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Article

Non-Conventional Yeasts as Sources of Ene-Reductases for the Bioreduction of Chalcones

by
Sara Filippucci
1,
Giorgia Tasselli
1,2,
Fatima-Zohra Kenza Labbani
3,4,
Benedetta Turchetti
1,
Maria Rita Cramarossa
5,
Pietro Buzzini
1 and
Luca Forti
5,*
1
Department of Agricultural, Food and Environmental Sciences, Industrial Yeasts Collection DBVPG, University of Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy
2
CIRIAF—Biomass Research Centre, University of Perugia, Via G. Duranti, 67, 06125 Perugia, Italy
3
Laboratory of Microbiological Engineering and Applications, Department of Biochemistry and Cell and Molecular Biology, Natural and Life Sciences Faculty, University of Mentouri, Route Ain El Bey, Constantine 25017, Algeria
4
Département des Sciences Naturelles, Ecole Normale Supérieure Assia Djebar de Constantine, Ville Universitaire, Ain El Bey Ali Mendjeli, Constantine 25000, Algeria
5
Department of Life Sciences, University of Modena and Reggio Emilia, via G. Campi 103, 41125 Modena, Italy
*
Author to whom correspondence should be addressed.
Fermentation 2020, 6(1), 29; https://doi.org/10.3390/fermentation6010029
Submission received: 9 October 2019 / Revised: 17 February 2020 / Accepted: 18 February 2020 / Published: 21 February 2020
(This article belongs to the Special Issue Biocatalysis and Fermentation—Enzyme Production and Whole Cell Biocatalysis)
Browse Figures
Versions Notes

Abstract

:
Thirteen Non-Conventional Yeasts (NCYs) have been investigated for their ability to reduce activated C=C bonds of chalcones to obtain the corresponding dihydrochalcones. A possible correlation between bioreducing capacity of the NCYs and the substrate structure was estimated. Generally, whole-cells of the NCYs were able to hydrogenate the C=C double bond occurring in (E)-1,3-diphenylprop-2-en-1-one, while worthy bioconversion yields were obtained when the substrate exhibited the presence of a deactivating electron-withdrawing Cl substituent on the B-ring. On the contrary, no conversion was generally found, with a few exceptions, in the presence of an activating electron-donating substituent OH. The bioreduction aptitude of the NCYs was apparently correlated to the logP value: Compounds characterized by a higher logP exhibited a superior aptitude to be reduced by the NCYs than compounds with a lower logP value.

Graphical Abstract

1. Introduction

The chemoselective bioreduction of α,β-unsaturated alkenes represents an important tool in the synthesis of a lot of fine chemicals and pharmaceuticals [1,2,3]. Ene-reductases (ERs) belong to the flavin-containing “Old Yellow Enzyme” family (OYE, EC 1.6.99.1), which includes a class of I flavin-dependent oxidoreductases, which have been extensively studied for their ability to catalyze the asymmetric reduction of electronically activated C=C bonds, possessing electron-withdrawing substituents in the presence of cofactor-recycling systems for NAD(P)H [4,5,6,7,8,9,10,11,12,13,14]. Intracellular ER homologues from bacteria, yeasts, filamentous fungi and higher plants have been isolated and characterized since the 1990s [15,16,17,18,19,20,21,22,23,24,25].
Chalcones represent an interesting class of bioactive open-chain flavonoids, exhibiting α,β-unsaturated carbonyl groups in their scaffolds. Some studies proved their important antitubercular [26], antioxidant [27], antifungal [28] and anticancer [29] activities.
Recently, whole-cells and enzymes catalyzing the C=C hydrogenation and C=O reduction of representative chalcones have been studied for obtaining compounds possessing noteworthy bioactivities. In particular, some dihydrochalcones (achieved via bioreduction of the C=C double bond) have been found to express antioxidant, UV-protective and pro-health activities, which could be interesting for pharmaceutical and cosmetic industries [25,30]. Moreover, their sweet taste make them attractive for producing sweeteners [31,32]. In addition, the dihydrochalcone obtained from bioreduction of (E)-1,3-diphenylprop-2-en-1-one has been isolated from the leaves of Leptoderris fasciculata [33], a woody liana used in traditional medicine for the treatment of dropsy, edema, pulmonary disorders and as a laxative [34].
Recent studies revealed that Non-Conventional Yeasts (NCYs) are able to express a number of promising biotechnological properties [35,36,37], including the ability to express important ERs activities. In this framework, due to the presence of cofactor-recycling systems for NAD(P)H at the level of cell metabolism, biotransformation processes catalyzed by whole-cells of NCYs could be considered as useful and cheaper alternatives in place of using purified enzymes for reducing α,β-unsaturated alkenes including chalcones [38,39,40,41,42].
Aiming to identify new possible substrates for the NCYs expressing ERs activity, the present paper reports a study on the ability of lyophilized cells of NCYs to bioreduce the activated C=C double bonds of chalcones. A Structure–Activity Relationship (SAR) approach was used.

