In recent years, the biotechnological production of carotenoids and lipids by yeasts emerges as a valuable strategy at the industrial level, also fitting the circular economy pillars when agri-food waste can be used as the main components of the culture media. In this study, bioprocesses employing red yeasts were developed using cost-effective agri-industrial residues, such as soy okara (a soybean industry byproduct). This low-cost substrate was investigated as a source of carbohydrates and essential nutrients, with its enzymatic pre-treatment optimized to create a balanced and efficient fermentation medium. The screening of a collection of red oleaginous yeasts identified Rhodotorula paludigena CBS 6565 and Rhodotorula diobovata CBS 324 as promising strains capable of efficiently producing both lipids and uncommon carotenoids. These two strains were cultivated in a lignocellulose hydrolysate-based medium supplemented with urea, validating the promising results of the screening. Urea, a cost-effective nitrogen source, was found to enhance carotenoid production compared to ammonium sulfate. Finally, soy okara was used as the fermentation medium for Rhodotorula paludigena CBS 6565. Soy okara underwent optimized enzymatic hydrolysis to maximize fermentable sugar release, while the addition of waste cooking oil and syrup from candied fruit processing significantly boosted carotenoid production, reaching 262.4 mg/L in 90 h. Among these, β-carotene and torularhodin contributed 140 mg/L and 72.5 mg/L, respectively. Furthermore, the yeast cells accumulated lipids, constituting 56

Synthesis of oximes and nitriles from alcohols traditionally requires high temperatures, toxic reagents, and unfriendly solvents. Herein, the combination of photo‐ and biocatalytic processes is disclosed by first performing a photocatalytic oxidative oximation of benzyl alcohols to produce benzaldehyde oximes in the presence of hydroxylamine hydrochloride (75%–99% conversion after 4 h). Subsequently, enzymatic dehydration was carried out using the double mutant of the aldoxime dehydratase OxdF1 (L318F/F306Y) in a stepwise manner, which allowed obtaining benzonitriles (50%–73% isolated yield).

This study focuses on the enzymatic modification of citrus pectin (CP) with stearic acid (SA) in a bi-phasic reaction system, with the aim of enhancing its application in packaging films. We employed a central composite design of experiments and principal component analysis to investigate the effects of pH, reaction temperature, and time on the modification process. The principal component analysis identified reaction time and temperature as critical factors influencing modification, leading to a maximum modification percentage of 43.35 ± 1.81 % at pH 7, 60 °C, and 72 h. The physical characteristics of modified citrus pectin (MCP) showed increased molecular weight and particle size, thereby influencing the packaging films' mechanical and barrier properties. Zeta potential measurements indicated improved dispersion stability, resulting in homogeneous film formation. Chemical analyses confirmed the structural modifications in MCP. The characterisation of MCP-based films revealed a trade-off between properties: a significant reduction in mechanical strength was observed, offset by substantial improvements in water and oxygen barrier performance. This work positions MCP as a functional biopolymer for developing advanced composite packaging materials with tailored barrier properties.

This study investigates the synthesis of aromatic nitriles using an evolved variant of OxdF1 (L318F/F306Y), an aldoxime dehydratase from Pseudomonas putida F1, engineered for improved catalytic efficiency toward benzaldehyde oxime. The double OxdF1 (L318F/F306Y) mutant effectively catalyzes the conversion of various benzaldoxime derivatives to the corresponding nitriles. Due to the enzyme's inherent instability, immobilized whole-cell systems are employed in a flow reactor to improve its stability and broaden its applicability, with the biotransformation of benzaldehyde oxime and 2,6-difluorobenzaldehyde oxime serving as case studies. The enzyme's stability is markedly improved, maintaining 87% yield even after 8 h of processing in the preparation of benzonitrile. Preparation of 2,6-difluorobenzontirile poses additional challenges due to the low water solubility of both the substrate, and even more so, the product, an important intermediate in various chemical applications. To overcome solubility limitations, a segmented liquid-liquid flow system (water/cyclohexane) was implemented, significantly improving the enzyme stability. The process was run continuously for 12 h, with a conversion of ≈70% by the end of the operation. Furthermore, 2,6-difluorobenzonitrile is selectively extracted in-line using a liquid-liquid extractor, thus, facilitating its efficient recovery and purification.

