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Article

Fungal Biocatalysis in Stereoselective Oxidation of 2-Phenylethanol

by
Agnieszka Raczyńska
*,
Beata Szmigiel-Merena
,
Małgorzata Brzezińska-Rodak
,
Magdalena Klimek-Ochab
and
Ewa Żymańczyk-Duda
*
Department of Biochemistry, Molecular Biology and Biotechnology, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże Stanisława Wyspiańskiego 27, 50-370 Wrocław, Poland
*
Authors to whom correspondence should be addressed.
Symmetry 2025, 17(1), 17; https://doi.org/10.3390/sym17010017
Submission received: 5 December 2024 / Revised: 22 December 2024 / Accepted: 24 December 2024 / Published: 26 December 2024
(This article belongs to the Special Issue Chemistry: Symmetry/Asymmetry—Feature Papers and Reviews)
Browse Figures
Versions Notes

Abstract

:
Three fungal strains were employed for the stereoselective oxidation of the cheap and commercially available substrate 2-phenylethanol, which resulted in chiral building blocks being received. The whole-cell biocatalysts were as follows: Beauveria bassiana DSM 1344, Beauveria brongniartii DSM 6651, and Rhizopus arrhizus DSM 1185. The main product of Beauveria bassiana bioconversion was 1-phenylethane-1,2-diol, obtained, depending on the form of the biocatalyst, as an R-enantiomer (e.g., 99.9%) with fresh biomass application or as a racemic mixture in cases of immobilization in agar-agar. The best and most innovative results for the synthesis of the R-enantiomer of diol were received under precisely defined conditions as a result of a scaling study conducted on an automatic batch reactor. This is a pioneering result, since, in previous studies, fresh mycelium of Aspergillus niger resulted in this product being received as the (S) enantiomer. Also, the use of Rhizopus arrhizus DSM 1185 (immobilized in polyurethane foams) presented important results, as the bioconversion of phenyl ethanol led, indeed, to the racemic mixture of 1-phenylethane-1,2-diol but was accompanied by a noticeable tyrosol synthesis, which had not been reported previously.

