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Study on green extraction of limonene from orange peel and cascade catalysis to produce carvol and carvone in deep eutectic solvents
Abstract
Carvol and carvone are oxidation products from the natural product limonene. They are important raw materials for the flavours and fragrances industry and also act as pharmaceutical active ingredients. Orange waste peels possibly represent an attractive source for limonene, but studies on valorizing orange peel wastes are rare. In this study, we report a new enzymatic cascade system for the in‐situ conversion of limonene from orange peel into valued‐added carvol and carvone. The use of deep eutectic solvents (DES) allows for efficient in‐situ extraction of limonene from waste orange peels. We propose a dual function use of DES as solvent for the extraction and the biocatalytic oxidation of limonene as well as cosubstrate to promote the oxidation reaction. Using ChCl‐Pro‐H2O DES for the extraction of limonene from waste orange peels, approximately 17 milligrams of limonene per gram of orange peel was achieved at 40°C for 24 h. Then, with ChCl‐Pro‐H2O DES as the extractant and reaction medium, a cascade reaction system of choline oxidase (ChOx) and unspecific peroxygenase (UPO) was established to catalyse the conversion of limonene into carvol and carvone. The concentration of the final products was up to about 1.6 mmol L−1. This study showed a biocatalytic transformation pathway and provides technical support for the high‐value utilization of waste in orange peel. Envisioned valorization of orange peel waste(OPW)‐derived limonene to carvone. Deep Eutectic Solvents (DESs) serve as extractant of limonene from OPW, as solvent and as sacrificial electron donor for the biocatalytic transformation to carvone.
... These results are very promising because they propose an efficient, enantioselective, and green method to synthetize chiral sulfoxides which are widely found in drugs, chiral intermediates as well as chiral ligands in catalysis. Another example of this synergistic work is reported in 2022 [33], where the dual function of ChCl/Pro/H2O 1:1:1 as solvent for the extraction of limonene from waste orange peels and for its biocatalytic oxidation as well as co-substrate (Table 3, Entry 5) was investigated. In this way, carvol and carvone are successfully obtained through a cascade reaction sequence. ...
During the last decade, a wide spectrum of applications and advantages in the use of deep eutectic solvents for promoting organic reactions has been well established among the scientific community. Among these synthetic methodologies, in recent years, various examples of biocatalyzed processes have been reported, making use of eutectic mixtures as reaction media, as an improvement in terms of selectivity and sustainability. This review aims to show the newly reported protocols in the field, subdivided by reaction class as a ‘toolbox’ guide for organic synthesis.
In this study, mandarin peel rich in limonene was the feedstock as citrus essential oil source. Box-Behnken design of Response Surface Method was used to optimize the solid mass, immersion time and washing time of the automatic solvent extraction for the maximum D-limonene yield. The proposed conditions as the optimum were calculated as 8.63 g mandarin peel, 8.5 min immersion time and 8 min washing time to achieve the greatest D-limonene yield (~ 75 mg/g). The proposed conditions were reliable depending on the difference between the actual and predicted values (1.7%). With solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS), 19 components were identified in the mandarin peel. D-limonene was the most abundant volatile compound (91.56%), whereas the other main volatile compounds were identifed as myrcene (4.93%), α-pinen (1.69%) and sabinene (0.85%) in the mandarin peel. Additionally, fourier transform infrared (FTIR) and nuclear magnetic resonance (¹H– and ¹³C-NMR) spectroscopies were also applied for the verification of GC-MS findings.