Tremendous quantities of textile waste generated and primarily landfilled annually represent a huge risk of contaminating the environment, together with loss of valuable resources. Especially, blended fabrics further pose a challenge for recycling and valorization strategies, while enzymatic hydrolysis offers a highly specific and environmentally friendly solution. In this study, we demonstrate that proteases specifically hydrolyze the wool components in blends with polyester, allowing recovery of pure polyester fibers as well as amino acids and peptides as platform molecules for further valorization. Recovered amino acids and peptides were successfully used as a nitrogen source for cultivation of Chlorella vulgaris and Rhodotorula mucilaginosa for the production of valuable biomolecules including pigments and lipids. Here, 11.3 mg/gCDW chlorophyll and 47% lipid content were obtained from algal biomass, while 1.1 mg/gCDW carotenoids and 35% lipids content were reached from the yeast grown on wool hydrolysate as the sole nitrogen source. These could be applied as natural dyes for textile applications or as biofuels to replace toxic synthetic compounds and fossil resources, respectively. The presented concept demonstrates feasibility of enzymatic recovery and microbial valorization of components of blended textile waste to support the development toward a circular bioeconomy.

In the last decade, phloretin (PHL) has attracted increasing attention due to its remarkable biological properties, including antimicrobial, antidiabetic, cardioprotective, anti-inflammatory, immunomodulatory, and antioxidant effects, becoming a leading ingredient in the cosmetic sector. In this work, an efficient, cost-effective, and highly productive biocatalytic strategy for the preparation of natural PHL has been developed starting from its glycosylated form, phloridzin, one of the main flavonoid components of apple processing waste (apple pomace). The process involved the use of the extremophilic β-glycosidase AHeGH1 immobilized on bacterial cellulose films in a two-liquid phase reaction system (water/2,2,5,5-tetramethyloxolane), allowing for the complete conversion of 5 g L-1 of substrate in 7 h of reaction (molar conversion >99%; isolated yield 95%). Since all the materials used in the biotransformation have been recovered and recycled (i.e., solvents, aqueous phase, and catalyst), this system can be considered a zero-waste reaction. Interestingly, a further leap forward in the overall bioprocess sustainability was achieved by producing bacterial cellulose, the support for enzyme immobilization, by fermentation of apple pomace. This allows for a biocatalytic process where both the substrate and the immobilization carrier derive from the same feedstock.

The one-shot green oxidation of different benzyl alcohol derivatives into the corresponding carboxylic acids was achieved by using Acetobacter malorum DSM 112354, a newly isolated strain. Oxidation of benzyl alcohol was initially optimized considering productivity after 8 h as response parameter. Under optimized conditions, a set of para-substituted benzyl alcohols was tested, showing the great versatility of Acetobacter malorum DSM 112354 which was able to oxidize all the substrates albeit with different rates and conversions. The effect of the substituent position was subsequently studied using 2-(hydroxymethyl)phenol, 3-(hydroxymethyl)phenol, and 4-(hydroxymethyl)phenol as starting materials, showing that all the substrates were efficiently transformed (74-98% molar conversion in 2-24 h). Finally, the oxidation of 1,4-phenylenedimethanol gave 4-(hydroxymethyl)benzoic acid proceeded with excellent regioselectivity, since <3% of terephthalic acid was observed. This regioselective reaction was optimized using Ba-alginate immobilized cells of Acetobacter malorum DSM 112354 in a continuous multiphasic flow reactor. The process continuously run for 24 h allowed the obtainment of 0.65 g of HMBA using a 14.4 mL reactor. This intensification was associated with upgraded biocatalyst productivity (37.1 mmolP/gcells dry weight) and space-time yield (45.1 mgP/mL d).

To advance sustainable packaging development, this study investigates modifying pectin-based films with citric acid cross-linking and nanocellulose reinforcement to enhance their functional properties. Cross-linked citrus pectin (CLCP) films were obtained from film-forming solutions containing different amounts of pectin (2.5-3.5 g 100 mL-1) and citric acid (1-2 g 100 mL-1), with glycerol (1-3 mL 100 mL-1), and nanocellulose (0-2 g 100 mL-1) added as a plasticizer and reinforcing agent, respectively. CLCP films were comprehensively characterized concerning structural, mechanical, and barrier properties. FTIR confirmed successful cross-linking, whereas the structural modifications occurred in the pectin network after cross-linking were elucidated for the first time using advanced 2D-NMR. Principal component analysis (PCA) revealed that nanocellulose concentration was the most significant factor in improving mechanical properties, with strong positive correlations with tensile strength (r = 0.586) and Young's modulus (r = 0.639). Conversely, glycerol concentration showed the strongest negative correlation (r = -0.746 and -0.741, respectively), confirming its plasticizing effect. Barrier properties were also significantly influenced: higher citric acid concentration and processing temperature improved the oxygen barrier. In contrast, higher pectin and nanocellulose concentrations decreased the oxygen transmission rate (r = -0.636 and r = -0.733, respectively). This systematic analysis provides a comprehensive design map for tuning film properties, demonstrating that co-modifying pectin with citric acid and nanocellulose is a highly effective strategy for developing advanced, sustainable packaging materials with tailored performance.