1. Introduction

Whole-cell biocatalysis based on the use of free or immobilized fungal, bacterial, or plant cells is an important method for obtaining compounds with broad applications in the chemical, biofuel, pharmaceutical, and cosmetic industries [1,2,3,4]. The possibility of carrying out selective transformations under mild conditions with reduced waste generation means that biocatalyzed reactions are an attractive alternative to classical chemical methods. The biotransformations of compounds with expanded steric hindrance are of special interest, including polycyclic compounds, where the introduction of functional groups at specific location or/and through specific configurations is particularly difficult by classical methods. Fungal biocatalysts are valuable for this type of modification, especially since it is possible to direct the selectivity of the reaction by simply manipulating the form of the biocatalyst or by engineering the reaction conditions [5].
Entomopathogenic fungi of the genus Beauveria sp. are known for their broad spectrum of enzymatic activities; e.g., Beauveria bassiana is one of the most common fungal biocatalysts [6], and it is well known in steroid [7,8,9,10] and flavonoid biotransformations [11,12,13]. Its hydroxylation, oxidation, glycosidation, Baeyer–Villiger oxidation, and ester hydrolysis activities [6] allow for the production of compounds that are difficult to synthesize by traditional chemical methods.
Another example is the Rhizopus arrhizus strain, which is also widely used in biotransformation processes due to its hydroxylation and selective reduction activity. Its biocatalytic capabilities have been described in the literature and feature, among other elements, reducing arylalkanones [14] and acetophenone [15] in the synthesis of chiral secondary carbinols and halohydrins [16] and in the biotransformation of betulonic acid [17]. Similarly to B. bassiana, it has found application in the biotransformation of steroids [18].
From a practical point of view, an important group of compounds are low-molecular-weight polyphenols such as hydroxyl derivatives of 2-phenylethanol (Scheme 1). These compounds are applied because of their biological activity. For example, tyrosol (4-(2-hydroxyethyl)phenol) is a very well-known cosmetic ingredient [19]. It is one of the main phenolic compounds naturally occurring in olive oil [20]. In biotransformation studies, it serves as a substrate for obtaining hydroxytyrosol [21,22], and those derivatives have been reported as ones that exhibit antioxidant, anticancer, antiviral, or anti-inflammatory activities [23,24,25]. 1-Phenylethane-1,2-diol (as a racemic mixture or R-enantiomer) is another example of a meaningful compound. Racemic 1-phenylethane-1,2-diol is a common substrate in terms of receiving chiral building blocks via oxidation carried out under kinetic control, which leads to optically pure S- and R-enantiomers [26,27]. As an example, the enantioselective oxidation of racemic diol yielded in (R)-(-)-mandelic acid results in a compound of high importance in terms of defining the absolute configuration of selected chiral compounds, e.g., secondary alcohols, or compounds that are valuable intermediates in the synthesis of biologically active compounds (e.g., antibiotics, anti-cholinergic medications). Moreover, derivatives of (R)-mandelic acid are also used as chiral synthons in the synthesis of anti-thrombotic, anti-tumor and anti-obesity agents [28,29,30]. (R)-1-phenyl-1,2-ethanediol is applied as a biomarker in the exposure of humans to styrene [31] and as an intermediate used in production in the pharmaceutical [32,33,34], agrochemical, and chemical industries [33,34]. The biocatalytic preparation of this compound, without the burden of the environment, is an alternative to organic synthesis. The substrates in (R)-1-phenyl-1,2-ethanediol synthesis via biocatalytic methods are mainly 2-hydroxyacetophenone, racemic styrene oxide, and phenylglyoxal [35,36,37,38,39].
As an alternative biocatalytic pathway, the biohydroxylations of cheap, commercially available substrate- 2-phenylethanol [40,41,42] may be considered.
Symmetry 17 00017 sch001
Scheme 1. Biotransformation products of 2-phenylethanol obtained in previous studies [41,42].
Scheme 1. Biotransformation products of 2-phenylethanol obtained in previous studies [41,42].
Symmetry 17 00017 sch001
So far, the biotransformations of 2-phenylethanol have resulted in compounds such as hydroxytyrosol, tyrosol, (S)-1-phenylethane-1,2-diol, and 4-hydroxyphenylacetic acid [41,42] (Scheme 1). All these chemical compounds are widely used in various industries. Previous studies demonstrated that, among others, Aspergillus niger IAFB 2301 cells and Aspergillus niger (OPI) spores are capable of bioconverting 2-phenylethanol to its derivatives [41,42]. However, bioconversion of 2-phenylethanol into the opposite enantiomer—(R)-1-phenylethane-1,2-diol—has not been achieved yet. This was an assumption to further research with the next fungal strains.
In this study, three different fungal strains were applied for the biotransformation of 2-phenylethanol: Beauveria bassiana DSM 1344, Beauveria brongniartii DSM 6651, and Rhizopus arrhizus DSM 1185.
The biocatalytic capacity of biocatalyst cells is influenced by its preparation for the process. Therefore, methods such as immobilization (e.g., polyurethane foams [41], agar-agar, and alginate [43]) or pre-incubation under starvation conditions were checked [44]. Such modifications usually affect the reaction efficiency and the optical purity of the obtained product [5]. This depends on the applied conditions, as once altering the permeability of cellular envelopes (immobilization) or, in case of nutrient deficiency, inducing the activity of the enzymes, which are knocked out in the cells cultivated under optimal conditions.
The research includes a number of experiments, finally resulting in the receiving of the optically pure (R)-1-phenylethane-1,2-diol and tyrosol and also forming the racemic mixture of diol (Scheme 2).

