β-Mannosidase from Cellulomonas fimi: Immobilization Study and Application in the β-Mannoside Synthesis

The β-d-mannopyranoside linkage is found in a number of biological structures, in particular, in the core trisaccharide of N-linked glycoproteins, as well as within the antigenic polysaccharides of Salmonella, yeasts, and glycolipids. The construction of this glycosydic bond by chemical approach is very challenging and requires cumbersome protection and activation steps prior to glycosylation. In this context, β-mannosidase from Cellulomonas fimi (Cf-β-Man) was immobilized for the first time, and it was employed in the synthesis of β-mannosides. Cf-β-Man immobilized on IDA-Co2+-agarose allows the synthesis of the disaccharide, cyanomethyl β-d-mannopyranosyl-(1→6)-2-acetamido-2-deoxy-1-thio-β-d-glucopyranoside, with a higher conversion compared to the soluble enzyme (20% vs. 5%) after 6 h under best conditions. This explorative work opens new scenarios concerning the design of engineered Cf-β-Man mutants and their immobilization in order to obtain a robust and recyclable biocatalyst for applications in chemoenzymatic glycan synthesis

Emulsifying properties of sugar-based surfactants prepared by chemoenzymatic synthesis

Sugar Fatty Acid Esters (SFAEs) are a class of non-ionic surfactants that can be synthesized from inexpensive natural resources. Depending on carbon chain length and nature of the sugar head group, SFAEs cover a wide range of hydrophilic–lipophilic balance (HLB) values, which result in tunable tenside properties and in turn relevant for a wide variety of industrial applications. Three sugar-based surfactants (6-O-lauroyl-, 6-O-palmitoyl- and 6-O-stearoyl-1-O-butyl glucopyranosides) have been prepared by a lipase-catalyzed esterification of isomeric mixture of n-butyl glucosides. Specifically, their interfacial features together with W/O emulsifying properties and stability over time have been finely evaluated (interfacial tension (IFT) values, W/O emulsion turbidity water droplet size distribution, first order kinetic constants of de-emulsification)

Discovery and characterization of new Ene-reductases

Seven new putative ene-reductases (ERs or OYEs) have been identified and selected using bioinformatics tools from different organisms: Galdieria sulphuraria (GsOYE), Chroococcidiopsis thermalis (CtOYE), Chloroflexus aggregans (CaOYE), Botryotinia fuckeliana (BfOYE1 and BfOYE4) and Aspergillus niger (AnOYE2 and AnOYE8). Based on most updated literature, both the photosynthetic organisms (Galdieria, Chroococcidiopsis and Chloroflexus) and the fungi (Botryotinia and Aspergillus) result very interesting sources for ERs, that have not been exploited until now. The cloning and expression strategy used was the same for all the seven putative sequences. After a first trial of expression in E. coli BL21(DE3 good over-expression and solubility were obtained for GsOYE, CtOYE, CaOYE and BfOYE1 while for the other three proteins further optimization was necessary. To overcome the low solubility of AnOYE2 and AnOYE8 and the low production of BfOYE4 many strategies have been performed such as lowering the temperature of expression and using chaperons, but also trying other expression hosts (i.e. P. pastoris). A biocatalytic characterization was carried out for all proteins. Once verified their activity as ene-reductases in vitro, a steady-state study was performed in order to obtain the kinetics parameters. For two enzymes, GsOYE and CtOYE, bioconversions were set-up at the Biocatalysis laboratories of Graz University headed by Prof. Kurt Faber, a main expert of biocatalysis and also of ene-reductases. Finally, a biochemical characterization was carried on in order to determine the thermal stability, pH tolerance and also the three-dimensional structure of the newly discovered enzymes. Moreover, their application for the hydride-independent isomerization of non-activated C=C-bonds and subsequent reduction is also discussed and the efforts in elucidating this new reactivity are shown in detail. The work presented in this thesis lead to the discovery of new ERs enlarging the possibility to find out novel and promising biocatalysts for C=C-bond bioreduction but also for other unexpected biocatalytic reactivities.Sette nuove ene-reduttasi putative sono state identificate attraverso mezzi bioinformatici con un approccio di “genome mining” da diversi organismi: Galdieria sulphuraria (GsOYE), Chroococcidiopsis thermalis (CtOYE), Chloroflexus aggregans (CaOYE), Botryotinia fuckeliana (BfOYE1 and BfOYE4) e Aspergillus niger (AnOYE2 and AnOYE8). In particolare gli organismi fotosintetici (Galdieria, Chroococcidiopsis e Chloroflexus) e i fungi (Botryotinia e Aspergillus) sono, ad oggi, fonti di ene-reduttasi rimaste inesplorate. Per il clonaggio e l’espressione di tutte e sette le sequenze codificanti le proteine di interesse è stata utilizzata una strategia comune. Inizialmente tutte le proteine sono state espresse utilizzando come ospite E. coli BL21(DE3); con questa strategia, però, sovraespressione e buona solubilità sono state ottenute solo per quattro delle sette proteine, GsOYE, CtOYE, CaOYE e BfOYE1. Per le altre tre proteine è stata necessaria un’ulteriore ottimizzazione. La bassa solubilità di AnOYE2 e AnOYE8 e la scarsa espressione di BfOYE4 sono state affrontate utilizzando diverse strategie, come la diminuzione della temperatura e l’utilizzo di chaperonine ma anche l’utilizzo di altri ospiti per l’espressione proteica (P. pastoris). Per tutte e sette le proteine ricombinanti è stata condotta una caratterizzazione biocatalitica. Una volta dimostrata la loro attività come ene-reduttasi in vitro, sono stati determinati anche i parametri cinetici per i substrati preferiti da ciascun enzima. Nel laboratorio del Professor Kurt Faber dell’Università di Graz, sono state messe a punto bioconversioni per i due enzimi GsOYE e CtOYE, al fine di capirne il profilo di selettività nei confronti di substrati standard. Infine, è stata condotta anche una caratterizzazione biochimica che ha permesso di determinare la stabilità termica e la tolleranza a diversi pH dei nuovi enzimi identificati; per i quali è stata anche ottenuta la struttura tridimensionale. Nella tesi è discusso l’utilizzo degli enzimi nell’isomerizzazione NADH-indipendente di substrati con legami C=C non attivati e la loro successiva riduzione, così come gli sforzi per chiarire il meccanismo d’azione di questa nuova reattività scoperta solo di recente. Il lavoro presentato ha portato alla scoperta di nuove ene-reduttasi, ampliando così il pannello dei biocatalizzatori disponibili per la riduzione del doppio legame C=C ma anche per reattività biocatalitiche inaspettate

