethanol and myristic acid under microwave radiation, the reaction time decreased from 24 h using conventional heating to 25 min (MW), and the productivity increased 20-fold compared with the reactions under conventional heating. In both assays, the temperature was maintained at 45 °C.

In comparison to the aforementioned studies, the PSFS is noteworthy not for its reaction rate, since other enzymes have been reported to achieve similar conversions within minutes, but rather for being prepared solely with agricultural by-products and still demonstrating 95% conversion in ester synthesis after 28 h of reaction (Table 3). To the best of our knowledge, this is the first report describing the production of fermented solids containing lipases from P. sanguineus with lipolytic activity and their application in catalyzing esterification reactions.

3.5. Kinetic Resolution of (RS)-1-phenyl-1-ethanol Catalyzed by the Lipases Contained in PSFS

The acyl donors ethyl acetate, isopropenyl acetate, vinyl acetate, vinyl propionate, and vinyl butyrate were evaluated for the transesterification of (RS)-1-phenyl-1-ethanol (

Table 5. Evaluation of acyl donors in the transesterification of (RS)-1-Phenyl-1-ethanol by lipases present in the dry fermented solid by P. sanguineus (PSFS).

Entry	Acyl Donor	c (%)	ee (%)		E
			Alcohol-(S)	Ester-(R)
1	Ethyl acetate	3	3	>99	>200
2	Isopropenyl acetate	12	14	>99	>200
3	Vinyl acetate	9	10	>99	>200
4	Vinyl propionate	16	18	>99	>200
5	Vinyl butyrate	13	12	>99	>200

Key: c = conversion to ester (%); ee_s = enantiomeric excess of alcohol (S) (%); ee_p = enantiomeric excess of ester (R) (%); E = enantiomeric ratio. Conversion (c) and E were calculated from ee_s and ee_p, according to Chen [32]. Reaction conditions: 0.6 U (triolein hydrolysis activity) of SFPS in 8 mL of reaction medium (12.5 mmol L⁻¹ (RS)-1-phenyl-1-ethanol and 50 mmol L ⁻¹ acyl donor) at 200 rpm for 72 h.

). When ethyl acetate was used as the acyl donor, the conversion was very low (3%), although the enantioselectivity (E) was high (>200). This poor performance is consistent with the known low reactivity of ethyl acetate in lipase-catalyzed resolutions, since the ethanol formed as a byproduct can reverse the equilibrium and reduce product formation. In contrast, vinyl and isopropenyl esters release acetaldehyde or acetone, respectively, which are unstable and help to shift the equilibrium towards ester production, explaining their comparatively higher conversions.

Given the high E-values obtained with vinyl acetate and isopropenyl, the acyl donor was altered with respect to the size of the acyl donor chain to improve the conversion. Vinyl butyrate yielded a conversion of 13% (Table 5, entry 5), as did isopropenyl acetate with a 12% conversion (Table 5, entry 2). The highest conversion, 16% after 72 h, was achieved using vinyl propionate as the acyl donor (Table 5, entry 4). This result suggests that, while enantioselectivity can be high with some acyl donors, the conversion rates vary significantly. The use of vinyl propionate appeared to offer the best compromise between conversion and enantioselectivity, showing the potential for optimizing the reaction conditions based on the choice of the acyl donor. Although the enantioselectivity values obtained were remarkable, the overall conversions were modest, with the best result being 16% after 72 h using vinyl propionate. This corresponds to a low mass productivity, which reflects the exploratory character of this study and the use of crude fermented solids as biocatalysts. Nevertheless, this is the first report of P. sanguineus-derived lipolytic activity applied to kinetic resolution, and further optimization of reaction parameters such as substrate loading, biocatalyst concentration, and reaction time will be necessary to improve productivity and evaluate the true industrial potential of this system.

Although enantioselectivity values were remarkably high (E > 200), the overall conversions were modest (3–16%). This may be attributed to substrate or product inhibition, mass transfer limitations associated with the crude fermented solid, low density of accessible active sites, or the limited reactivity of some acyl donors under the tested conditions. Further optimization of substrate concentration, biocatalyst loading, and water activity will be required to overcome these limitations and enhance the productivity of the system.

The lipases contained in the PSFS showed a preference for the (R)-enantiomer at the kinetic resolution of (RS)-1-phenyl-ethanol. Typically, enantioselective lipases show a preference for the (R)-enantiomer [53]. (RS)-1-phenyl-1-ethanol is commonly used as a standard compound to assess the enantioselectivity of lipases toward secondary aromatic alcohols. In the case of (R)-enantioselective lipases, this enantiopreference was consistent with that of other substrates [21,58]. The use of lipases to catalyze the kinetic resolution of secondary alcohol racemates is being recognized as an alternative route to obtain pure chiral products [19,59]. Enantiomerically pure secondary alcohols have significant applications in the synthesis of both natural and synthetic compounds, such as chiral drugs, fragrances, and pheromones, and their production has broad implications for industries, including pharmaceuticals and cosmetics [19,60].

Despite the growing interest in using lipases for catalyzing transesterification reactions to obtain enantiomerically pure alcohols, establishing commercial processes requires low-cost biocatalysts [19,61]. In this context, the use of lipolytic fermented solids as biocatalysts for esterification and transesterification has emerged as a promising and economically viable alternative. This approach minimizes the need for enzyme extraction, purification, and immobilization steps, significantly reducing production costs [15,62,63]. Nagy et al. [20] studied the production of fermented solids containing wheat bran (9 g) and olive oil (1 g), moistened with saline solution, and produced during the screening of 38 fungi. Dried fermented solids with lipolytic activity were used for the kinetic resolution of the secondary alcohols (RS)-1-phenyl-1-ethanol, (RS)-1-cyclohexyl-1-ethanol, and (RS)-1-naphthyl-2-ethanol. Almost all fermented solids showed enantiopreference for the (R)-enantiomer at the resolution of (RS)-1-phenyl-1-ethanol, with conversions of approximately 50% and enantioselectivity ratios (E) greater than 200. In this study, the P. sanguineus SFPS offered an advantage in terms of simplicity of preparation, as it was produced using only wheat bran without any additional supplementation. This biocatalyst effectively catalyzed the kinetic resolution of (RS)-1-phenyl-1-ethanol via transesterification, exhibiting clear enantioselectivity toward the (R)-enantiomer, with a 16% conversion of vinyl propionate and an enantiomeric ratio (E) exceeding 200 (Table 5). These results highlight the potential of SFPS for the synthesis of enantiomerically pure secondary alcohols.

4. Conclusions

The filamentous fungus P. sanguineus successfully produced lipases via SSF, with optimal enzyme production achieved using wheat bran as the sole substrate without the need for additional inducers or nutrients. Under conditions of 60% initial moisture and 96 h of fermentation, an enzymatic activity of 24 U mL⁻¹ was obtained. The resulting fermented solid demonstrated catalytic efficiency in esterification reactions, reaching a 95% conversion of capric acid to ethyl caprate within 28 h of the reaction. Additionally, it catalyzed the kinetic resolution of (RS)-1-phenyl ethanol through transesterification, showing high enantioselectivity for the (R)-enantiomer, with 16% conversion of vinyl propionate after 72 h and an enantiomeric ratio (E) exceeding 200. These findings highlight the potential of P. sanguineus fermented solids for application in ester synthesis and the production of enantiomerically pure secondary alcohols. This is the first study to report lipase production by P. sanguineus in fermented solids and its application in enzymatic catalysis.