Enzymatic degradation of polylactic acid (PLA).

Environmental concerns arising from the increasing use of polluting plastics highlight polylactic acid (PLA) as a promising eco-friendly alternative. PLA is a biodegradable polyester that can be produced through the fermentation of renewable resources. Together with its excellent properties, suitable for a wide range of applications, the use of PLA has increased significantly over the years and is expected to further grow. However, insufficient degradability under natural conditions emphasizes the need for the exploration of biodegradation mechanisms, intending to develop more efficient techniques for waste disposal and recycling or upcycling. Biodegradation occurs through the secretion of depolymerizing enzymes, mainly proteases, lipases, cutinases, and esterases, by various microorganisms. This review focuses on the enzymatic degradation of PLA and presents different enzymes that were isolated and purified from natural PLA-degrading microorganisms, or recombinantly expressed. The review depicts the main characteristics of the enzymes, including recent advances and analytical methods used to evaluate enantiopurity and depolymerizing activity. While complete degradation of solid PLA particles is still difficult to achieve, future research and improvement of enzyme properties may provide an avenue for the development of advanced procedures for PLA degradation and upcycling, utilizing its building blocks for further applications as envisaged by circular economy principles. KEY POINTS: • Enzymes can be promisingly utilized for PLA upcycling. • Natural and recombinant PLA depolymerases and methods for activity evaluation are summarized. • Approaches to improve enzymatic degradation of PLA are discussed.

Shifting the balance: soluble ADAM10 as a potential treatment for Alzheimer's disease.

Introduction: Accumulation of amyloid ß in the brain is regarded as a key initiator of Alzheimer's disease pathology. Processing of the amyloid precursor protein (APP) in the amyloidogenic pathway yields neurotoxic amyloid ß species. In the non-amyloidogenic pathway, APP is processed by membrane-bound ADAM10, the main α-secretase in the nervous system. Here we present a new enzymatic approach for the potential treatment of Alzheimer's disease using a soluble form of ADAM10. Methods: The ability of the soluble ADAM10 to shed overexpressed and endogenous APP was determined with an ADAM10 knockout cell line and a human neuroblastoma cell line, respectively. We further examined its effect on amyloid ß aggregation by thioflavin T fluorescence, HPLC, and confocal microscopy. Using N-terminal and C-terminal enrichment proteomic approaches, we identified soluble ADAM10 substrates. Finally, a truncated soluble ADAM10, based on the catalytic domain, was expressed in Escherichia coli for the first time, and its activity was evaluated. Results: The soluble enzyme hydrolyzes APP and releases the neuroprotective soluble APPα when exogenously added to cell cultures. The soluble ADAM10 inhibits the formation and aggregation of characteristic amyloid ß extracellular neuronal aggregates. The proteomic investigation identified new and verified known substrates, such as VGF and N-cadherin, respectively. The truncated variant also exhibited α-secretase capacity as shown with a specific ADAM10 fluorescent substrate in addition to shedding overexpressed and endogenous APP. Discussion: Our in vitro study demonstrates that exogenous treatment with a soluble variant of ADAM10 would shift the balance toward the non-amyloidogenic pathway, thus utilizing its natural neuroprotective effect and inhibiting the main neurotoxic amyloid ß species. The potential of such a treatment for Alzheimer's disease needs to be further evaluated in vivo.

Cultured meat platform developed through the structuring of edible microcarrier-derived microtissues with oleogel-based fat substitute.

With the increasing global demand for meat, cultured meat technologies are emerging, offering more sustainable solutions that aim to evade a future shortage of meat. Here, we demonstrate a cultured meat platform composed of edible microcarriers and an oleogel-based fat substitute. Scalable expansion of bovine mesenchymal stem cells on edible chitosan-collagen microcarriers is optimized to generate cellularized microtissues. In parallel, an oleogel system incorporated with plant protein is developed as a fat substitute, which is comparable to beef fat in appearance and texture. Combining the cellularized microtissues with the developed fat substitute, two types of cultured meat prototypes are introduced: layered cultured meat and burger-like cultured meat. While the layered prototype benefits enhanced stiffness, the burger-like prototype has a marbling meat-like appearance and a softer texture. Overall, this platform and the established technological basis may contribute to the development of different cultured meat products and promote their commercial production.