Graphical abstract
Food factories seek the application of natural products, green feedstock and eco-friendly processes, which minimally affect the properties of the food item and products. Today, water and conventional polar solvents are used in many areas of food science and technology. As modern chemistry evolves, new green items for building eco-friendly processes are being developed. This is the case of deep eutectic solvents (DESs), named the next generation of green solvents, which can be involved in many food industries. In this review, we timely analyzed the progress on applying DES toward the development of formulations, extraction of target biomolecules, food processing, extraction of undesired molecules, analysis and determination of specific analytes in food samples (heavy metals, pesticides), food microbiology, and synthesis of new packaging materials, among many other applications. For this, the latest developments (over the last 2-3 years) have been discussed emphasizing innovative ideas and outcomes. Relevantly, we discuss the hypothesis and the key features of using DES in the mentioned applications. To some extent, the advantages and limitations of implementing DES in the food industry are also elucidated. Finally, based on the findings of this review, the perspectives, research gaps and potentialities of DESs are stated.
The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L–1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H2O2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L–1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO).
Styrofoam is widely used for food packaging because it has good weight, stability, and stiffness but it cannot be decomposed naturally by microorganisms. D-Limonene, one of the monoterpene derivatives, is an environmentally friendly styrofoam solvent. This monoterpene is the main component of essential oil that can be extracted from orange peel. The aim of this study is to destruct styrofoam wastes (foods packaging, electronics packaging, foam boards and cup noodles) using d-limonene from orange (Citrus Maxima) peel extraction by water distillation process. The orange peel after size reduction is extracted by water distillation process at 94°C for 7 hours to produce essential oils. It was used to destruct styrofoam waste at the different volume ratio (1:0:0; 1:0:3; 1:1:2; 1:2:1) of the essential oil mixture (essential oil : ethanol: water). The result of this study shows that styrofoam wastes can be destructed using d-limonene from orange (Citrus Maxima) peel extraction. D-limonene of essential oil from orange (Citrus Maxima) peel can be used as destructive agent of styrofoam waste. The fastest destruction time of styrofoam waste occurs at electronic packaging with essential oil mixture by volume ratio of 1:1:2.
Peroxygenases are an emerging new class of enzymes allowing selective oxyfunctionalisation reactions in a cofactor-independent way different from well-known P450 monooxygenases. Herein, we focused on recent developments from organic synthesis, molecular biotechnology and reaction engineering viewpoints that are devoted to bring these enzymes in industrial applications. This covers natural diversity from different sources, protein engineering strategies for expression, substrate scope, activity and selectivity, stabilisation of enzymes via immobilisation, and the use of peroxygenases in low water media. We believe that peroxygenases have much to offer for selective oxyfunctionalisations and we have much to study to explore the full potential of these versatile biocatalysts in organic synthesis.
A bienzymatic cascade for selective sulfoxidation is presented. The evolved recombinant peroxygenase from Agrocybe aegeritra catalyses the enantioselective sulfoxidation of thioanisole whereas the choline oxidase from Arthrobacter nicotianae provides the H2O2 necessary via reductive activation of ambient oxygen. The reactions are performed in choline chloride‐based deep eutectic solvents serving as co‐solvent and stoichiometric reductant at the same time. Very promising product concentrations (up to 15 mM enantiopure sulfoxide) and catalyst performances (turnover numbers of 150,000 and 2100 for the peroxygenase and oxidase, respectively) have been achieved.
Natural deep eutectic solvents (NADES) are proposed as alternative solvents for peroxygenase‐catalysed oxyfunctionalization reactions. Choline chloride‐based NADES are of particular interest as they can serve as solvent, enzyme‐stabiliser and sacrificial electron donor for the in situ H2O2 generation. This report provides the first proof‐of‐concept and basic characterisation of this new reaction system. Highly promising turnover numbers for the biocatalysts of up to 200,000 have been achieved.
The use of natural deep eutectic solvents (NADES) as multifunctional solvents for limonene bioprocessing was reported. NADES were used for the extraction of limonene from orange peel wastes, as solvent for the chemoenzymatic epoxidation of limonene, and as sacrificial electron donor for the in situ generation of H2O2 to promote the epoxidation reaction. The proof‐of‐concept for this multifunctional use was provided, and the scope and current limitations of the concept were outlined.
