Fully Biocatalytic Rearrangement of Furans to Spirolactones.

A multienzymatic pathway enables the preparation of optically pure spirolactone building blocks. In a streamlined one-pot reaction cascade, the combination of chloroperoxidase, an oxidase, and an alcohol dehydrogenase renders an efficient reaction cascade for the conversion of hydroxy-functionalized furans to the spirocyclic products. The fully biocatalytic method is successfully employed in the total synthesis of the bioactive natural product (+)-crassalactone D, and as the key module in a chemoenzymatic route yielding lanceolactone A.

Peroxidase-induced C-N bond formation via nitroso ene and Diels-Alder reactions.

The formation of new carbon-nitrogen bonds is indisputably one of the most important tasks in synthetic organic chemistry. Here, nitroso compounds offer a highly interesting reactivity that complements traditional amination strategies, allowing for the introduction of nitrogen functionalities via ene-type reactions or Diels-Alder cycloadditions. In this study, we highlight the potential of horseradish peroxidase as biological mediator for the generation of reactive nitroso species under environmentally benign conditions. Exploiting a non-natural peroxidase reactivity, in combination with glucose oxidase as oxygen-activating biocatalyst, aerobic activation of a broad range of N-hydroxycarbamates and hydroxamic acids is achieved. Thus both intra- and intermolecular nitroso-ene as well as nitroso-Diels-Alder reactions are performed with high efficiency. Relying on a commercial and robust enzyme system, the aqueous catalyst solution can be recycled over numerous reaction cycles without significant loss of activity. Overall, this green and scalable C-N bond-forming strategy enables the production of allylic amides and various N-heterocyclic building blocks utilizing only air and glucose as sacrificial reagents.

An Artificial In Vitro Metabolism to Angiopterlactone B Inspired by Traditional Retrosynthesis.

Nature's way to construct highly complex molecular entities as part of biosynthetic pathways is unmatched by any chemical synthesis. Yet, relying on a cascade of native enzymatic transformations to achieve a certain target structure, biosynthesis is also significantly limited in its scope. In this study, non-natural biocatalytic modules, a peroxidase-mediated Achmatowicz rearrangement and a dehydrogenase-catalyzed borrowing-hydrogen-type isomerization were successfully incorporated into an artificial metabolism, combining the benefits of traditional retrosynthesis with the elegance and efficacy of biosynthetic networks. In a highly streamlined process, the total synthesis of tricyclic angiopterlactone B was achieved in two steps operating entirely in an aqueous environment while relying mainly on enzymes as key reaction mediators.

Aerobic C-N Bond Formation through Enzymatic Nitroso-Ene-Type Reactions.

The biocatalytic oxidation of acylated hydroxylamines enables the direct and selective introduction of nitrogen functionalities by activation of allylic C-H bonds. Utilizing either laccases or an oxidase/peroxidase couple for the formal dehydrogenation of N-hydroxycarbamates and hydroxamic acids with air as the terminal oxidant, acylnitroso species are generated under particularly mild aqueous conditions. The reactive intermediates undergo C-N bond formation through an ene-type mechanism and provide high yields both in intramolecular and intermolecular enzymatic aminations. Investigations on different pathways of the two biocatalytic systems and labelling studies provide more insight into this unprecedented promiscuity of classical oxidoreductases as catalysts for nitroso-based transformations.

Coding Synthetic Chemistry Strategies for Furan Valorization into Bacterial Designer Cells.

Following a synthetic chemistry blueprint for the valorization of lignocellulosic platform chemicals, this study showcases a so far unprecedented approach to implement non-natural enzyme modules in vivo. For the design of a novel functional whole cell tool, two purely abiotic transformations, a styrene monooxygenase-catalyzed Achmatowicz rearrangement and an alcohol dehydrogenase-mediated borrowing hydrogen redox isomerization, were incorporated into a recombinant bacterial host. Introducing this type of chemistry otherwise unknown in biosynthesis, the cellular factories were enabled to produce complex lactone building blocks in good yield from bio-based furan substrates. This whole cell system streamlined the synthetic cascade, eliminated isolation and purification steps, and provided a high degree of stereoselectivity that has so far been elusive in the chemical methodology.