Resorcinol-based hemiindigoid derivatives as human tyrosinase inhibitors and melanogenesis suppressors in human melanoma cells.

Human tyrosinase (hsTYR) catalyzes the key steps of melanogenesis, making it a privileged target for reducing melanin production in vivo. However, very few hsTYR inhibitors have been reported so far in the literature, whereas thousands of mushroom tyrosinase (abTYR) inhibitors are known. Yet, as these enzymes are actually very different, including at their active sites, there is an urgent need for new true hsTYR inhibitors in order to enable human-directed pharmacological and dermocosmetic applications without encountering the inefficiency and toxicity issues currently triggered by kojic acid or hydroquinone. Starting from the two most active compounds reported to date, i.e. a 2-hydroxypyridine-embedded aurone and thiamidol, we combined herein key structural elements and developed new nanomolar hsTYR inhibitors with cell-based activity. From a complete series of thirty-eight synthesized derivatives, excellent inhibition values were obtained for two compounds in both human melanoma cell lysates and purified hsTYR assays, and a promising improvement was observed in whole cell experiments.

Lipase Catalyzed Transesterification of Model Long-Chain Molecules in Double-Shell Cellulose-Coated Oil-in-Water Emulsion Particles as Microbioreactors.

Lipase-catalyzed transesterification is prevalent in industrial production and is an effective alternative to chemical catalysis. However, due to lipases' unique structure, the reaction requires a biphasic system, which suffers from a low reaction efficiency caused by a limited interfacial area. The use of emulsion particles was found to be an effective way to increase the surface area and activity. This research focuses on cellulose as a natural surfactant for oil-in-water emulsions and evaluates the ability of lipase, introduced into the emulsion's aqueous phase, to integrate with the emulsion microparticles and catalyze the transesterification reaction of high molecular weight esters dissolved in the particles' cores. Cellulose-coated emulsion particles' morphology was investigated by light, fluorescence and cryogenic scanning electron microscopy, which reveal the complex emulsion structure. Lipase activity was evaluated by measuring the hydrolysis of emulsified p-nitrophenyl dodecanoate and by the transesterification of emulsified methyl laurate and oleyl alcohol dissolved in decane. Both experiments demonstrated that lipase introduced in the aqueous medium can penetrate the emulsion particles, localize at the inner oil core interface and perform effective catalysis. Furthermore, in this system, lipase successfully catalyzed a transesterification reaction rather than hydrolysis, despite the dominant presence of water.

Whole cell evaluation and the enzymatic kinetic study of urease from ureolytic bacteria affected by potentially toxic elements.

Microbially induced carbonate precipitation (MICP) is a biomineralization process that has various applications in environmental pollution remediation and restoration of a range of building materials. In this study, a ureolytic bacterium, Lysinibacillus sp. GY3, isolated from an E-waste site, was found as a promising catalyst for remediation of heavy metals via the MICP process. This bacterial isolate produced significant amounts of urease and showed a great persistence in immobilization of potentially toxic elements. A reference ureolytic strain, Bacillus megaterium VS1, was selected in order to compare the efficiency of Lysinibacillus sp. GY3. Study on urease localization indicated 80 % more urease activity secreted extracellularly as for Lysinibacillus sp. GY3 compared to B. megaterium VS1. From the investigation on effects of metals on both intra- and extra-cellular urease, it was clear that Lysinibacillus sp. GY3 produced the most stable urease under conditions of metal pressure, especially retaining more than 70 % activity in the presence of 1 g/L Pb2+ and Zn2+. These results suggest that this isolated microorganism could be promisingly introduced in the MICP process to stabilize complex heavy metal pollutions, with reference to the regulating ability under harsh conditions to stabilize urease activity. This species is so important both for its biological features and environmental impacts. In addition, the present study will bring new insight in the field of metal remediation coupled with enzyme engineered biotechnology.