2. Materials and Methods

2.1. Chemicals and Culture Media

A set of chalcones were used as substrate for bioreduction by NCYs. A Structure–Activity Relationship (SAR) approach was used by means of different substituents on the B-ring: 1a = (E)-1,3-diphenylprop-2-en-1-one; 2a = (E)-1-(4-chlorophenyl)-3-phenylprop-2-en-1-one; 3a = (E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one (4-hydroxychalcone); (Figure 1A). Besides, two α,β-unsatured ketones, i.e., (3E)-4-phenylbut-3-en-2-one (4a) and (3E)-4-(4-chlorophenyl)but-3-en-2-one (5a), were used for comparison (Figure 1B). They were from Sigma-Aldrich Co, USA.
The following microbiological culture media were used: YEPG: yeast extract 10 g L−1, peptone 10 g L−1, glucose 20 g L−1 and agar 15 g L−1; and Carvone Medium (CM) [39]: yeast extract 3 g L−1, malt extract 3 g L−1, peptone 5 g L−1, glucose 10 g L−1 and pH 6.5. Ingredients of the culture media were from Difco (Franklin Lakes, NJ, USA).

2.2. Yeast Strain

Thirteen NCY strains belonging to ascomycetous and basidiomycetous species (genera Candida, Cyberlindnera, Goffeauzyma, Hanseniaspora, Kazachstania, Naganishia, Pichia, Scheffersomyces, Solicoccozyma and Wickerhamomyces) were used. They were preliminarily selected from a few hundred of environmental strains isolated worldwide for their ability to catalyze the biotransformation of α,β-unsaturated alkenes [39,40,41]. All strains are conserved at the Industrial Yeasts Collection DBVPG of the University of Perugia, Italy. Salient information on strains are reported in Table 1 and are available on the DBVPG website (www.dbvpg.unipg.it). NCY strains were maintained in frozen form (−80 °C), while working cultures were routinely grown on YEPG agar slants at 20 or 25 °C, depending on their psychrophilic or mesophilic aptitudes.

2.3. Preparation of the Lyophilized NCYs Whole-Cells Biocatalyst

Lyophilized NCYs whole-cells were obtained as previously reported [40]. Briefly, aliquots (200 μL) of 24 h cell suspensions, calibrated to A580 = 0.5 (approx. 106 cells mL−1), were used to inoculate 110 mL of CM [39]. After incubation for 48 h at 20 °C, the NCYs’ biomass and supernatants were separately harvested. Cells were washed 3 times by using 50 mM phosphate buffer (pH 6.5), centrifuged each time for 15 min at 4000 rpm, snap frozen (−80 °C) and lyophilized for 48 h in a Lyophilizer Modulyo (Edwards, Irvine, CA, USA).

2.4. Bio-Reduction Reactions

A total of 30 mg of lyophilized NCYs cells were resuspended in 25 mL sterile vials containing 4.5 mL of 50 mM phosphate buffer (pH 6.5). A total of 0.5 mL of 10% w/v glucose, acting as a cofactor-recycling system, was also added. As a final point, chalcone was added at a final concentration of 5 mM and the vials were incubated on an orbital shaker (120 rpm) at 20 or 25 °C (depending on their psychrophilic or mesophilic status) for 120 h. In order to determine whether chalcone was spontaneously reduced in the absence of the NCY cells, blank (cell-free) vials containing 50 mM phosphate buffer + 50 mM glucose and each chalcone were analyzed at 120 h. After incubation, vials were sealed and frozen (−30 °C) until GC–MS analysis.