A New Thermophilic Ene-Reductase from the Filamentous Anoxygenic Phototrophic Bacterium Chloroflexus aggregans

Aiming at expanding the biocatalytic toolbox of ene-reductase enzymes, we decided to explore photosynthetic extremophile microorganisms as unique reservoir of (new) biocatalytic activities. We selected a new thermophilic ene-reductase homologue in Chloroflexus aggregans, a peculiar filamentous bacterium. We report here on the functional and structural characterization of this new enzyme, which we called CaOYE. Produced in high yields in recombinant form, it proved to be a robust biocatalyst showing high thermostability, good solvent tolerance and a wide range of pH optimum. In a preliminary screening, CaOYE displayed a restricted substrate spectrum (with generally lower activities compared to other ene-reductases); however, given the amazing metabolic ductility and versatility of Chloroflexus aggregans, further investigations could pinpoint peculiar chemical activities. X-ray crystal structure has been determined, revealing conserved features of Class III (or thermophilic-like group) of the family of Old Yellow Enzymes: in the crystal packing, the enzyme was found to assemble as dimer even if it behaves as a monomer in solution. The description of CaOYE catalytic properties and crystal structure provides new details useful for enlarging knowledge, development and application of this class of enzymes

A Comprehensive Guide to Enzyme Immobilization: All You Need to Know

Enzyme immobilization plays a critical role in enhancing the efficiency and sustainability of biocatalysis, addressing key challenges such as limited enzyme stability, short shelf life, and difficulties in recovery and recycling, which are pivotal for green chemistry and industrial applications. Classical approaches, including adsorption, entrapment, encapsulation, and covalent bonding, as well as advanced site-specific methods that integrate enzyme engineering and bio-orthogonal chemistry, were discussed. These techniques enable precise control over enzyme orientation and interaction with carriers, optimizing catalytic activity and reusability. Key findings highlight the impact of immobilization on improving enzyme performance under various operational conditions and its role in reducing process costs through enhanced stability and recyclability. The review presents numerous practical applications of immobilized enzymes, including their use in the pharmaceutical industry for drug synthesis, in the food sector for dairy processing, and in environmental biotechnology for wastewater treatment and dye degradation. Despite the significant advantages, challenges such as activity loss due to conformational changes and mass transfer limitations remain, necessitating tailored immobilization protocols for specific applications. The integration of immobilization with modern biotechnological advancements, such as site-directed mutagenesis and recombinant DNA technology, offers a promising pathway for developing robust, efficient, and sustainable biocatalytic systems. This comprehensive guide aims to support researchers and industries in selecting and optimizing immobilization techniques for diverse applications in pharmaceuticals, food processing, and fine chemicals