The Structure of Bilirubin Oxidase from Bacillus pumilus Reveals a Unique Disulfide Bond for Site-Specific Direct Electron Transfer.

Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications. Bilirubin oxidase (BOD) is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials. BOD has been extensively investigated over the last few decades. While the enzyme's internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated, a crystal structure of BOD from bacterial origin has never been determined. Here we present the first crystal structure of BOD from Bacillus pumilus (BpBOD) at 3.5 Å resolution. Overall, BpBOD shows high homology with the fungal enzymes; however, it holds a unique surface-exposed disulfide bond between Cys229 and Cys322 residues. We present methodologies to orient the T1 site towards the electrode by coupling the reduced disulfide bond with maleimide moiety on the electrodes. The developed configurations were further investigated and revealed improved direct electron transfer rates with the electrodes. The work presented here may contribute to the construction of rationally designed bioanodes or biocathode configurations that are based on redox-active enzymes.

Yeast-derived potato patatins: Biochemical and biophysical characterization.

Potato patatin is considered a valuable plant protein by the food industry for its exceptional functional properties and nutritional value. Nonetheless, it has not been widely used due to its low abundance in potatoes and high cost. Pichia pastoris was utilized for expression of patatin to overcome agricultural limitations. Biochemical and biophysical characterization of Patatin-B2 (rPatB2) and Patatin-17 (rPat17) is described. rPatB2 and rPat17 had higher zeta potential and superior solubility at various pH conditions in comparison with commercial patatin, whereas particle size distribution was similar. Inflection temperatures were higher than potato isolated patatins. Antioxidant capacity of rPatB2 and rPat17 was similar to that of commercial patatin and the specific enzymatic activity of rPatB2 was 5-fold higher than rPat17 and patatins isolated from potato. Results indicate yeast-derived patatin properties are comparable to patatins from potatoes, suggesting their potential use in various plant-based products such as meat and dairy analogues.

Ureolytic bacteria from electronic waste area, their biological robustness against potentially toxic elements and underlying mechanisms.

Ureolytic bacteria can be a promising mediator used for the immobilization of potentially toxic elements via microbially-induced carbonate precipitation (MICP) process from biodegradable ions to carbonate form. Electronic waste (E-waste) environment is very complex compared to general metal contaminated soil, however, MICP has not been studied under such an environment. In this study, three bacterial strains were successfully isolated from an E-waste area in Guiyu, China, and indicated to have positive ureolytic behavior with significant heavy metal resistance (specific to Cu and Pb), among which, a strain of Lysinibacillus sp. was proven to show a great persistence in heavy metal immobilization. This featured strain can tolerate up to 100 ppm copper and 1000 ppm lead according to minimal inhibitory concentration (MIC) results, and its urease activity was well-adapted to metal effects. Results also revealed the positive correlation (R2 = 0.9819) between metal concentrations and surface layer protein content present in bacterial cells. The underlying mechanism on the role of S-layer protein in heavy metal immobilization during biocalcification was elucidated. The metabolic system of heavy metal resistance for these E-waste derived isolates is novel and represents a point of interest for possible environmental applications to immobilize toxic heavy metals from electronic waste sites.

The maternal foam plug constitutes a reservoir for the desert locust's bacterial symbionts.

