Caulobacter segnis Dioxygenase CsO2: A Practical Biocatalyst for Stilbenoid Ozonolysis.

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.

Characterization, genome analysis and genetic tractability studies of a new nanocellulose producing Komagataeibacter intermedius isolate.

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.

Mycobacterium smegmatis acyltransferase: The big new player in biocatalysis.

After several decades during which proteases and after lipases took the biotransformation world scene as the predominant biocatalysts, a new, promising enzyme was discovered and characterized. The acyltransferase from Mycobacterium smegmatis (MsAcT) has in fact an extraordinary activity for a wide array of reactions, such as trans-esterification, amidation, trans-amidation and perhydrolysis, both in water and solvent media, giving rise to a series of interesting compounds including APIs (i.e., active pharmaceutical ingredients), natural flavors and fragrances, monomers for polymer synthesis, and peracids employed as disinfectants or antimicrobials. Although the most used acylating agent has been ethyl acetate (EtOAc), depending on the reaction type also acetamide, dimethyl carbonate and a variety of other esters, have been reported. The best yields were reached using very reactive donors such as vinyl or isopropenyl esters (almost complete conversion in rapid reaction times and water media for condensation reactions). In this review article the most innovative scientific advances on MsAcT, its mechanism and engineering are summarized, putting a particular focus on the different kind of processes (batch and flow) that it is possible to carry out using this enzyme as free or immobilized form. In conclusion, the author personal view on the unexplored reaction possibilities using MsAcT is reported as a window on the future of the topic.

Whole cells of recombinant CYP153A6-E. coli as biocatalyst for regioselective hydroxylation of monoterpenes.

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.

Biocatalytic Approaches for an Efficient and Sustainable Preparation of Polyphenols and Their Derivatives.

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.

Characterization of Komagataeibacter Isolate Reveals New Prospects in Waste Stream Valorization for Bacterial Cellulose Production.

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.

Structural insights into the desymmetrization of bulky 1,2-dicarbonyls through enzymatic monoreduction.

Benzil reductases are dehydrogenases preferentially active on aromatic 1,2-diketones, but the reasons for this peculiar substrate recognition have not yet been clarified. The benzil reductase (KRED1-Pglu) from the non-conventional yeast Pichia glucozyma showed excellent activity and stereoselectivity in the monoreduction of space-demanding aromatic 1,2-dicarbonyls, making this enzyme attractive as biocatalyst in organic chemistry. Structural insights into the stereoselective monoreduction of 1,2-diketones catalyzed by KRED1-Pglu were investigated starting from its 1.77 Å resolution crystal structure, followed by QM and classical calculations; this study allowed for the identification and characterization of the KRED1-Pglu reactive site. Once identified the recognition elements involved in the stereoselective desymmetrization of bulky 1,2-dicarbonyls mediated by KRED1-Pglu, a mechanism was proposed together with an in silico prediction of substrates reactivity.

Influence of Lytic Polysaccharide Monooxygenase Active Site Segments on Activity and Affinity.

In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or absence of a family 1 carbohydrate binding module (CBM1) connected via a linker to the C-terminus of the LPMO. This study investigates the changes in activity induced by shortening the second active site segment (Seg2) or removing the CBM1 from Neurospora crassa LPMO9C. NcLPMO9C and generated variants have been tested on regenerated amorphous cellulose (RAC), carboxymethyl cellulose (CMC) and xyloglucan (XG) using activity assays, conversion experiments and surface plasmon resonance spectroscopy. The absence of CBM1 reduced the binding affinity and activity of NcLPMO9C, but did not affect its regioselectivity. The linker was found important for the thermal stability of NcLPMO9C and the CBM1 is necessary for efficient binding to RAC. Wild-type NcLPMO9C exhibited the highest activity and strongest substrate binding. Shortening of Seg2 greatly reduced the activity on RAC and CMC and completely abolished the activity on XG. This demonstrates that Seg2 is indispensable for substrate recognition and the formation of productive enzyme-substrate complexes.