Deep eutectic solvents (DES) are a class of neoteric solvents used in multiple applications amongst which biocatalytic processes. Due to its simple preparation, low cost and inherent biodegradable properties, its use as a non-volatile biocompatible co-solvent with both whole cells and isolated enzymes has displayed increased enzyme activity and stability translating to higher product conversions and a surprising higher enantioselectivity in a range of biotransformations. This review lays out the latest updates on the use of DES in redox biocatalytic reactions. With that purpose, a clear division has been made to summarize the application of DES in bioreduction reactions using whole cells and purified alcohol dehydrogenases, oxidations involving alcohol dehydrogenases, heme-dependent enzymes, peroxidases, laccases and catalases, ozonolysis reactions, and finally mention the application of lipase in the mediation of chemoenzymatic epoxidation and Baeyer-Villiger reactions.
Limonene is present in orange peel wastes and is known as an antimicrobial agent, which impedes biogas production when digesting the peels. In this work, pretreatment of the peels to remove limonene under mild condition was proposed by leaching of limonene using hexane as solvent. The pretreatments were carried out with homogenized or chopped orange peel at 20-40°C with orange peel waste and hexane ratio (w/v) ranging from 1 : 2 to 1 : 12 for 10 to 300 min. The pretreated peels were then digested in batch reactors for 33 days. The highest biogas production was achieved by treating chopped orange peel waste and hexane ratio of 12 : 1 at 20°C for 10 min corresponding to more than threefold increase of biogas production from 0.061 to 0.217 m(3) methane/kg VS. The solvent recovery was 90% using vacuum filtration and needs further separation using evaporation. The hexane residue in the peel had a negative impact on biogas production as shown by 28.6% reduction of methane and lower methane production of pretreated orange peel waste in semicontinuous digestion system compared to that of untreated peel.
The production of orange juice on an industrial level leads to a considerable quantity of solid and liquid residue (around 8–20 million tons y−1 globally), which is still considered as waste or used as a complement in agriculture. In general, orange residues have no economic value, even though their composition is rich in soluble sugars, cellulose, hemicellulose, pectin and essential oils that could form the basis of several industrial processes. In this study, information was collected on the technological potential of the solid and liquid residues generated in the processing of orange juice. Possible applications include human consumption, fertilizer, animal feed, charcoal, adsorption of chemical compounds, bio-oil production and extraction of essential oils and pectin. In this preliminary study, alternatives are proposed for the minimization and recovery of solid and liquid residues generated in the production of orange juice with a view to the implantation of industrial plants which can reuse this material, in order to add value to this solid and liquid waste and provide environmental benefits. The alternatives were proposed based on information and data available in the literature and the concepts of clean technologies.
1,4-Butanediol is shown as an efficient cosubstrate to promote NAD(P)H-dependent redox biocatalysis. The thermodynamically and kinetically inert lactone coproduct makes the regeneration reaction irreversible. Thereby not only the molar surplus of cosubstrate is dramatically reduced but also faster reaction rates are obtained.
In order to engineer the choline oxidase from Arthrobacter nicotianae (An_CodA) for the potential application as biological bleach in detergents, the specific activity of the enzyme toward the synthetic substrate tris-(2-hydroxyethyl)-methylammonium methylsulfate (MTEA) was improved by methods of directed evolution and rational design. The best mutants (up to 520% wt-activity with MTEA) revealed mutations in the FAD- (A21V, G62D, I69V) and substrate-binding site (S348L, V349L, F351Y). In a separate screening of a library comprising of randomly mutagenised An_CodA, with the natural substrate choline, four mutations were identified, which were further combined in one clone. The constructed clone showed improved activity towards both substrates, MTEA and choline. Mapping these mutation sites onto the structural model of An_CodA revealed that Phe351 is positioned right in the active site of An_CodA and very likely interacts with the bound substrate. Ala21 is part of an alpha-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme.
Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). A. aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H(2)O(2) (2 mM) for optimum activity. The apparent K(m) values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H(2)O(2) were 37, 298, 1,001, 2,367 and 1,313 microM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils.
The aqueous substrate and product toxicity thresholds in the microbial biotransformation of (-)-trans-carveol to the fragrance/flavor compound (R)-(-)-carvone by Rhodococcus erythropolis were determined. Above aqueous phase concentrations of approx. 500 mg carveol/l and 200-600 mg carvone/l, the biotransformation activity of the biocatalyst was inhibited. This biotransformation was undertaken in a single aqueous phase 3 l [corrected] reactor in which a total of 5 ml carveol (mixture of isomers) was added before the biotransformation rate decreased significantly. The carvone volumetric productivity was 31 mg/lh. Although the growth of the organism post-exposure was not affected, dramatic morphological changes in response to the accumulation of the inhibitory substrate and product were observed.
Recently, cost-effective production of highly value-added furan chemicals from abundant and renewable bioresources has gained much attentions via chemoenzymatic approach in environmentally-friendly reaction system. In this work, chemoenzymatic cascade reaction...
Caraway is a widespread agricultural crop that has been utilized by humans since ancient times. It is known as a kitchen spice, medicinal plant in phytotherapy and, due to its high essential oil content, as a fragrant plant and raw material for terpene production. Limonene and carvone, which in turn are used as precursors for the synthesis of further flavorings and fine chemicals, account for more than 90 % of the essential oil. However, the quality of essential oils can quickly deteriorate due to inadequate storage and by external impacts such as heat, oxygen, and light. In the present study, caraway seeds, caraway essential oil as well as pure standards of limonene and carvone were stored in parallel under real-time conditions at 25 °C and 40 °C to assess the effect of the plant matrix and the natural blend of compounds on essential oil stability. GC and HPLC analyses were used to show that the volatile compounds were well protected in the caraway seeds as no significant changes were determined. In comparison, a remarkable loss of limonene (22.4 %) due to oxidation-induced alterations was observed for the pure substance, while carvone contents remained almost constant over the entire storage period. The decline of limonene (9.2 %) as part of the essential oil was much lower while carvone again remained unchanged. The formation of novel minor compounds such as limonene-1,2-epoxide, carveol, and limonene-1,2-diol was mainly due to limonene downstream oxidation and increased with elevated storage temperatures. Volatile compounds were best protected in the intact plant seeds, and storage of the entire essential oil is preferable compared with isolated terpenes due to higher stability as representatively demonstrated for the major compound limonene.
Orange, one of the most important fruit categories to be consumed across the world, when processed produces 50% of its weight as waste. Current waste management options for orange peel waste are inadequate to use the waste in wholesome and its disposal might lead to other environmental concerns. Here, we present microwave hydrothermal carbonization as an alternative to utilize the orange peel waste. Further, using citric acid to catalyze the microwave hydrothermal carbonization resulted in 30% higher maximal yield of hydrochar, and the hydrochar produced had better elemental, proximate and energy properties than hydrochar made during uncatalyzed microwave hydrothermal carbonization. Further, structural analysis revealed that citric acid promoted the formation of nanospheres during microwave hydrothermal carbonization. Taken together, microwave hydrothermal carbonization of orange peel waste using citric acid as a catalyst might not only help address the waste management concerns for orange peel waste, but also can produce end products of potential commercial value.