In this work we developed an eco-friendly valorisation of Citrus wastes (CWs), through a solvent-assisted ultrasonication extraction technique, thus having access to a wide range of bio-active compounds and polysaccharides, extremely useful in different industrial sectors (food, cosmetics, nutraceutical). Water-based low-amplitude ultrasonication was examined as a potential method for pectin extraction as well as polar and non-polar citrus extractives (CEs), among which hesperidin and triglycerides of 18 carbon fatty acids were found to be the most representative ones. In addition, citric acid:glycerol (1:4)-based deep eutectic solvent (DES) in combination with ultrasonic extraction was utilized to extract microcellulose (CMC), from which stable cellulose nanocrystals (CNCs) with glycerol-assisted high amplitude ultrasonication were obtained. The physical and chemical properties of the extracted polysaccharides (pectin, micro and nanocellulose) were analysed through DLS, ζ-potential, XRD, HP-SEC, SEM, AFM, TGA-DSC, FTIR, NMR, and PMP-HPLC analyses. The putative structure of the extracted citrus pectin (CP) was analysed and elucidated through enzyme-assisted hydrolysis in correlation with ESI-MS and monosaccharide composition. The developed extraction methods are expected to influence the industrial process for the valorisation of CWs and implement the circular bio-economy.

The current work aims to produce nanoparticle-infused starch-based bioactive thermoplastic packaging films. The FeO and ZnO nanoparticles were examined to be potential active ingredients for the production of nanoparticle-infused bioactive thermoplastic packaging films. The bio-thermoplastic films infused with FeO and ZnO nanoparticles showed high oxygen scavenging and antimicrobial activity, respectively. Consecutively, both films were combined to form a double-layer Nano-Biothermoplastic packaging system for food preservation. The distribution and diffusion of nanoparticles in starch-based films were examined to be influenced by the amorphous character of starch and the swelling index of the film, respectively. The amorphous property of starch molecules showed a masking effect on the crystalline characteristics of nanoparticles in Nano-Biothermoplastic films. The diffusion of nanoparticles from the Nano-Biothermoplastic packaging system was found to influence the microbial, chemical, and color characteristics of mutton and chicken meat stored at 4 °C.

The present work deals with the development of cellulose-reinforced starch-based bioactive thermoplastic packaging films, from complete recycling of banana peel waste. The nanocellulose fibers, starch and bioactive compounds from banana peel were extracted and reconstituted to produce cellulose-reinforced starch-based bioactive thermoplastic packaging films. The banana peel starch was examined to have an abundance of amylopectin (88.55 & PLUSMN; 0.28% (w/w)) and high thermal stability (-295 degrees C maximum degradation temperature), to serve as a matrix for the thermoplastic films. The ethanolic extract of the banana peel with major active compounds of & beta;-sitosterol, and 1, 2 Benzenedicarboxylic acid mono (2-ethyl hexyl ester) was examined to be having high antioxidant (74.43 & PLUSMN; 0.26% DPPH inhibition) and antimicrobial properties, to serve as a potential bioactive ingredient for the development of bioactive thermoplastic films. The addition of banana peel-based nanocellulose fiber improved the mechanical (6-fold increase in tensile strength) and barrier properties (0.6-fold reduction in O2 permeability) of banana peel thermoplastic films (BPT). The developed bioactive BPT films were examined to have a UV blocking capacity of-98%. The produced Bioactive BPT films were found to be effective in the shelf life extension of bread by 10 days. The proposed methodology for the complete recycling of banana peel will take the state of research in agro-waste management one step closer to a green bio-circle economy.

This study investigates the effect of the hydrolysis process on bacterial cellulose (BC) to obtain bacterial cellulose nanocrystals (BCNCs) used to create high oxygen barrier nanocomposite coatings for food packaging applications.

Ozonolysis is a useful as well as dangerous reaction for performing alkene cleavage. On the other hand, enzymes are considered a more sustainable and safer alternative. Among them, Caulobacter segnis dioxygenase (CsO2) known so far for its ability to catalyze the coenzyme-free oxidation of vinylguaiacol into vanillin, was selected and its substrate scope evaluated towards diverse natural and synthetic stilbenoids. Under optimized conditions, CsO2 catalyzed the oxidative cleavage of the C=C double bonds of various trans-stilbenes, providing that a hydroxyl moiety was necessary in para-position of the phenyl group (e. g., resveratrol and its derivatives) for the reaction to take place, which was confirmed by modelling studies. The reactions occurred rapidly (0.5-3 h) with high conversions (95-99 %) and without formation of by-products. The resveratrol biotransformation was carried out on 50-mL scale thus confirming the feasibility of the biocatalytic system as a preparative method.