2. Materials and Methods

2.1. Chemicals

Cultivation medium as recommended by DSMZ-German Collection of Microorganisms and Cell Cultures GmbH for B. brongniartii: 4 g of yeast extract, 15 g of soluble starch, 1 g of K2HPO4, and 0.5 g of MgSO4 × 7 H2O per liter; and for B. bassiana: 30 g of malt extract and 3 g of soya peptone per liter. Medium for R. arrhizus: potato dextrose broth (PDB): 4 g of potato extract and 20 g of glucose per liter.
Commercially available standards of (R)-1-phenylethane-1,2-diol, (S)-1-phenylethane-1,2-diol, 1-phenylethane-1,2-diol, tyrosol (4-(2-hydroxyethyl)phenol), 2-phenylethanol, Triton X-100, and sodium alginate were purchased from Sigma-Aldrich.
Ethyl acetate, anhydrous magnesium sulfate, ethanol (99.8%), n-hexane (HPLC), 2-propanol (HPLC), acetonitrile (HPLC), formic acid, glucose, agar-agar, and dipotassium phosphate were purchased from Avantor.
Agar-agar, soya peptone, malt extract, Potato Dextrose Broth (PDB), and Potato Dextrose Agar (PDA) were purchased from Biocorp; soluble starch was purchased from POCH, and yeast extract from VWR Chemicals.
Calcium chloride, barium chloride, and magnesium sulfate heptahydrate were purchased from Chempur.
Polyurethane foams (porosity of 740–1040 µm, 1060–1600 µm, or 2300–3300 µm) were purchased from Bogmar.

2.2. Microorganisms

Beauveria bassiana (DSM 1344), Beauveria brongniartii (DSM 6651), and Rhizopus arrhizus (DSM 1185) were purchased from the German Collection of Microorganisms and Cell Cultures.

2.3. Cultivation Conditions

2.3.1. Inoculum

Inoculum for microorganism cultivation consisted of their spores, which were recovered from sporulated Petri dishes with solid medium (PDA). Spores were washed with 10 mL of sterile Triton X-100 water solution (0.05%), suspended, and then transferred to conical flasks. The concentration of the spore solution was adjusted to about 1.4 × 106 spores/mL for R. arrhizus, 3 × 109 spores/mL for B. bassiana, and 4 × 107 spores/mL for B. brongniartii.

2.3.2. Cultivation Conditions—Fresh Biomass of Microorganisms

All cultures were grown in 250 mL flasks containing 100 mL of growth medium on a rotary shaker (135 rpm) at 24 °C for 3 days (for R. arrhizus), 5 days (for B. bassiana), and 6 days (for B. brongniartii) until the mid-log growth phase was reached. The average amount of biomass grown per 100 mL of culture medium was 10 g (R. arrhizus), 26 g (B. bassiana), and 16 g (B. brongniartii). Then, these amounts were transferred each into 50 mL of biotransformation medium (distilled water).

2.3.3. Immobilization Procedure—Growth of the R. arrhizus and B. brongniartii in the Presence of Polyurethane Foams with Different Porosity

The polyurethane foams were cut into 1 cm cubes (porosity of 740–1040 µm or 1060–1600 µm or 2300–3300 µm), washed with sterile water, and autoclaved in 250 mL Erlenmeyer’s flasks containing 100 mL of growth medium. Inoculum containing spores solution (500 µL) was transferred aseptically to sterile medium with polyurethane foams (20 cubes in each flask). The cultures were incubated at the same conditions as fresh biomass for 3 days (R. arrhizus) and 6 days (B. brongniartii).

2.3.4. Immobilization of B. bassiana Biomass in Agar-Agar

After incubation (6 days), fresh biomass was separated from growth medium by centrifugation (4000 rpm, 10 min, 4 °C), then washed with distilled water, centrifuged again, and then was used in the immobilization process. Agar-agar solution (6.0%) was added to fresh biomass in a 1:1 ratio. Then, the solidifying fungal cells immobilized in agar-agar were divided into small pieces.