This research investigates the use of seven natural deep eutectic solvents (NADESs) for valorisation of orange peel waste, with the final goal to propose a unique NADES for integrated biorefinery. Initial screening of NADESs revealed the excellent ability of cholinium-based NADES with ethylene glycol as hydrogen bond donor (ChEg50) to serve as a medium for orange peel-catalysed kinetic resolution (hydrolysis) of (R,S)-1-phenylethyl acetate with high enantioselectivity (ee = 83.2%, X = 35%), as well as it’s stabilizing effect on the hydrolytic enzymes (hydrolytic enzymes within ChEg50 peel extract were stabile during 20 days at 4 °C). The ChEg50 also showed a satisfactory capacity to extract D-limonene (0.5 mg gFW⁻¹), and excellent capacity to extract polyphenols (45.7 mg gFW⁻¹), and proteins (7.7 mg gFW⁻¹) from the peel. Based on the obtained results, the integrated biorefinery of orange peel waste using ChEg50 in a multistep process was performed. Firstly, enantioselective kinetic resolution was performed (step I; ee = 83.2%, X = 35%), followed by isolation of the product 1-phenylethanol (step II; h = 82.2%) and extraction of polyphenols (step III; h = 86.8%) from impoverished medium. Finally, the residual orange peel was analysed for sugar and lignin content, and results revealed the potential of waste peel for the anaerobic co-digestion process. The main bottlenecks and futures perspective of NADES-assisted integrated biorefinery of orange peel waste were outlined through SWOT analysis.
The acquisition of resveratrol from Polygonum cuspidatum is complicated and costs organic solvents due to extraction and hydrolysis of its corresponding glycoside (polydatin). In this work, a novel one-pot method based on deep eutectic solvent (DES) was developed for simultaneous extraction and conversion of polydatin to resveratrol from Polygonum cuspidatum for the first time. The extraction yield of resveratrol by DES-based one-pot method were significantly higher than that of water, methanol and ethanol. After optimization by One-Variable-at-a-Time and response surface methodology, the extraction yield of resveratrol reached 12.26 ± 0.14 mg/g within 80 min. The conversation efficiency of polydatin to resveratrol in Polygonum cuspidatum from five different origins was more than 96.3%. Scanning electron microscope results indicated the selected DES disrupted plant cell walls to enhance the yield of resveratrol. The results indicated that one green method was successfully established for efficient extraction and conversion of polydatin to resveratrol from Polygonum cuspidatum.
Peroxygenases are attractive biocatalysts for the selective introduction of oxygen into organic molecules under mild conditions with hydrogen peroxide as the oxygen source. In addition to the identification of primary peroxygenases, different classes of enzymes were shown to display promiscuous peroxygenase activity. Even though enzymes with peroxygenase activity are promising industrial biocatalysts, further optimization of their properties is required for their effective use in industrial applications. Here we give a comprehensive overview of enzymes with peroxygenase activity and review diverse strategies, including directed evolution, rational approaches and the assistance of small functional molecules to improve the expression, catalytic activity, substrate scope or selectivity of these promising enzymes. Furthermore, we discuss the exploration of modified or unnatural cofactors to design artificial peroxygenases for desired reactions. The rapidly expanding field of hydrogen peroxide-utilizing enzymes bears a great potential to provide biocatalysts for selective oxyfunctionalization chemistry, contributing to the development of environmentally friendly and sustainable oxidation processes.
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Sigmund, M., Poelarends, G.J. Current state and future perspectives of engineered and artificial peroxygenases for the oxyfunctionalization of organic molecules. Nat Catal 3, 690–702 (2020). 10.1038/s41929-020-00507-8.