Cascade Catalysis Through Bifunctional Lipase Metal Biohybrids for the Synthesis of Enantioenriched O-Heterocycles from Allenes.

Lipase/metal nanobiohybrids, generated by growth of silver or gold nanoparticles on protein matrixes are used as highly effective dual-activity heterogeneous catalysts for the production of enantiomerically enriched 2,5-dihydrofurans from allenic acetates in a one-pot cascade process combining a lipase-mediated hydrolytic kinetic resolution with a metal-catalyzed allene cycloisomerization. Incorporating a novel strategy based on enzyme-polymer bioconjugates in the nanobiohybrid preparation enables excellent conversions in the process. Candida antarctica lipase B (CALB) in combination with a dextran-based polymer modifier (DexAsp) proved to be most efficient when merged with silver nanoparticles. A range of hybrid materials were produced, combining Ag or Au metals with Thermomyces lanuginosus lipase (TLL) or CALB and its DexAsp or polyethyleneimine polymer bioconjugates. The wider applicability of the biohybrids is demonstrated by their use in allenic alcohol cyclizations, where a variety of dihydrofurans are obtained using a CALB/gold nanomaterial. These results underline the potential of the nanobiohybrid catalysis as promising approach to intricate one-pot synthetic strategies.

Statistical optimization of media components for production of extracellular lipase from edible mushroom Cantharellus cibarius.

Cantharellus cibarius is a wild edible mushrooms and considered as a plethora of compounds with potential biotechnological applications. This study highlighted the utilization of C. cibarius mushroom in the production of extracellular lipase under submerged fermentation, representing the first report on lipase production by this mushroom. Various physicochemical factors were optimized via one-factor-at-a time (OFAT) method. Maximum enzyme production was recorded when the mushroom mycelium was grown at 30 °C on pH 6.0 for 96 h in the medium supplemented with 1% [(v/v)] olive oil. Productivity of enzyme was affected by variation in the nitrogen sources, carbon sources, metal ions and NaCl salt. Glucose and peptone significantly enhanced enzyme production as carbon and nitrogen sources, respectively. Stimulatory and inhibitory effects were found by Ca2+ and Zn2+ ions, respectively. Furthermore, Box-Behnken Design (BBD) of Response Surface Methodology (RSM) was employed to optimize the interactive effects of specific media components like glucose, olive oil and CaCl2. The regression model was significant with a coefficient of determination (R2) value of 0.9483. Statistically optimized design (RSM) resulted approximately two-fold increase (23.5-42.283 UmL-1) of lipase production than classical optimization method (OFAT), confirmed the validation of model. The kinetic parameters for p-nitrophenyl palmitate hydrolysis, Km and Vmax were 5.24 mM and 0.768 mmol/min/mg respectively, established a high affinity for the substrate.

Synthesis of silver and gold nanoparticles-enzyme-polymer conjugate hybrids as dual-activity catalysts for chemoenzymatic cascade reactions.

Novel hybrids containing silver or gold nanoparticles have been synthesized in aqueous media and at room temperature using enzymes or tailor-made enzyme-polymer conjugates, which directly induced the formation of inorganic silver or gold species. The choice of pH, protein, or bioconjugate strongly affected the final metallic nanoparticles hybrid formation. Using Candida antarctica lipase (CALB) in a solution, nanobiohybrids containing Ag2O nanoparticles of 9 nm average diameter were obtained. The use of tailor-made bioconjugates, for example, the CALB modified with dextran-aspartic acid polymer (Dext6kDa), resulted in a nanobiohybrid containing smaller Ag(0)/Ag2O nanoparticles. In the case of nanobiohybrids based on gold, Au(0) species were found in all cases. The Au-CALB hybrid contained spherical nanoparticles with 18 nm average diameter size, with a minor range of larger ones (>100 nm) while the AuNPs-CALB-Dext6kDa hybrid was formed by much smaller nanoparticles (9 nm, minor range of 22 nm), and also nanorods of 20-30/40-50 nm length. Using Thermomyces lanuginosus lipase (TLL), apart from the nanoparticle formation, nanoflowers with a diameter range of 100-200 nm were obtained. All nanobiohybrids maintained (dual) enzymatic and metallic activities. For instance, these nanobiohybrids exhibited exquisite dual-activity for hydrolysis/cycloisomerization cascades starting from allenic acetates. By merging the transition metal reactivity with the inherent lipase catalysis, allenic acetates directly converted to the corresponding O-heterocycles in enantiopure form catalysed by AgNPs-CALB-Dext6kDa, taking advantage of a kinetic resolution/cyclization pathway. These results showed the high applicability of these novel hybrids, offering new opportunities for the design of novel reaction cascades.