2.3.5. Immobilization of B. bassiana Biomass in Calcium Alginate

After incubation, fresh fungal biomass (26 g) was separated from growth medium by centrifugation (at 4000 rpm for 10 min at 24 °C), washed with distilled water, centrifuged again, and then was used in the immobilization process. Distilled water was added to fresh biomass (in a ratio of 1:4.8) and mixed with an equal portion of sodium alginate solution (2.0%). The mixture was added dropwise to a 1% CaCl2 water solution and left for 30 min. Fungal cells immobilized in calcium alginate beads were separated by filtration using a Büchner funnel and transferred to a 0.8% BaCl2 water solution for the next 10 min. Immobilized fungal cells were separated again by filtration using a Büchner funnel and then transferred to 250 mL conical flasks containing 50 mL of distilled water as a biotransformation medium.

2.3.6. Pre-Incubation of Fresh Biomass of R. arrhizus

Pre-incubation of mycelium of R. arrhizus under starvation conditions was performed in 250 mL conical flasks containing 50 mL of distilled water on a rotary shaker (135 rpm) at room temperature for 24 h. After that, the biocatalyst was used for a biotransformation reaction.

2.4. Procedures of Biotransformation

After cultivation, the biocatalyst (fresh biomass or immobilized ones) was transferred to conical flasks (250 mL) with biotransformation media (50 mL of distilled water), and the different amounts (depending on the outcome) of the 2-phenylethanol were added as a substrate: 15 mg (2.5 mM), 30 mg (5 mM), or 60 mg (10 mM). Flasks were shaken at 135 rpm at room temperature. Biotransformations were carried out for 3 h for 1–5 days. Experiments were performed in triplicate. After each incubation period, the biocatalyst was removed by filtration (R. arrhizus) or centrifugation at 4000 rpm for 10 min at 4 °C (B. bassiana, B. brongniartii) and the supernatant was extracted twice with ethyl acetate. Organic layers were dried over anhydrous MgSO4 and evaporated under reducing pressure. Final products mixture was dissolved in ethanol (99.8%) or HPLC eluent mixture (acidic H2O (0.1% formic acid) and acetonitrile, 91:9) and analyzed by HPLC method.

2.5. Semi-Preparative Biotransformation Procedures

2.5.1. Simplified Flow Bioreactor for R. arrhizus

A simplified bioreactor model (Figure 1) was packed with immobilized cells (1060–1600 µm polyurethane foams, 20 cubes) and filled with biotransformation medium (150 mL of distilled water) containing substrate (5 mM) flowing continuously from the inside. The circulation of the medium through the column has been forced by a peristaltic pump (3 mL min−1). Progress of bioconversion was monitored by HPLC analysis after 3 h and after 1 to 5 days in the same time intervals (24 h).

2.5.2. Batch Bioreactor for B. bassiana

The microbiological bioreactor (Applikon Biotechnology my-Control, capacity of 1 L, Aplikon Biotechnology, Getinge, Sweden) was packed with fresh biomass (100 g/600 mL biotransformation medium-distilled water). Substrate in the amount of 600 mg (8.36 mM) was added, and biotransformation proceeded for 5 days. The process was carried out with stirring at 400–700 rpm, a temperature of 25 °C, and aeration (1 L min−1). Samples were collected within 24-h time intervals and analyzed by the HPLC method.