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Bacterial nanocellulose (BC) is a highly versatile biopolymer currently pursued as a material of choice in varied themes of biomedical and material science research fields. With the aim to extend the biotechnological applications, the genetic tractability of the BC producers within the Komagataeibacter genus and its potential as an alternative host chassis in synthetic biology have been extensively studied. However, such studies have been largely focused on the model Komagataeibacter spp. Here, we present a novel K. intermedius strain capable of utilizing glucose, and glycerol sources for biomass and BC synthesis. Genome assembly identified one bacterial cellulose synthetase ( bcs ) operon containing the complete gene set encoding the BC biogenesis machinery ( bcsI ) and three additional copies ( bcsII–IV ). Investigations on the genetic tractability confirmed plasmid transformation, propagation of vectors with pBBR1 and p15A origin of replications and constitutive and inducible induction of recombinant protein in K. intermedius ENS15. This study provides the first report on the genetic tractability of K. intermedius , serving as starting point towards future genetic engineering of this strain.

Microbial carboxylesterases are valuable biocatalysts that can selectively hydrolyze a wide spectrum of esters and may find their own applicability at industrial scale. In this work, we report the calorimetric study of an atypical enantioselective carboxylesterase from Bacillus coagulans (BCE). The influence of different pH conditions (from pH 6 to 9) on BCE thermal stability was investigated through Differential Scanning Calorimetry (DSC). A complete thermodynamic analysis of the system in combination with specific activity measurements was performed for the assessment of the best working conditions for such an enzyme. The overall results indicate that the kinetic benefits deriving from the temperature rise overwhelm the thermodynamic disadvantages in terms of protein stability, being aware that the working temperature should always be kept well below the onset of the denaturation process, which may trigger aggregation effects. Although the results concern this specific enzyme, the methodological approach has a general validity and may be useful as a guideline to design enzyme optimal working conditions through calorimetric methods

Optimized recombinant whole cells of E. coli bearing CYP153A6 were employed for catalyzing the hydroxylation of different monoterpene derivatives. In most cases, high selectivity was observed with exclusive hydroxylation of the allylic methyl group bound to the aliphatic ring. In the case of (R)- and (S)-carvone, hydroxylation occurred also on the other allylic methyl group, although to a lesser extent. Biotransformations carried out in fed-batch mode on (S)-limonene and α-terpineol showed that recombinant whole cells retained activity for at least 24 h, allowing for the recovery of 3.25 mg mL−1 of (S)-perillyl alcohol and 5.45 mg mL−1 of 7-hydroxy-α-terpineol, respectively.

Many sectors of industry, such as food, cosmetics, nutraceuticals, and pharmaceuticals, have increased their interest in polyphenols due to their beneficial properties. These molecules are widely found in Nature (plants) and can be obtained through direct extraction from vegetable matrices. Polyphenols introduced through the diet may be metabolized in the human body via different biotransformations leading to compounds having different bioactivities. In this context, enzyme-catalyzed reactions are the most suitable approach to produce modified polyphenols that not only can be studied for their bioactivity but also can be labeled as green, natural products. This review aims to give an overview of the potential of biocatalysis as a powerful tool for the modification of polyphenols to enhance their bioaccessibility, bioavailability, biological activity or modification of their physicochemical properties. The main polyphenol transformations occurring during their metabolism in the human body have been also presented.

Komagataeibacter spp. has been used for the bioconversion of industrial wastes and lignocellulosic hydrolysates to bacterial cellulose (BC). Recently, studies have demonstrated the capacity of Komagataeibacter spp. in the biotransformation of inhibitors found in lignocellulosic hydrolysates, aromatic lignin-derived monomers (LDMs) and acetate. In general, detoxification and BC synthesis from lignocellulosic inhibitors requires a carbon flow from acetyl-coA towards tricarboxylic acid and gluconeogenesis, respectively. However, the related molecular aspects have not yet been identified in Komagataeibacter spp. In this study, we isolated a cellulose-producing bacterium capable of synthesizing BC in a minimal medium containing crude glycerol, a by-product from the biodiesel production process. The isolate, affiliated to Komagataeibacter genus, synthesized cellulose in a minimal medium containing glucose (3.3 ± 0.3 g/L), pure glycerol (2.2 ± 0.1 g/L) and crude glycerol (2.1 ± 0.1 g/L). Genome assembly and annotation identified four copies of bacterial cellulose synthase operon and genes for redirecting the carbon from the central metabolic pathway to gluconeogenesis. According to the genome annotations, a BC production route from acetyl-CoA, a central metabolic intermediate, was hypothesized and was validated using acetate. We identified that when K. rhaeticus ENS9b was grown in a minimal medium supplemented with acetate, BC production was not observed. However, in the presence of readily utilizable substrates, such as spent yeast hydrolysate, acetate supplementation improved BC synthesis.