2.5. GC–MS Analyses

Products obtained after bioconversion were detected via GC–MS after extraction with a solution of octanol (as internal standard) in ethyl acetate 0.1% v/v (5 mL). GC–MS analyses were performed on a Hewlett Packard (USA) G1800C Series II gas chromatograph–mass spectrometer equipped with a HP-5 column (25 m × 0.2 mm, 0.5 µm film thickness) coated with (5%)-diphenyl-(95%)-dimethylpolysiloxane copolymer. Compounds derived from biotransformation of substrates were identified by comparing their respective mass fragmentation patterns (EI, 70 eV) with the database library NIST05 (MS Library Software Varian, USA). Temperature program: 1a, 4a and 5a: 120 °C, hold for 2 min, 10 °C/min to 250 °C, hold for 5 min, Detector 280 °C, Injector 270 °C; 2a: 100 °C, hold for 3 min, 15 °C/min to 180 °C, hold for 3 min, 20 °C/min to 240 °C, hold for 5 min Detector 280 °C, Injector 270 °C; 3a: 120 °C, hold for 2 min, 15 °C/min to 270 °C, hold for 15 min, Detector 280 °C, Injector 270 °C. Retention times (min): (E)-1,3-diphenylprop-2-en-1-one 1a (12.06), 1,3-diphenylpropan-1-one 1b (10.70); (E)-1-(4-chlorophenyl)-3-phenylprop-2-en-1-one 2a (15.74), 1-(4-chlorophenyl)-3-phenylpropan-1-one 2b (14.35); (E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one 3a (21.07), 1-(4-hydroxyphenyl)-3-phenylpropan-1-one 3b (17.90); (3E)-4-phenylbut-3-en-2-one 4a (4.56), 4-phenylbutan-2-one 4b (3.29); (3E)-4-(4-chlorophenyl)but-3-en-2-one 5a (6.87), 4-(4-chlorophenyl)butan-2-one 5b (5.60).
All the results were expressed as biotransformation yield, i.e., a % of the substrate converted to a given derivative. The concentration of the substrate and product were measured by an internal standard method. All the results represented the average of three independent experiments, and the statistical significance of these average data was assessed via ANOVA.

2.6. LogP Calculation

The logP values of chalcones were calculated by the ACD/LogP v.14.06 program in the software package for ACD/Labs 2016 2.2 (Advanced Chemistry Development).