2.6. Analytical Methods

1-Phenylethane-1,2-diol and tyrosol were detected by HPLC (Beckman System Gold 126 Solvent Module, Supelcosil LC-18 column, 25 cm × 4.6 mm, 5 µm). Acidic H2O (0.1% formic acid) and acetonitrile were used as the mobile phase at a flow rate of 1 mL min−1 and a temperature of 24 °C. The percentage of eluent composition of % acidic H2O:% acetonitrile was 91:9. Injections were administered in a full loop (20 µL). Products were detected with λ = 276 nm. Retention times were as follows: products—tyrosol (12.2 min) and 1-phenylethane-1,2-diol (13.2 min); substrate—2-phenylethanol (26.6 min)
(R)-1-phenylethane-1,2-diol was detected by HPLC (AccQPrep HP125 UV-Vis, Teledyne ISCO, Inc., Lincoln, NE, USA; CHIRALCEL—OB column, particle size 10 μm, 4.6 mm × 250 mm). The analysis was carried out using n-hexane/2-propanol (96:4) as an eluent at a flow rate of 1.0 mL min−1 and a temperature of 24 °C. Product was detected with λ = 254 nm. Retention times were as follows: product—(R)-1-phenylethane-1,2-diol (16.9 min); (S)-1-phenylethane-1,2-diol (20.9 min); and substrate—2-phenylethanol (13.3 min).
Results were confirmed by comparison to commercially available standards, and the amount of the products was determined using calibration curves.