A hallmark of the desert locust's ancient and deserved reputation as a devastating agricultural pest is that of the long-distance, multi-generational migration of locust swarms to new habitats. The bacterial symbionts that reside within the locust gut comprise a key aspect of its biology, augmenting its immunity and having also been reported to be involved in the swarming phenomenon through the emission of attractant volatiles. However, it is still unclear whether and how these beneficial symbionts are transmitted vertically from parent to offspring. Using comparative 16S rRNA amplicon sequencing and direct experiments with engineered bacteria, we provide evidence for vertical transmission of locust gut bacteria. The females may perform this activity by way of inoculation of the egg-pod's foam plug, through which the larvae pass upon hatching. Furthermore, analysis of the composition of the foam revealed chitin to be its major component, along with immunity-related proteins such as lysozyme, which could be responsible for the inhibition of some bacteria in the foam while allowing other, more beneficial, strains to proliferate. Our findings reveal a potential vector for the transgenerational transmission of symbionts in locusts, which contributes to the locust swarm's ability to invade and survive in new territories.

Effect of Maternal Diet and Milk Lipid Composition on the Infant Gut and Maternal Milk Microbiomes.

Inter-subject variability in human milk microbiome is well known; however, its origins and possible relationship to the mother's diet are still debated. We investigated associations between maternal nutrition, milk fatty acids composition and microbiomes in mother-infant dyads. Breast milk and infant fecal samples were collected across three time points (one week, one month and three months postpartum) from 22 mother-infant pairs. Food frequency questionnaires for the months of pregnancy and three months postpartum were collected. Milk fatty acids were analyzed by GC-MS and the microbiome in breast milk and infant feces was determined by 16S rRNA sequencing. Statistical interactions were computed using Spearman's method and corrected for multiple comparisons. We found significant negative correlation between Streptococcus relative abundance in maternal milk and intake of unsaturated fatty acids and folic acid at one month postpartum. At three months postpartum, vitamin B-12 consumption was significantly associated with a single operational taxonomic unit belonging to Streptococcus. Comparison between milk microbiome and lipid composition showed, one-month postpartum, significant negative correlation between Streptococcus relative abundance and the abundance of oleic acid. Additional correlations were detected between Staphylococcus hominis and two medium-chain saturated fatty acids. Our results reinforce the hypothesis that maternal nutrition may affect milk microbiome.

A coupled enzymatic reaction of tyrosinase and glucose dehydrogenase for the production of hydroxytyrosol.

Hydroxytyrosol (HT) is a diphenolic compound prevalent mainly in olives with pronounced antioxidant activity and proven benefits for human health. Current production limitations have motivated studies concerning the hydroxylation of tyrosol to HT with tyrosinase; however, accumulation of the diphenol is restricted due to its rapid subsequent oxidation to 3,4-quinone-phenylethanol. In this study, a continuous two-enzyme reaction system of sol-gel-immobilized tyrosinase and glucose dehydrogenase (GDH) was developed for the synthesis of HT. Purified tyrosinase from Bacillus megaterium (TyrBm) and E. coli cell extract expressing GDH from B. megaterium were encapsulated in a sol-gel matrix based on triethoxysilane precursors. While tyrosinase oxidized tyrosol to 3,4-quinone-phenylethanol, GDH catalyzed the simultaneous reduction of the cofactor NAD+ to NADH, which was the reducing agent enabling the accumulation of HT. Using 50 mM tyrosol, the immobilized system under optimized conditions, enabled a final HT yield of 7.68 g/L with productivity of 2.30 mg HT/mg TyrBm beads. Furthermore, the immobilized bi-enzyme system showed the feasibility for HT production from 1 mM tyrosol using a 0.5-L bioreactor as well as stable activity over 8 repeated cycles. The production of other diphenols with commercial importance such as L-dopa (3,4-dihydroxyphenylalanine) or piceatannol may be synthesized with this efficient approach.

The COP9 signalosome mediates the Spt23 regulated fatty acid desaturation and ergosterol biosynthesis.