3. Results and Discussion

Figure 1 reports the chemical structures of the substrates used for checking the NCYs’ ERs activity. The first substrate, namely (E)-1,3-diphenylprop-2-en-1-one (1a, Figure 1A), was used as model compound to screen the ability of the lyophilized cells of the NCYs to reduce the α,β C=C double bond. The presence of the conjugate C=O double bond was also considered for assessing the chemoselectivity of the reduction.
The reaction scheme of the bioreduction of chalcones (E)-1,3-diphenylprop-2-en-1-one (1a), (E)-1-(4-chlorophenyl)-3-phenylprop-2-en-1-one (2a), and (E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one (3a) into dihydrochalcones 1,3-diphenylpropan-1-one (1b), 1-(4-chlorophenyl)-3-phenylpropan-1-one (2b) and 1-(4-hydroxyphenyl)-3-phenylpropan-1-one (3b), respectively, catalyzed by the lyophilized cells of the NCYs is reported in Figure 2. The results of the screening, in terms of conversion yields expressed as molar percentage, are reported in Table 2.
With few exceptions, whole-cells of NCYs were able to hydrogenate the C=C double bond occurring in the (E)-1,3-diphenylprop-2-en-1-one 1a scaffold: in particular, nine strains, namely Cyberlindnera amylophila DBVPG 6346, Goffeauzyma gastrica DBVPG 4709, Hanseniaspora guillermondii DBVPG 6790, Kazachstania exigua DBVPG 6469, Kazachstania spencerorum DBVPG 6746, Kluyveromyces lactis DBVPG 6854, Naganishia diffluens DBVPG 6237, Scheffersomyces shehatae DBVPG 6850 and Wickerhamomyces canadensis DBVPG 6211 exhibited bioconversion yields ≥ 94%, with an excellent repeatability and a low standard deviation (Table 2). On the contrary, three strains (i.e., Candida freyschussii DBVPG 6208, Goffeauzyma gilvescens DBVPG 4712 and Pichia kluyveri DBVPG 5826) showed lower bioconversion yields (from 0.7 to 11.5%), but with low repeatability of the bioreduction process (Table 2).
One of the main drawbacks of using whole-cells as catalysts is sometimes their poor chemoselectivity when C=C versus C=O bond reduction is catalyzed. It is worth noting that in any case, the screened NCYs did not exhibit any reduction of the C=O bond (Figure 2). In a previous study, we observed that many of the yeasts herein screened possess carbonyl reductase activity (alcohol dehydrogenase, ADH), being able to reduce the carbonyl group of molecules such as ketoisophorone [40]. The high chemoselectivity in the bioreduction of chalcones observed in the present study is probably not due to the lack of ADH activity in the yeast strains, rather to the steric hindrance given by the bulky phenyl substituents or to enzyme localization (not accessible to the substrates). Anyway, the bioreduction of C=O bond in ketones bearing bulky substituents was found in bacteria whole cells [43], as well as in other microorganism [44,45,46,47] such as algae, filamentous fungi, yeasts and in plant tissues [48].
Considering these encouraging results, the ability of NCYs to bioreduce chalcones substituted with both deactivating and activating groups on the B-ring ((E)-1-(4-chlorophenyl)-3-phenylprop-2-en-1-one 2a and (E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one 3a, respectively) was also checked. The results are reported in Table 2. Worthy bioconversion yields were obtained when the substrate exhibited the presence of a deactivating electron-withdrawing Cl substituent on the B-ring (2a): all the NCYs exhibited the ability to reduce C=C double bond of 2a with bioconversion yields ranging from 8% to 98% (Table 2). On the contrary, in the presence of the activating electron-donating substituent OH (3a), no conversion was generally found, with the sole exception of Goffeauzyma gilvescens DBVPG 4712 (yield = 47.8%), and Cyberlindera amylophila DBVPG 6346, Kaz. spencerorum DBVPG 6746 and K. lactis DBVPG 6854, which totally reduced the chalcone 3a (yield = 100%) (Table 1). Interestingly, the last three NCYs also exhibited worthy bioconversion yields of 1a and 2a (falling into the range from 94.0% to 100% and from 48.4% to 88.3%, respectively). On the contrary, Pichia kluyveri DBVPG 5826 showed a bioconversion yield of the chlorocalcone 2a ≥ 95%, but no or very low activity versus 1a and 3a. Taking into account the above few exceptions, the bioreduction aptitude of NCYs was apparently correlated to the logP value, which is an indirect measure of the lipophilic degree of a given compound: The substrates 1a and 2a, which were characterized by a higher logP (4.01 and 4.78, respectively), exhibited a superior aptitude to be reduced by NCYs than the chalcone 3a (logP = 3.65). This trend could be justified by considering how the different molecules can go across the yeast cell membrane. Due to the lyophilized nature of the whole cells of the NCYs herein used, the passage of molecules, including 1a, 2a and 3a, across the cell membrane to reach the intracellular ERs should be much simpler. In fact, some studies reported that dehydration–rehydration cycles can determine a significant decrease of cell sizes together with a strong folding of membranes, thus leading to an increased permeability in lyophilized cells [49,50]. In this framework, the hypothesis postulated by some authors [51,52] that higher lipophilic molecules (characterized by a higher logP, i.e., (E)-1,3-diphenylprop-2-en-1-one 1a and (E)-1-(4-chlorophenyl)-3-phenylprop-2-en-1-one 2a) could more easily go across yeast plasma membranes by using the free diffusion mechanism (thus easily reaching cytoplasm ERs) differently from the lesser lipophilic ones (characterized by a lower logP, i.e., (E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one 3a) could justify the superior aptitude of 1a and 2a to be bioreduced by ERs occurring at the cytoplasm level of the lyophilized cells of the NCYs. In addition, the presence of an OH substituent on the B-ring of 3a (Figure 1) could affect its capability to form hydrogen bonds with hydrophilic components occurring in the cell surface, thus reducing the rate of passage across the membrane.
The degree of lipophilicity of a given molecule is just one of the parameters determining its passage across the membrane and its interaction with enzymes. Thus, the α,β-unsaturated ketones (3E)-4-phenylbut-3-en-2-one (4a) and (3E)-4-(4-chlorophenyl)but-3-en-2-one (5a) (Figure 1B), exhibiting lower logP (2.17 and 2.70, respectively), but also lesser steric hindrance and substituents unable to form hydrogen bonds, were studied for their ability to be bioreduced by NCYs (Figure 3). The results are reported in Table 2. Overall, with the sole remarkable exception of K. lactis DBVPG 6854 on substrate 4a (bioreduction yield = 99.1%), the aptitude of both substrates 4a and 5a to be bioreduced is significantly lower than those of the substrates 1a–3a. These results seem to confirm the importance of the lipophilicity and that the steric hindrance is a less important factor in the determining the aptitude to be bioreduced.
In close analogy with previous literature [53], the results herein reported underline that the use of lyophilized yeast cells could be considered preferable for enhancing the permeability of a membrane to the substrate. Indeed, as supposed by some authors [39,54], the ERs that reduced the α,β-unsatured carbonyl compounds could be exclusively associated with yeast biomass, with no release of extracellular enzymes.