3. Results and Discussion

An unquestionable advantage of biotransformations is the ability to direct the bioconversion of a particular substrate into the desired product by simply altering the reaction conditions and modifying the form or mode of the biocatalyst. Among fungal strains, even well-studied ones, the diversity of enzymatic activities involved in metabolism is remarkable. These differences are either strain-specific or influenced by external factors such as temperature, pH, or the presence of xenobiotics. Consequently, altering a single parameter in the reaction can be a “game changer”, enabling the achievement of satisfactory results. The appropriate selection of reaction conditions and immobilization methods can significantly enhance the biocatalyst’s resistance to external factors [45], as well as improve reaction efficiency and enantioselectivity [5].
Three fungal strains (Beauveria bassiana DSM 1344, Beauveria brongniartii DSM 6651, and Rhizopus arrhizus DSM 1185) were applied for the biotransformation of 2-phenylethanol. To optimize the biocatalytic performance of these whole-cell biocatalysts, various conditions were introduced, including pre-incubation under starvation conditions, varying process durations, and different substrate concentrations. Additionally, several immobilization methods, such as polyurethane foams, agar-agar, and calcium alginate, were evaluated.
The results, summarized in Table 1, demonstrate that depending on the conditions, bioconversion with B. bassiana DSM 1344 and R. arrhizus DSM 1185 enabled the formation of (R)-1-phenylethane-1,2-diol. Additionally, R. arrhizus also facilitated the production of tyrosol, albeit as part of a product’s mixture.
A key parameter to consider in a biocatalytic process is the appropriate substrate concentration, as it critically influences the reaction yield by affecting the biocatalyst cells. To evaluate biomass sensitivity, varying concentrations of 2-phenylethanol (15, 30, and 60 mg per 50 mL flask system) were tested.
This parameter is particularly crucial, as demonstrated in Scheme 3, which correlates the biocatalyst form, reaction duration, and substrate concentration with the progress of 2-phenylethanol biotransformation catalyzed by R. arrhizus.
This reaction primarily results in a mixture of products, including racemic 1-phenylethane-1,2-diol as the main product and up to 5% tyrosol in the product mixture. In the initial phase of biotransformation (3 h), the substrate concentration had minimal effect on the reaction (Scheme 3). However, extending the reaction time revealed that using 30 mg of substrate in 50 mL of medium resulted in better bioconversion progress after three days (1.23 mg of product). In contrast, increasing the substrate concentration to 60 mg yielded more product (1.85 mg) after two days. Unfortunately, extending the reaction time beyond this point led to a decline in product formation, likely due to the toxic effects of the xenobiotic substrate [46,47] or further metabolic transformations of the resulting derivatives.
In comparison, for the Beauveria bassiana strain, the optimal bioconversion time for 60 mg of 2-phenylethanol per 50 mL was also the third day. While the total product yield was lower (0.38 mg) compared to R. arrhizus, the reaction exhibited high enantioselectivity, producing exclusively (R)-1-phenylethane-1,2-diol with an enantiomeric excess (e.e.) of 99.9%. Notably, strict control of the biotransformation time was necessary, as extending the reaction beyond three days resulted in a decrease in enantiomeric excess, likely due to the increased activity of enzymes with opposite enantioselectivity.
These findings formed the basis for further studies on the use of B. bassiana as a biocatalyst. Laboratory-scale experiments (50 mL biotransformation medium) demonstrated the potential for producing pure (R)-1-phenylethane-1,2-diol. Scaling up the reaction in a 1 L bioreactor under controlled conditions resulted in a 12-fold increase in production. As with the laboratory scale, the third day was optimal for (R)-1-phenylethane-1,2-diol production, yielding 28.8 mg in 600 mL of medium (Table 2). Importantly, while product formation decreased on subsequent days, no reduction in enantioselectivity was observed, suggesting the dominance of R-selective enzymes under controlled conditions.
In contrast, maintaining consistent aeration in the flask system proved challenging, which likely influenced enzyme activity during substrate oxy-functionalization. These unfavorable conditions may have triggered secondary metabolic pathways, reducing the optical purity of the product due to the activation of other enzyme groups.
Although both fungal strains (R. arrhizus and B. bassiana) catalyzed the biotransformation of 2-phenylethanol, the reactions exhibited differing enantioselectivity. In both cases, the main or sole product was 1-phenylethane-1,2-diol. The formation of this derivative might be attributed to the activity of unspecific peroxygenases (EC 1.11.2.1) secreted by some fungal cells [48,49], or a multi-step reaction involving dehydrogenation, epoxidation (mediated by monooxygenase activity), epoxide hydrolysis, and diol formation [50].
For B. bassiana, the spatial structure of the enzyme’s active or binding site may enable the specific arrangement of 2-phenylethanol or its intermediate, favoring the oxirane ring opening to form the R-diol. Conversely, in R. arrhizus, the biocatalytic pathway may involve enzymes physiologically suited for substrates with expanded steric hindrance (e.g., steroids). Consequently, substrate binding or processing of the oxidized intermediate might occur randomly, resulting in a racemic product mixture. Additional enzyme groups, such as monooxygenases and epoxide hydrolases with differing enantioselectivity, likely contribute to the reaction, as evidenced by the presence of small quantities of tyrosol in the post-reaction mixture.