The COP9 signalosome (CSN) is a conserved eukaryotic complex, essential for vitality in all multicellular organisms and critical for the turnover of key cellular proteins through catalytic and non-catalytic activities. Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of the CSN complex, since it includes a conserved enzymatic core but lacks non-catalytic activities, probably explaining its non-essentiality for life. A previous transcriptomic analysis of an S. cerevisiae strain deleted in the CSN5/RRI1 gene, encoding to the CSN catalytic subunit, revealed a downregulation of genes involved in lipid metabolism. We now show that the S. cerevisiae CSN holocomplex is essential for cellular lipid homeostasis. Defects in CSN assembly or activity lead to decreased quantities of ergosterol and unsaturated fatty acids (UFA); vacuole defects; diminished lipid droplets (LDs) size; and to accumulation of endoplasmic reticulum (ER) stress. The molecular mechanism behind these findings depends on CSN involvement in upregulating mRNA expression of SPT23. Spt23 is a novel activator of lipid desaturation and ergosterol biosynthesis. Our data reveal for the first time a functional link between the CSN holocomplex and Spt23. Moreover, CSN-dependent upregulation of SPT23 transcription is necessary for the fine-tuning of lipid homeostasis and for cellular health.

Transglutaminase modifies the physical stability and digestibility of chickpea protein-stabilized oil-in-water emulsions.

This study sought to utilize enzymatic crosslinking to modulate the properties of chickpea-stabilized o/w emulsions and determine its effect on digestibility. Oil-in water emulsions were produced from 40% corn oil and 6% chickpea protein (w/w) with/without addition of transglutaminase (TG). Crosslinking increased the particle size and poly-dispersity, and led to the formation of a gel-like structure (G' > G″) with 1.5 order of magnitude higher G' compared to the non-crosslinked emulsion. Enzyme addition improved the emulsion physical stability (over a month) compared to the non-crosslinked emulsion that showed phase separation after two weeks of storage. Results of in vitro digestion showed decreased digestibility of TG-crosslinked chickpea-stabilized emulsions, while proteomic analysis revealed that the crosslinked emulsion is a source of bioactive peptides that are liberated by human digestive enzymes. Overall, application of TG can rationally modify the functionality and digestibility of o/w emulsions towards positive effects on human health.

Exploiting the 1-(4-fluorobenzyl)piperazine fragment for the development of novel tyrosinase inhibitors as anti-melanogenic agents: Design, synthesis, structural insights and biological profile.

The development of Tyrosinase inhibitors (TYRIs) could represent an efficacious strategy for pharmacological intervention on skin pathologies related to aberrant production of melanin. Based on in silico studies we designed and tested a library of twenty-four compounds bearing the 4-(4-fluorobenzyl)piperazin-1-yl]-fragment. As result, we identified several compounds with excellent inhibit effects at low micromolar concentration against TYR from Agaricus bisporus (TyM). Among them, compound 25 (IC50 = 0.96 µM) proved to be ∼20-fold more potent than the reference compound kojic acid (IC50 = 17.76 µM) having wide applications in the cosmetics and pharmaceutical industries. The mode of interaction of active inhibitor 25 was deciphered by means of crystallography as well as molecular docking and these results were consistent with kinetic experiments. Moreover, the identified compound 25 exhibited no considerable cytotoxicity and showed anti-melanogenic effects on B16F10 melanoma cells. Therefore, a combination of computational and biochemical approaches could represent a rational guidelines for further structural modification of this class of compounds as future anti-melanogenic agents.

Tyrosinase-crosslinked pea protein emulsions: Impact of zein incorporation.