4. Conclusions

Our study has shown that lyophilized NCYs whole cells are useful biocatalysts to obtain dihydrochalcones from corresponding chalcones. In particular, we have identified nine strains (namely, Cyberlindnera amylophila DBVPG 6346, Goffeauzyma gastrica DBVPG 4709, Hanseniaspora guillermondii DBVPG 6790, Kazachstania exigua DBVPG 6469, Kazachstania spencerorum DBVPG 6746, Kluyveromyces lactis DBVPG 6854, Naganishia diffluens DBVPG 6237, Scheffersomyces shehatae DBVPG 6850 and Wickerhamomyces canadensis DBVPG 6211) that exhibited high bioconversion yields, with an excellent repeatability and a low standard deviation
The bioreduction aptitude of the NCYs was affected by both the structure of chalcones and the logP value of tested substrates: NCYs were able to better reduce compounds characterized by a higher logP than ones with a lower logP value. Due to their sweet taste, dihydrochalcones are interesting derivatives for the food industry for the production of new sweeteners. Furthermore, some dihydrochalcones have been found to express interesting biological activities, which could be important for the pharmaceutical and cosmetic industries.

Supplementary Materials

The following are available online at https://www.mdpi.com/2311-5637/6/1/29/s1, Figure S1: chromatograms for biotransformations.