The next approaches to improve the results were focused on the other parameters strongly influencing the progress and selectivity of biotransformations, such as the deficiency of nutrients. This is a very well-known strategy introduced in the biotechnological processes relying on the activities of secondary pathways. However, in presented studies, such an approach resulted in the activation of the pathways (Beauveria strains), which enabled the fast and effective degradation of the substrate. In the research, 2-phenylethanol was considered a xenobiotic, which can be converted by enzymatic systems involved in the secondary metabolism, switched on by starvation conditions. Obviously, environmental stress factors can be different, such as deficiency of carbon or nitrogen sources, changes in temperature, light, and pH [51,52,53]. In case of discussing processes, the pre-incubation of the biocatalysts under starvation conditions was implemented to force the bioconversion of the substrate [54,55]. However, studies conducted with R. arrhizus cells showed that the starvation step did not affect the substrate bioconversion. In this case, the activity or synthesis of enzymes involved in substrate bioconversion is inhibited by stress conditions. An extremely different situation was observed in the case of Beauveria strains, where the use of pre-incubation under starvation conditions resulted in the activation of degradative pathways and the gradual utilization of 2-phenylethanol for cellular purposes within a maximum of 72 h.
These conclusions were the basis for the modifications of the form of the biocatalyst, which is another common method applied in biotransformations, as a solution allowed protecting the viable microbes. Immobilization methods, in particular, can influence membrane transport mechanisms and enzyme activities associated with the cell envelope, potentially altering selectivity. Choosing an appropriate immobilization method requires tailoring it to the microorganism’s growth characteristics.
This approach proved highly effective for R. arrhizus. Immobilization on polyurethane foams with two porosity ranges (740–1040 µm and 1060–1600 µm) allowed extensive fungal mycelium overgrowth in both cases. Biotransformation of 2-phenylethanol by immobilized R. arrhizus cells produced the same product mixture (tyrosol and primarily racemic 1-phenylethane-1,2-diol) but in higher amounts than with fresh mycelium. Foams with 740–1040 µm porosity yielded significantly more product in 50 mL medium. After one day, product formation reached 1.55 mg/50 mL, with a maximum of 2 mg after three days. The primary product, 1-phenylethane-1,2-diol, constituted 1.85 mg, and tyrosol accounted for 0.15 mg (Scheme 3).
The effectiveness of this immobilization approach is attributable to the filamentous growth form of R. arrhizus, which facilitates foam overgrowth and enhances resistance to external factors [56,57,58]. Immobilized biocatalysts produced comparable (4th day) or higher (3rd and 5th days) product yields when transforming 30 mg of substrate, compared to fresh mycelium transforming 60 mg (Scheme 3—green and blue series). However, scaling attempts using a simplified continuous-flow bioreactor (Figure 1) revealed partial catalyst washout from polyurethane foams, rendering results irrelevant for scaling.
For B. brongniartii DSM 6651, immobilization on polyurethane foams was also successful. However, both fresh and immobilized biomass degraded 2-phenylethanol (Table 1), with fresh biomass completing degradation faster (48 h) than immobilized forms (72 h) due to mass exchange limitations. These results suggest B. brongniartii utilizes 2-phenylethanol as an energy and carbon source [59,60]. Such degradative activity has industrial applications, particularly in pharmaceutical waste disposal, which is critical for aquatic ecosystem protection [61].
By contrast, B. bassiana mycelium could not efficiently overgrow foam pores, necessitating alternative immobilization methods like calcium alginate and agar-agar matrices. Immobilization in agar-agar slightly improved resistance to substrate or product toxicity, yielding 0.43 mg of product in 50 mL, compared to 0.38 mg with fresh mycelium. However, immobilization negatively impacted enantioselectivity, producing racemic 1-phenylethane-1,2-diol. Increasing substrate concentration to 60 mg (10 mM) inhibited bioconversion, likely due to enhanced toxicity.
Agar encapsulation may limit substrate-mycelium contact and partially deactivate certain membrane-associated enzymes involved in bioconversion, favoring the activity of less selective enzymes. Agar-agar immobilization may also alter enantioselectivity by modifying transport selectivity across the cell envelope. Previous studies demonstrate that different immobilization methods can completely reverse enantioselectivity, yielding S- or R-enantiomers depending on the carrier material [43].
Entrapment in calcium alginate further confirmed these findings. Alginate beads prevented substrate contact with the biocatalyst, resulting in no biotransformation or degradation of 2-phenylethanol, regardless of substrate concentration (5 mM or 10 mM). This carrier tightly seals fungal cells, blocking enzymatic access to the substrate.
In earlier studies, semi-preparative biotransformation of 2-phenylethanol with A. niger yielded optically pure (S)-1-phenylethane-1,2-diol [41]. In the present work, the (R)-enantiomer was formed using B. bassiana. These findings are significant, as both optical isomers can be obtained through the bioconversion of inexpensive and widely available 2-phenylethanol.