Enzymatic Bromocyclization of α- and γ-Allenols by Chloroperoxidase from Curvularia inaequalis.

Vanadate-dependent chloroperoxidase from Curvularia inaequalis catalyzes 5-endo-trig bromocyclizations of α-allenols to produce valuable halofunctionalized furans as versatile synthetic building blocks. In contrast to other haloperoxidases, also the more challenging 5-exo-trig halocyclizations of γ-allenols succeed with this system even though the scope still remains more narrow. Benefitting from the vanadate chloroperoxidase's high resiliency towards oxidative conditions, cyclization-inducing reactive hypohalite species are generated in situ from bromide salts and hydrogen peroxide. Crucial requirements for high conversions are aqueous biphasic emulsions as reaction media, stabilized by either cationic or non-ionic surfactants.

Anti-Angiogenic Effect of Cantharellus cibarius Extracts, its Correlation with Lipoxygenase Inhibition, and Role of the Bioactives Therein.

Angiogenesis is a complex physiological process that cannot be treated with single agent therapy. Several edible fungi have been known to encompass bioactive compounds, and are promising sources of multi-component drugs. One such widely consumed edible fungi is Cantharellus cibarius, which has been explored for its biological activities. The present study focused on assessing the anti-angiogenic activity of petroleum ether and ethanol extracts of C. cibarius using chick chorioallantoic membrane (CAM) assay. Both the extracts showed a dose-dependent response which was compared with the anti-angiogenic activity of the positive controls silibinin, and lenalidomide. The extracts were also studied for their lipoxygenase (LOX) inhibitory potential and compared to ascorbic acid as the positive control. The IC50 values of the petroleum ether extract, ethanol extract, and ascorbic acid for LOX inhibition assay were 135.4, 113.1, and 41.5 µg/mL, respectively. Although both the extracts showed similar responses in CAM assay, ethanol extract proved to be more potent in LOX inhibition assay. Finally, the extracts were investigated for their chemical composition using GC-MS. A correlation between LOX inhibition and anti-angiogenic potential was established at the molecular level. A meticulous literature search was carried out to correlate the biochemical composition of the extracts to their anti-angiogenic activity.

Methanol-Driven Oxidative Rearrangement of Biogenic Furans - Enzyme Cascades vs. Photobiocatalysis.

The oxidative ring expansion of bio-derived furfuryl alcohols to densely functionalized six-membered O-heterocycles represents an attractive strategy in the growing network of valorization routes to synthetic building blocks out of the lignocellulosic biorefinery feed. In this study, two scenarios for the biocatalytic Achmatowicz-type rearrangement using methanol as terminal sacrificial reagent have been evaluated, comparing multienzymatic cascade designs with a photo-bio-coupled activation pathway.

Investigation on mycelial growth requirements of Cantharellus cibarius under laboratory conditions.

The golden chanterelle represents one of the commonly found, edible mushrooms that is highly valued in various cuisines. The present study focused on assessing the requirements of Cantharellus cibarius such as pH, temperature, as well as the carbon and nitrogen sources for mycelial growth. Optimization of the growth parameters was carried out by one-factor-at-a-time method. The optimal pH and temperature were determined to be 6.0 and 22.5 °C, respectively. Among the various carbon sources studied, sucrose at a concentration of 2% gave maximum mycelial growth and proved to be the most suitable one. Amongst the nitrogen sources studied, peptone, ammonium sulphate, and sodium nitrate, gave the maximum mycelial growth at an optimized concentration of 0.5%. In the presence of beef extract and yeast extract, a change in colony pigmentation from yellow to dark grey was observed. Finally, the carbon to nitrogen ratio of 2:0.5 proved to be optimal for mycelial growth. This study is the first report on the optimisation of in vitro growth requirements of C. cibarius.