Author Contributions

Conceptualization, L.F. and P.B.; methodology, L.F. and P.B.; formal analysis, S.F., G.T., F.-Z.K.L., B.T. and M.R.C.; data curation, S.F., G.T., F.-Z.K.L., B.T. and M.R.C.; writing—original draft preparation, all authors; writing—review and editing, L.F. and P.B.; supervision, L.F. and P.B.; project administration, L.F. and P.B. All authors have read and agree to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 06 00029 g001 550
Figure 1. Chemical structures of substrates (from 1a to 5a: A, chalcones; B, α,β-unsatured ketones) with different substituents (R) used for checking NCY ene-reductase activity.
Figure 1. Chemical structures of substrates (from 1a to 5a: A, chalcones; B, α,β-unsatured ketones) with different substituents (R) used for checking NCY ene-reductase activity.
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Figure 2. Reaction scheme of the bioreduction of chalcones 1a, 2a and 3a into dihydrochalcones 1b, 2b and 3b, respectively, by NCYs.
Figure 2. Reaction scheme of the bioreduction of chalcones 1a, 2a and 3a into dihydrochalcones 1b, 2b and 3b, respectively, by NCYs.
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Figure 3. Reaction scheme of the bioreduction of substrates 4a and 5a to products 4b and 5b, respectively, by NCYs.
Figure 3. Reaction scheme of the bioreduction of substrates 4a and 5a to products 4b and 5b, respectively, by NCYs.
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Table
Table 1. Salient information on the Non-Conventional Yeast (NCY) strains used in the present study.
Table 1. Salient information on the Non-Conventional Yeast (NCY) strains used in the present study.
SpeciesOriginLocation
Candida freyschussii DBVPG 6208Wood pulpSweden
Cyberlindnera amylophila DBVPG 6346Frass of Pinus taeda (loblolly pine)USA
Goffeauzyma gastrica DBVPG 4709Sub-glacial debris of the Sforzellina glacierItaly
Goffeauzyma gilvescens DBVPG 4712Supra-glacial debris of the Sforzellina glacierItaly
Hanseniaspora guilliermondii DBVPG 6790Trachea of beeFrance
Kazachstania exigua DBVPG 6469SoilSouth Africa
Kazachstania naganishii DBVPG 7133Decaying leavesJapan
Kazachstania spencerorum DBVPG 6746SoilSouth Africa
Kluyveromyces lactis DBVPG 6854Rain forest drosophilidsBrazil
Naganishia diffluens DBVPG 6237Soil of vineyardHungary
Pichia kluyveri DBVPG 5826Soil close to plum treeAlgeria
Scheffersomyces shehatae DBVPG 6850Rain forest drosophilidsBrazil
Wickerhamomyces canadensis DBVPG 6211Ground wood pulpSweden
Table
Table 2. Bioreduction yield of substrates 1a–5a to products 1b–5b by NCYs.(*)
Table 2. Bioreduction yield of substrates 1a–5a to products 1b–5b by NCYs.(*)
DBVPG Accession NumbersSpecies1a Conversion mol% (± SD)2a Conversion mol% (± SD)3a Conversion mol% (± SD)4a Conversion mol% (± SD)5a Conversion mol% (± SD)
6208Candida freyschussii2.9 ± 1.523.2 ± 13.50.0 ± 0.00.0 ± 0.03.1 ± 0.5
6346Cyberlindnera amylophila94.0 ± 2.888.3 ± 10.2100.0 ± 0.00.0 ± 0.00.0 ± 0.0
4709Goffeauzyma gastrica100.0 ± 0.065.2 ± 56.50.0 ± 0.03.2 ± 0.62.7 ± 1.0
4712Goffeauzyma gilvescens11. 5 ± 5.678.0 ± 6.147.8 ± 11.07.4 ± 4.016.9 ± 1.4
6790Hanseniaspora guilliermondii96.2 ± 1.676.4 ± 22.20.0 ± 0.00.0 ± 0.01.8 ± 1.1
6469Kazachstania exigua96.3 ± 0.817.4 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0
7133Kazachstania naganishii0.0 ± 0.083.5 ± 3.60.0 ± 0.03.2 ± 0.42.7 ± 1.2
6746Kazachstania spencerorum95.2 ± 2.048.4 ± 4.3100.0 ± 0.046.9 ± 7.44.7 ± 0.4
6854Kluyveromyces lactis100.0 ± 0.073.3 ± 24.3100.0 ± 0.099.1 ± 0.80.0 ± 0.0
6237Naganishia diffluens99.1 ± 1.620.9 ± 20.90.0 ± 0.015.6 ± 8.321.2 ± 4.7
5826Pichia kluyveri0.7 ± 0.798.1 ± 3.40.0 ± 0.00.0 ± 0.01.5 ± 0.5
6850Scheffersomyces shehatae97.5 ± 4.351.6 ± 11.50.0 ± 0.013.5 ± 3.90.0 ± 0.0
6211Wickerhamomyces canadensis100.0 ± 0.08.0 ± 11.30.0 ± 0.00.0 ± 0.00.0 ± 0.0
(*) Chromatograms for biotransformations are available in the supplementary materials.

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MDPI and ACS Style

Filippucci, S.; Tasselli, G.; Kenza Labbani, F.-Z.; Turchetti, B.; Cramarossa, M.R.; Buzzini, P.; Forti, L. Non-Conventional Yeasts as Sources of Ene-Reductases for the Bioreduction of Chalcones. Fermentation 2020, 6, 29. https://doi.org/10.3390/fermentation6010029

AMA Style

Filippucci S, Tasselli G, Kenza Labbani F-Z, Turchetti B, Cramarossa MR, Buzzini P, Forti L. Non-Conventional Yeasts as Sources of Ene-Reductases for the Bioreduction of Chalcones. Fermentation. 2020; 6(1):29. https://doi.org/10.3390/fermentation6010029

Chicago/Turabian Style

Filippucci, Sara, Giorgia Tasselli, Fatima-Zohra Kenza Labbani, Benedetta Turchetti, Maria Rita Cramarossa, Pietro Buzzini, and Luca Forti. 2020. "Non-Conventional Yeasts as Sources of Ene-Reductases for the Bioreduction of Chalcones" Fermentation 6, no. 1: 29. https://doi.org/10.3390/fermentation6010029

APA Style

Filippucci, S., Tasselli, G., Kenza Labbani, F. -Z., Turchetti, B., Cramarossa, M. R., Buzzini, P., & Forti, L. (2020). Non-Conventional Yeasts as Sources of Ene-Reductases for the Bioreduction of Chalcones. Fermentation, 6(1), 29. https://doi.org/10.3390/fermentation6010029

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