Author Contributions

A.R., B.S.-M., M.B.-R., and E.Ż.-D. conceptualized the idea and methodology; A.R., M.B.-R., and E.Ż.-D. wrote the original drafted manuscript; M.K.-O., E.Ż.-D., and M.B.-R. edited and revised the manuscript; E.Ż.-D. and M.B.-R. supervised the experiments and writing. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported in part by the project Minigrants for doctoral students at the Wroclaw University of Science and Technology, number 50SD/0068/24.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Symmetry 17 00017 sch002
Scheme 2. Biotransformations of 2-phenylethanol carried out by Beauveria bassiana DSM 1344 and Rhizopus arrhizus DSM 1185.
Scheme 2. Biotransformations of 2-phenylethanol carried out by Beauveria bassiana DSM 1344 and Rhizopus arrhizus DSM 1185.
Symmetry 17 00017 sch002
Symmetry 17 00017 g001
Figure 1. General work scheme in semi-preparative experiment—simplified flow bioreactor model.
Figure 1. General work scheme in semi-preparative experiment—simplified flow bioreactor model.
Symmetry 17 00017 g001
Symmetry 17 00017 sch003
Scheme 3. The amount of the mixture of products received after biotransformation of 2-phenylethanol with Rhizopus arrhizus DSM 1185 presented as a function of substrate concentration (mg/50 mL) and form of biocatalyst.
Scheme 3. The amount of the mixture of products received after biotransformation of 2-phenylethanol with Rhizopus arrhizus DSM 1185 presented as a function of substrate concentration (mg/50 mL) and form of biocatalyst.
Symmetry 17 00017 sch003
Table 1. Biotransformation products depending on the selected strain and form of biocatalyst.
Table 1. Biotransformation products depending on the selected strain and form of biocatalyst.
Product (R)-1-Phenylethane-1,2-diol(R,S)-1-Phenylethane-1,2-diolTyrosol
Biocatalyst
B. bassiana
DSM 1344		fresh biomass in flasksimmobilized biomass
(agar-agar)
fresh biomass in bioreactor
R. arrhizus
DSM 1185		 fresh biomassfresh biomass
immobilized biomass
(polyurethane foams)		immobilized biomass
(polyurethane foams)
B. brongniartii
DSM 6651		2-phenylethanol degradation—no desired product formation
Table 2. Amount of (R)-1-phenylethane-1,2-diol [mg] produced during the biotransformation of 2-phenylethanol (600 mg) in a batch bioreactor (600 mL medium).
Table 2. Amount of (R)-1-phenylethane-1,2-diol [mg] produced during the biotransformation of 2-phenylethanol (600 mg) in a batch bioreactor (600 mL medium).
(R)-1-Phenylethane-1,2-diol (e.e. 99.9%) [mg/600 mL]
StrainBiotransformation Time [Days]Product [mg]
B. bassiana (fresh cells)112.9 (±0.095)
217.6 (±0.110)
328.8 (±0.078)
410.0 (±0.130)
55.1 (±0.075)
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Raczyńska, A.; Szmigiel-Merena, B.; Brzezińska-Rodak, M.; Klimek-Ochab, M.; Żymańczyk-Duda, E. Fungal Biocatalysis in Stereoselective Oxidation of 2-Phenylethanol. Symmetry 2025, 17, 17. https://doi.org/10.3390/sym17010017

AMA Style

Raczyńska A, Szmigiel-Merena B, Brzezińska-Rodak M, Klimek-Ochab M, Żymańczyk-Duda E. Fungal Biocatalysis in Stereoselective Oxidation of 2-Phenylethanol. Symmetry. 2025; 17(1):17. https://doi.org/10.3390/sym17010017

Chicago/Turabian Style

Raczyńska, Agnieszka, Beata Szmigiel-Merena, Małgorzata Brzezińska-Rodak, Magdalena Klimek-Ochab, and Ewa Żymańczyk-Duda. 2025. "Fungal Biocatalysis in Stereoselective Oxidation of 2-Phenylethanol" Symmetry 17, no. 1: 17. https://doi.org/10.3390/sym17010017

APA Style

Raczyńska, A., Szmigiel-Merena, B., Brzezińska-Rodak, M., Klimek-Ochab, M., & Żymańczyk-Duda, E. (2025). Fungal Biocatalysis in Stereoselective Oxidation of 2-Phenylethanol. Symmetry, 17(1), 17. https://doi.org/10.3390/sym17010017

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