A Multi-Enzymatic Cascade Reaction for the Synthesis of Vidarabine 5′-Monophosphate

We here described a three-step multi-enzymatic reaction for the one-pot synthesis of vidarabine 5′-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade), and adenosine triphosphate (ATP) as precursors. To this aim, three enzymes involved in the biosynthesis of nucleosides and nucleotides were used in a cascade mode after immobilization: uridine phosphorylase from Clostridium perfringens (CpUP), a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), and deoxyadenosine kinase from Dictyostelium discoideum (DddAK). Specifically, CpUP catalyzes the phosphorolysis of araU thus generating uracil and α-d-arabinose-1-phosphate. AhPNP catalyzes the coupling between this latter compound and Ade to form araA (vidarabine). This nucleoside becomes the substrate of DddAK, which produces the 5′-mononucleotide counterpart (araA-MP) using ATP as the phosphate donor. Reaction conditions (i.e., medium, temperature, immobilization carriers) and biocatalyst stability have been balanced to achieve the highest conversion of vidarabine 5′-monophosphate (≥95.5%). The combination of the nucleoside phosphorylases twosome with deoxyadenosine kinase in a one-pot cascade allowed (i) a complete shift in the equilibrium-controlled synthesis of the nucleoside towards the product formation; and (ii) to overcome the solubility constraints of araA in aqueous medium, thus providing a new route to the highly productive synthesis of araA-MP.</jats:p

A New Thermophilic Ene-Reductase from the Filamentous Anoxygenic Phototrophic Bacterium Chloroflexus aggregans

Aiming at expanding the biocatalytic toolbox of ene-reductase enzymes, we decided to explore photosynthetic extremophile microorganisms as unique reservoir of (new) biocatalytic activities. We selected a new thermophilic ene-reductase homologue in Chloroflexus aggregans, a peculiar filamentous bacterium. We report here on the functional and structural characterization of this new enzyme, which we called CaOYE. Produced in high yields in recombinant form, it proved to be a robust biocatalyst showing high thermostability, good solvent tolerance and a wide range of pH optimum. In a preliminary screening, CaOYE displayed a restricted substrate spectrum (with generally lower activities compared to other ene-reductases); however, given the amazing metabolic ductility and versatility of Chloroflexus aggregans, further investigations could pinpoint peculiar chemical activities. X-ray crystal structure has been determined, revealing conserved features of Class III (or thermophilic-like group) of the family of Old Yellow Enzymes: in the crystal packing, the enzyme was found to assemble as dimer even if it behaves as a monomer in solution. The description of CaOYE catalytic properties and crystal structure provides new details useful for enlarging knowledge, development and application of this class of enzymes.</jats:p

A Multi-Enzymatic Cascade Reaction for the Synthesis of Vidarabine 5′-Monophosphate

We here described a three-step multi-enzymatic reaction for the one-pot synthesis of vidarabine 5&prime;-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade), and adenosine triphosphate (ATP) as precursors. To this aim, three enzymes involved in the biosynthesis of nucleosides and nucleotides were used in a cascade mode after immobilization: uridine phosphorylase from Clostridium perfringens (CpUP), a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), and deoxyadenosine kinase from Dictyostelium discoideum (DddAK). Specifically, CpUP catalyzes the phosphorolysis of araU thus generating uracil and &alpha;-d-arabinose-1-phosphate. AhPNP catalyzes the coupling between this latter compound and Ade to form araA (vidarabine). This nucleoside becomes the substrate of DddAK, which produces the 5&prime;-mononucleotide counterpart (araA-MP) using ATP as the phosphate donor. Reaction conditions (i.e., medium, temperature, immobilization carriers) and biocatalyst stability have been balanced to achieve the highest conversion of vidarabine 5&prime;-monophosphate (&ge;95.5%). The combination of the nucleoside phosphorylases twosome with deoxyadenosine kinase in a one-pot cascade allowed (i) a complete shift in the equilibrium-controlled synthesis of the nucleoside towards the product formation; and (ii) to overcome the solubility constraints of araA in aqueous medium, thus providing a new route to the highly productive synthesis of araA-MP