Orange peel waste (OPW) can be an effective feedstock for extraction of natural bioactive components such as limonene, a high value-added chemical broadly exploited for food, pharmaceutical, and cosmetic industrial applications. Extraction of limonene from OPW has been conventionally performed via solvent extraction using hexane, a hazardous petrochemical solvent currently restricted under international regulations. In this work, we have conducted a comparative assessment of the performance of a variety of green solvents for sustainable valorisation of OPW through limonene extraction. In particular, cyclopentyl methyl ether (CPME), ethyl lactate (EL), isopropyl alcohol (IPA), polyethylene glycol 300 (PEG 300), isopropyl acetate (IAc), dimethyl carbonate (DMC), methyl ethyl ketone (MEK), 2-methyl-tetrahydrofuran (2-MeTHF) and ethyl acetate (EAc) have been evaluated as hexane replacement for the recovery of limonene from OPW. Initially, a preliminary solvent screening was carried out using the COnductor-like Screening MOdel for Real Solvents (COSMO-RS) to estimate the solubility of limonene in the proposed solvents and rank their theoretical extraction performance. Afterwards, experimental studies were performed to determine the limonene extraction yields and optimize the operating conditions (temperature, time and solvent load) for limonene recovery from OPW using the various solvents, as confirmed by gas chromatography mass spectrometry (GC–MS) analysis. Overall, results support that CPME and 2-MeTHF bio-based solvents significantly outperform the benchmark petrochemical solvent hexane by increasing limonene extraction yields up to 80% and 40% respectively at optimum operating conditions. Moreover, recovery and reuse of these solvents in consecutive extraction cycles was successfully accomplished, while scanning electron microscopy analysis (SEM) suggests that solvent effects on biomass structure disruption could be beneficial for further bioprocessing.
Extraction of polyphenolic compounds from orange peel (OP) via solid-liquid extraction (SLE) using deep eutectic solvents (DES) was investigated in this work as a means to develop sustainable separation processes to recover natural antioxidants from food waste biomass. In particular, choline chloride-based DES paired with glycerol and ethylene glycol were explored as potential extractants to evaluate the effect of the solvent structure (i.e. number of hydroxyl groups and hydrogen bond acceptor/donor ratio) on the polyphenol extraction efficiency. Afterwards, the effect of different operating parameters (i.e. solvent concentration, temperature, time and solid/liquid ratio) were further evaluated to determine the optimal extraction conditions for polyphenols recovery in terms of total phenolic content (TPC) and antioxidant activity based on DPPH radical scavenging method. Under optimum conditions (DES 10 wt.% water, temperature of 333.15 K, 1:10 solid/liquid ratio and extraction time of 100 min), choline chloride-based DES outperformed the benchmark solvent (aqueous (aq.) ethanol 30 wt.% water) for the extraction of polyphenolic compounds from OP, with [Ch]Cl:EG 1:4 providing the highest TPC (3.61 mg gallic acid equivalent per gram of orange peel (GAE/g OP) and antioxidant potential (30.6 µg/ml). Although ethylene glycol (EG) has provided the highest TPC (5.84 mg GAE/g OP), DES have proved more selective towards target polyphenolic compounds. Additionally, Scanning Electron Microscopy (SEM) was performed to illustrate the structural modifications occurred on the biomass before and after the extraction process, supporting DES as efficient solvents for cell wall dissolution. Lastly, characterization and quantification of individual polyphenolic profiles in the extracts were completed via high performance liquid chromatography (HPLC), displaying ferulic acid as the most abundant compound followed by ρ-coumaric acid and gallic acid.
DES (deep eutectic solvents) are a new class of ionic liquids that have excellent properties. The strength of interaction between molecules in the DES affects their properties and applications. In this work, the strength of molecular interactions between components in the betaine monohydrate salt and polyol (glycerol or/and propylene glycol) eutectic mixtures was studied by experimental and computational studies. The melting point and fusion enthalpy of the mixtures were measured using STA (Simultaneous Thermal Analyzer). The nature and strength of intermolecular interactions were observed by FT-IR and NMR spectroscopy. The molecular dynamics simulation used to determine the number of H-bonds, percent occupancy, and radial distribution functions in the eutectic mixtures. The interaction between betaine monohydrate and polyol is following order: betaine monohydrate-glycerol-propylene glycol > betaine monohydrate-glycerol > betaine monohydrate-propylene glycol, where the latter is the eutectic mixture with the lowest stability, strength and extent of the hydrogen bonding interactions between component molecules. The presence of intra-molecular hydrogen bonding interactions, the inter-molecular hydrogen bonding interactions between betaine molecule and polyol, and also interactions between polyol and H2O of betaine monohydrate in the eutectic mixtures.