In silico characterization of bacterial chitinase: illuminating its relationship with archaeal and eukaryotic cousins.

BACKGROUND: Chitin is one of the most abundant biopolymers on Earth, only trailing second after cellulose. The enzyme chitinase is responsible for the degradation of chitin. Chitinases are found to be produced by wide range of organisms ranging from archaea to higher plants. Though chitin is a major component of fungal cell walls and invertebrate exoskeletons, bacterial chitinase can be industrially generated at low cost, in facile downstream processes at high production rate. Microbial chitinases are more stable, active, and economically practicable compared to the plant- and animal-derived enzymes. RESULTS: In the present study, computationally obtained results showed functional characteristics of chitinase with particular emphasis on bacterial chitinase which is fulfilling all the required qualities needed for commercial production. Sixty-two chitinase sequences from four different groups of organisms were collected from the RCSB Protein Data Bank. Considering one suitable exemplary sequence from each group is being compared with others. Primary, secondary, and tertiary structures are determined by in silico models. Different physical parameters, viz., pI, molecular weight, instability index, aliphatic index, GRAVY, and presence of functional motifs, are determined, and a phylogenetic tree has been constructed to elucidate relationships with other groups of organisms. CONCLUSIONS: This study provides novel insights into distribution of chitinase among four groups and their characterization. The results represent valuable information toward bacterial chitinase in terms of the catalytic properties and structural features, can be exploited to produce a range of chitin-derived products.

Anti-angiogenic and anti-inflammatory activity of the summer truffle (Tuber aestivum Vittad.) extracts and a correlation with the chemical constituents identified therein.

Fungi are a huge source of unexplored bioactive compounds. Owing to their biological activities, several fungi have shown commercial application in the health industry. Tuber aestivum Vittad. is one such edible fungi with an immense scope for practical biological applications. In the present study, the anti-angiogenic activity of petroleum ether and ethanol extracts of T. aestivum was investigated using the chick chorioallantoic membrane assay and compared to the positive controls silibinin and lenalidomide. Both the extracts showed a dose-dependent anti-angiogenic response. The extracts were also assessed for their anti-inflammatory potential by lipoxygenase-inhibition assay. The IC50 values for LOX inhibition assay, computed by the Boltzmann plot, were 368.5, 147.3 and 40.2 µg/mL, for the petroleum ether extract, ethanol extract, and the positive control ascorbic acid, respectively. The ethanol extract of T. aestivum showed superior anti-angiogenic and anti-inflammatory activity than the petroleum ether extract. Compositional investigation of the extracts by GC-MS revealed the presence of various bioactive compounds. The compounds were correlated to their anti-angiogenic and anti-inflammatory activity based on a meticulous literature search.

Shape and Phase Transitions in a PEGylated Phospholipid System.

Poly(ethylene glycol) (PEG) polymers and PEG-conjugated lipids are widely used in bioengineering and drug transport applications. A PEG layer in a drug carrier increases hydrophilic repulsion, inhibits membrane fusion and serum opsonin interactions, and prolongs the storage and circulation time. It can also change the carrier shape and have an influence on many properties related to the content release of the carrier. In this paper, we focus on the physicochemical effects of PEGylation in the lipid bilayer. We introduce laurdanC as a fluorophore for shape recognition and phase transition detection. Together with laurdanC, cryogenic transmission electron microscopy, differential scanning calorimetry, molecular dynamics simulations, and small-angle X-ray scattering/wide-angle X-ray scattering, we acquire information of the particle/bilayer morphology and phase behavior in systems containing 1,2-dipalmitoyl- sn-glycero-3-phosphocholine:1,2-distearoyl- sn-glycero-3-phosphoethanolamine-PEG(2000) with different fractions. We find that PEGylation leads to two important and potentially usable features of the system. (1) Spherical vesicles present a window of elevated chain-melting temperatures and (2) lipid packing shape-controlled liposome-to-bicelle transition. The first finding is significant for targets requiring multiple release sequences and the second enables tuning the release by composition and the PEG polymer length. Besides drug delivery systems, the findings can be used in other smart soft materials with trigger-polymers as well.