The effect of tyrosinase-crosslinking of pea protein and pea-zein complexes on the properties of concentrated o/w emulsions was studied in the present work. Emulsions comprising 2% pea protein (w/w) solubilized in the aqueous phase (60% w/w) with or without zein solubilized in the oil phase (40% w/v), were fabricated by using high pressure homogenization. Tyrosinase treated emulsions (TyrBm-crosslinked) and non-crosslinked emulsions were evaluated after 2 h of incubation. Crosslinked pea protein stabilized emulsions led to better stability, larger particle size, increased viscosity and a paste-like structure, compared to non-crosslinked pea protein stabilized emulsions. Zein incorporation in the crosslinked pea-zein stabilized emulsions, contributed to significant improvement of stability and an increase in G' concurrently with a gel-like structure formation (G' > G″), compared to the non-crosslinked pea-zein and crosslinked pea protein stabilized emulsions. In general, crosslinked emulsions showed higher protein adsorption percentage compared to non-crosslinked emulsions, while the fraction adsorbed at the oil/water interface contained crosslinked convicilin/vicilin and zein fractions. Altogether, results demonstrate that enzymatic covalent bond formation in pea protein or zein-pea protein complexes is a useful approach to design and formulate sauces, cheese and meat replacements, and other vegetarian or vegan emulsion based foods. In addition, this work represents a step forward in application of functionalized zein in concentrated oil-in-water-emulsions.

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol.

An enhanced stability of enzymes in organic solvents is desirable under industrial conditions. The potential of lipases as biocatalysts is mainly limited by their denaturation in polar alcohols. In this study, we focused on selected solvent tunnels in lipase from Geobacillus stearothermophilus T6 to improve its stability in methanol during biodiesel synthesis. Using rational mutagenesis, bulky aromatic residues were incorporated to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. The chemical and structural characteristics of each solvent tunnel were systematically analyzed. Selected residues were replaced with Phe, Tyr, or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed an enhanced stability up to 81-fold compared with that of the wild type. All stabilizing mutations were found in the longest tunnel detected in the "closed-lid" X-ray structure. The combination of Phe substitutions in an A187F/L360F double mutant resulted in an increase in unfolding temperature (Tm ) of 7°C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. A kinetic analysis with p-nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates (kcat), though their stability properties mostly determined the transesterification capability. Seven crystal structures of different variants were solved, disclosing new π-π or CH/π intramolecular interactions and emphasizing the significance of aromatic interactions for improved solvent stability. This rational approach could be implemented for the stabilization of other enzymes in organic solvents.IMPORTANCE Enzymatic synthesis in organic solvents holds increasing industrial opportunities in many fields; however, one major obstacle is the limited stability of biocatalysts in such a denaturing environment. Aromatic interactions play a major role in protein folding and stability, and we were inspired by this to redesign enzyme voids. The rational protein engineering of solvent tunnels of lipase from Geobacillus stearothermophilus is presented here, offering a promising approach to introduce new aromatic interactions within the enzyme core. We discovered that longer tunnels leading from the surface to the enzyme active site were more beneficial targets for mutagenesis for improving lipase stability in methanol during biodiesel biosynthesis. A structural analysis of the variants confirmed the generation of new interactions involving aromatic residues. This work provides insights into stability-driven enzyme design by targeting the solvent channel void.

Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering.

Polyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome of Thermothelomyces thermophila and expressed in Pichia pastoris The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein. TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model of TtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant of TtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCE A novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

Targeting Tyrosinase: Development and Structural Insights of Novel Inhibitors Bearing Arylpiperidine and Arylpiperazine Fragments.

The inhibition of tyrosinase (Ty, EC 1.14.18.1) represents an efficient strategy of decreasing melanogenesis and skin hyperpigmentation. A combination of crystallographic and docking studies on two different tyrosinases, that from Bacillus megaterium (TyBm) and that from a mushroom (TyM), has contributed to increasing our knowledge about their structural information and translating that information to the most druggable human Ty (TyH) isozyme. In particular, we designed and synthesized a series of 1-(4-fluorobenzyl)piperazine and 1-(4-fluorobenzyl)piperidine derivatives showing inhibitory activities on TyM at micromolar ranges and more potency than that of the reference compound, kojic acid. The crystal structures of TyBm with inhibitor 3 (IC50 value of 25.11 µM) and 16 (IC50 value of 5.25 µM) were solved, confirming the binding poses hypothesized by in silico studies and revealing the main molecular determinants for the binding recognition of the inhibitors.