Chemical syntheses in contemporary process industries today are predominantly conducted using organic solvents, which are potentially hazardous to humans and the environment alike. Green chemistry was developed as a means to overcome this hazard and it also holds enormous potential for designing clean, safe and sustainable processes. The present work incorporates the concepts of green chemistry in its design of a lipase-mediated epoxidation process for monoterpenes; the process uses alternative reaction media, namely deep eutectic solvents (DESs), which have not been reported for such an application before. Choline chloride (ChCl), in combination with a variety of hydrogen bond donors (HBD) at certain molar ratios, was screened and tested for this purpose. The process was optimized through the design of experiments (DoE) using the Taguchi method for four controllable parameters (temperature, enzyme amount, peroxide amount and type of substrate) and one uncontrollable parameter (DES reaction media) in a crossed-array design. Two distinct DESs, namely glycerol:choline chloride (GlCh) and sorbitol:choline chloride (SoCh), were found to be the best systems and they resulted in a complete conversion of the substrates within 8 h. Impurities (esters) were found to form in both the DESs, which was a concern; as such, we developed a novel minimal DES system that incorporated a co-substrate into the DES so that this issue could be overcome. The minimal DES consisted of urea·H2O2 (U·H2O2) and ChCl and exhibited better results than both the GlCh and SoCh systems; complete conversions were achieved within 2 h for 3-carene and within 3 h for both limonene and α-pinene. Product isolation with a simple water/ethyl acetate based procedure gave isolated yields of 87.2 ± 2.4%, 77.0 ± 5.0% and 84.6 ± 3.7% for 3-carene, limonene and α-pinene respectively.
A chemoenzymatic method for the production of epoxidized vegetable oils was developed. The unique combination of the commercial lipase G from Penicillieum camembertii with certain deep eutectic solvents enabled the efficient production of epoxidized vegetable oils.
The enormous quantity of food wastes discarded annually forces a look into alternatives for this interesting feedstock. Thus, food bio-waste valorisation is one of the current imperatives of society. This review is the most comprehensive overview of currently existing technologies and processes in this field. It tackles classical and innovative physical, physico-chemical and chemical methods of food waste pre-treatment and extraction for the recovery of added value compounds and detection by modern technologies and is an outcome of the COST Action EUBIS, TD1203 Food Waste Valorisation for Sustainable Chemicals, Materials and Fuels.
In this proof of concept study we demonstrate direct utilization of limonene containing waste product orange peel as starting material for a biocatalytic cascade reaction. The product of this cascade is chiral carvolactone, a promising building block for thermoplastic polymers. Four different concepts were applied to augment limonene availability based on either water extraction solely, addition of extraction enhancers or biomass dissolution.
Unspecific peroxygenase (UPO) is a highly efficient biocatalyst with a peroxide dependent monooxygenase activity and many biotechnological applications, but the absence of suitable heterologous expression systems has precluded its use in different industrial settings. Recently, the UPO from Agrocybe aegerita was evolved for secretion and activity in Saccharomyces cerevisiae [8]. In the current work, we describe a tandem-yeast expression system for UPO engineering and large scale production. By harnessing the directed evolution process in S. cerevisiae, the beneficial mutations for secretion enabled Pichia pastoris to express the evolved UPO under the control of the methanol inducible alcohol oxidase 1 promoter. Whilst secretion levels were found similar for both yeasts in flask fermentation (∼8mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217mg/L). The P. pastoris UPO variant maintained similar biochemical properties of the S. cerevisiae counterpart in terms of catalytic constants, pH activity profiles and thermostability. Thus, this tandem-yeast expression system ensures the engineering of UPOs to use them in future industrial applications as well as large scale production.