Chemoenzymatic Hydrogen Production from Methanol through the Interplay of Metal Complexes and Biocatalysts.

Microbial methylotrophic organisms can serve as great inspiration in the development of biomimetic strategies for the dehydrogenative conversion of C1 molecules under ambient conditions. In this Concept article, a concise personal perspective on the recent advancements in the field of biomimetic catalytic models for methanol and formaldehyde conversion, in the presence and absence of enzymes and co-factors, towards the formation of hydrogen under ambient conditions is given. In particular, formaldehyde dehydrogenase mimics have been introduced in stand-alone C1 -interconversion networks. Recently, coupled systems with alcohol oxidase and dehydrogenase enzymes have been also developed for in situ formation and decomposition of formaldehyde and/or reduced/oxidized nicotinamide adenine dinucleotide (NADH/ NAD+ ). Although C1 molecules are already used in many industries for hydrogen production, these conceptual bioinspired low-temperature energy conversion processes may lead one day to more efficient energy storage systems enabling renewable and sustainable hydrogen generation for hydrogen fuel cells under ambient conditions using C1 molecules as fuels for mobile and miniaturized energy storage solutions in which harsh conditions like those in industrial plants are not applicable.

Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades.

Plant-derived carbohydrates are an abundant renewable resource. Transformation of carbohydrates into new products, including amine-functionalized building blocks for biomaterials applications, can lower reliance on fossil resources. Herein, biocatalytic production routes to amino carbohydrates, including oligosaccharides, are demonstrated. In each case, two-step biocatalysis was performed to functionalize d-galactose-containing carbohydrates by employing the galactose oxidase from Fusarium graminearum or a pyranose dehydrogenase from Agaricus bisporus followed by the ω-transaminase from Chromobacterium violaceum (Cvi-ω-TA). Formation of 6-amino-6-deoxy-d-galactose, 2-amino-2-deoxy-d-galactose, and 2-amino-2-deoxy-6-aldo-d-galactose was confirmed by mass spectrometry. The activity of Cvi-ω-TA was highest towards 6-aldo-d-galactose, for which the highest yield of 6-amino-6-deoxy-d-galactose (67 %) was achieved in reactions permitting simultaneous oxidation of d-galactose and transamination of the resulting 6-aldo-d-galactose.

Enantioconvergent Biocatalytic Redox Isomerization.

Alcohol dehydrogenases can act as powerful catalysts in the preparation of optically pure γ-hydroxy-δ-lactones by means of an enantioconvergent dynamic redox isomerization of readily available Achmatowicz-type pyranones. Imitating the traditionally metal-mediated "borrowing hydrogen" approach to shuffle hydrides across molecular architectures and interconvert functional groups, this chemoinspired and purely biocatalytic interpretation effectively expands the enzymatic toolbox and provides new opportunities in the assembly of multienzyme cascades and tailor-made cellular factories.

Fermentative production of extracellular amylase from novel amylase producer, Tuber maculatum mycelium, and its characterization.

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22 ± 2 °C) under static conditions which resulted in 1.41 U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 98% of the maximal activity after 100 min of incubation. The amylase activity was marginally enhanced in presence of Cu2+ and Na+ and slightly reduced by K+, Ca2+, Fe2+, Mg2+, Co2+, Zn2+, and Mn2+ ions at 1 mM concentration.

Kirmse-Doyle- and Stevens-Type Rearrangements of Glutarate-Derived Oxonium Ylides.

A novel chemoenzymatic synthetic cascade enables the preparation of densely decorated tetrahydrofuran building blocks. Here, the lipase-catalyzed desymmetrization of 3-alkoxyglutarates renders highly enantioenriched carboxylic acid intermediates, whose subsequent activation and oxonium ylide rearrangement by means of rhodium or copper complexes furnishes functionalized O-heterocycles with excellent diastereoselectivity. The two-step protocol offers a streamlined and flexible synthesis of tetrahydrofuranones bearing different benzylic, allylic or allenylic side chains with full control over multiple stereogenic centers.