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Deep eutectic solvents, as a new type of eco-friendly solvent, have attracted increasing attention in chemistry for the extraction and separation of target compounds from various samples. To summarize the application of deep eutectic solvents, this review highlights some of the unique properties of deep eutectic solvents and deep-eutectic-solvent-based materials, as well as their applications in extraction and separation. In this paper, the available data and references in this field are reviewed to summarize the application developments of deep eutectic solvents. Based on the development of deep eutectic solvents, the exploitation of new deep eutectic solvents and deep-eutectic-solvent-based materials are expected to diversify into extraction and separation.This article is protected by copyright. All rights reserved
Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1 encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein and the resulting products tested. Over 9,000 clones were screened using an ad-hoc dual-colorimetric assay that assessed both peroxidative and oxygen-transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant: the evolved leader increased functional expression 27-fold whereas a 18-fold improvement in the kcat/Km for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic co-solvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/L, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme-access channel. Overall, the directed evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.
Deep eutectic solvents (DESs) are emerging rapidly as a new type of green solvent instead of an ionic liquid (IL), and are typically formed by mixing choline chloride with hydrogen bond donors. Few studies have applied DESs to the extraction and determination of bioactive compounds. Therefore, in the present study, DESs were used to extract flavonoids (myricetin and amentoflavone), which are well known and widely used antioxidants, to extend their applications. A range of alcohol-based DESs with different alcohols to choline chloride (ChCl) mixing ratios were used for extraction using several extraction methods. Other factors, such as temperature, time, water addition and solid/liquid ratio, were examined systematically using a response surface methodology (RSM). A total of 0.031 and 0.518mgg-1 of myricetin and amentoflavone were extracted under the optimized conditions: 35vol% of water in ChCl/1,4-butanediol (1/5) at 70.0°C for 40.0min and a solid/liquid ratio of 1/1 (g10mL-1). Good linearity was obtained from 0.1×10-3 to 0.1mgmL-1 (r2>0.999). The excellent properties of DESs highlight their potential as promising green solvents for the extraction and determination of a range of bioactive compounds or drugs.
The antimicrobial activity of the essential oils of Mentha spicata L. and Anethum sowa Roxb. (Indian dill) were studied. The major chemical constituents of the hydrodistilled essential oils and their major isolates from cultivated M. spicata and A. sowa were identified by IR, 1H- and 13C-NMR and GC: (S)-(−)-limonene (27.3%) and (S)-(−)-carvone (56.6%) (representing 83.9% of the spearmint oil) and (R)-(+)-limonene (21.4%), dihydrocarvone (5.0%), (R)-(+)-carvone (50.4%) and dillapiole (17.7%) (together 76.9% in Indian dill oil), respectively. In vitro bioactivity evaluation of the isolated oil components revealed that both the optical isomers of carvone were active against a wide spectrum of human pathogenic fungi and bacteria tested. (R)-(+)-limonene showed comparable bioactivity profile over the (S)-(−)-isomer. The activity of these monoterpene enantiomers was found to be comparable to the bioactivity of the oils in which they occurred. Copyright © 2001 John Wiley & Sons, Ltd.
d-Carvone or (4S)-(+)-carvone has a caraway/dill odour and is the main constituent of caraway (Carum carvi) and dill (Anethum graveolens) seed oils whilst l-Carvone or (4R)-(−)-carvone has a sweet spearmint odour and is the main constituent of spearmint (Mentha spicata). Carvone is produced by both extraction and purification of essential oils from caraway, dill and spearmint seeds and by chemical and biotechnological synthesis.The several applications of carvone as fragrance and flavour, potato sprouting inhibitor, antimicrobial agent, building block and biochemical environmental indicator, along with its relevancy in the medical field, justify the interest in this monoterpene.
In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K
M = 1.51 ± 0.09 mM and V
max = 42.73 ± 0.42 mU/min for choline chloride and K
M = 4.77 ± 0.76 mM and V
max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.
Life cycle assessment of orange peel waste management
- V Negro
- B Ruggeri
- D Fino
- D Tonini
Negro V, Ruggeri B, Fino D, Tonini D. Life cycle assessment of orange
peel waste management. Resour Conserv Recycl. 2017;127:148-158.