Production and Characterization of New Baeyer-Villiger Monooxygenases

The Baeyer-Villiger reaction is a very important oxidative reaction in organic synthesis that leads to the production of optically pure esters and lactones from ketones. It is typically performed by peroxyacids, oxydative reagents that are toxic, hazardous and lacking selectivity. By using "green chemistry", it is possible to perform the same reaction using "bio-catalyzers", enzymes named Baeyer-Villiger monoxygenases (BVMOs), a group of avin dependent monooxygenases, that use NAD(P)H and molecular oxygen in order to catalyze chemo-, regio-, and enantio-selective oxidative reactions. BVMOs have been identied in a large number of bacteria and fungi but only a restricted number of them is available in recombinant form. In order to discover novel BVMOs, new sources of enzymes will become important. Using bioinformatics tools, we have identied and selected ve new putative type I BVMOs from dierent organisms: Oryza sativa (Os; plant), Physcomitrella patens (Pp; moss), Cyanidioschyzon merolae (Cm; red alga), Trichodesmium erythraeum (Te; cyanobacterium), Haloterrigena turkmenica (Ht; archeabacterium). In particular, basing on most updated literature, the photosynthetic eukaryotes Oryza, Physcomitrella and Cyanidioschyzon would result very uncommon sources for BVMOs. The cloning and expression strategy used was the same for all the ve putative sequences. The very low solubility of the expressed proteins was the real bottleneck of the work. To overcome the solubility problem many strategies have been performed lowering the inducer concentration, the growth temperature and using chaperones. Adding on the culture an excess of ribo avin, the precursor of the FAD, results successful to obtain soluble avoproteins from Physcomitrella patens and Cyanidioschyzon merolae, showing the importance of the avin cofactor in the folding process. A biocatalytic characterisation of both expressed proteins was made at the Molecular Enzymology group of the Groningen Biomolecular Science and Biotechnology Institute (GBB), University of Groningen (The Netheralnds) headed by Prof. Dr. M.W. Fraaije, a main expert in avoenzymes and particularly in Baeyer-Villiger monooxygenses. Once veried their Baeyer-Villiger monooxyge-3 nase activity a condition optimization (pH, temperature and stability) has been performed; then the two proteins were tested to investigate their substrate speci-city. A steady-state study was carried out in order to obtain kinetics parameters and many conversions were made to determine their selectivity prole. The work presented led to discover new type I BVMOs enlarging the possibility to nd out novel and promising biocatalysts for oxidative reactions

Chemo-enzymatic synthesis of new resveratrol-related dimers containing the benzo[b]furan framework and evaluation of their radical scavenger activities

The chemo-enzymatic synthesis and the evaluation of radical scavenger performance of resveratrolrelated dimers with antioxidative power have been investigated. The dimeric compounds, containing the benzo[b]furan framework, were prepared via an oxidative dimerization catalyzed by a laccase from Trametes versicolor, followed by a treatment with the organic oxidant DDQ. This methodology can be useful for the synthesis of various 2,3-diarylbenzo[b]furans derivates, a class of compound that exhibits a wide range of biological activities

Use of insulin degludec in pregnancy: two case reports and a literature review

Abstract As of now, insulin Degludec has no indication for use in pregnancy, because of the lack of studies that prove its safety for foetus. However it isn't infrequent that some women conceive while treating with insulin Degludec. So, before deciding to change the type of insulin therapy during pregnancy, an evaluation of the risk associated to a possible temporary worsening of glycaemic control, due to that insulin replacement, is needed. Referring to case series reported in the scientific literature could provide a support when a clinical decision need to be taken. We report two cases of women affected by type 1 diabetes who had unplanned pregnancies during treatment with insulin Degludec. In order to avoid the risk of a possible worsening of glycaemic control due to insulin switch, we decided to continue the treatment with Degludec during their pregnancies, after obtaining the patients' written informed consent. Daily insulin requirement gradually increased for both women pregnancy progressed, and glycated haemoglobin (HbA1c) values improved from the first observation to delivery: 55 mmol/mol (7.2%) at 9 weeks to 47 mmol/mol (6.5%) at 36 weeks, in Patient 1 (P1); 44 mmol/mol (6.2%) at 8 weeks to 33 mmol/mol (5.2%) at 36 weeks, in Patient 2 (P2). P1 delivered at week 37 with a caesarean section due to failed induction. The newborn, a girl of 3398 g at birth, developed neonatal hypoglycaemia and respiratory distress (Apgar 6-6). Six days after birth she underwent colectomy because of necrotizing enterocolitis and was finally diagnosed with atypical cystic fibrosis. P2 gave birth to a healthy girl (weight 2745g at birth, Apgar 7-9) at 37 weeks, undergoing a caesarean section for maternal cervical dystocia, without neonatal complications. Our experience provides additional evidence on the safety of insulin Degludec in pregnancy without any maternal or neonatal outcome suggesting its toxicity

POLYPYRROLE SYNTHESIS VIA CARBOXYMETHYLCELLULOSE-IRON COMPLEXES

Polypyrrole (PPy) was chemically synthesised at two pH levels (pH = 2 and unadjusted pH, i.e. 6.6) using pre-formed carboxymethylcellulose-iron (CMC-Fe) complexes. The CMC-Fe complexes were prepared at a fixed CMC concentration, i.e. 5.5x10-5 mol/L, and with an increasing FeCl3 amount (from 4x10-3 to 5x10-2 mol/L). The quantity of iron bound to CMC was determined by the inductively coupled plasma (ICP-MS) method. In order to understand the interactions between CMC and iron, speciation of the systems was simulated by Phreeqc software. SEM analysis showed that, in some conditions (particularly at pH = 2), Py polymerised within the CMC-Fe complexes, forming particles with size ranging between 300 and 600 nm. In order to evaluate polymer electric conductivity, films were prepared by direct casting of the PPy-CMC-Fe dispersions with and without addition of film-forming CMC, and bulky PPy-CMC-Fe pellets were obtained by compression. Despite the different arrangement PPy-CMC-Fe particles in dry films, the amount of iron bound to CMC during the formation of CMC-Fe complexes was found to be the dominant parameter affecting polymer conductivity

From the Amelioration of a NADP+-dependent Formate Dehydrogenase to the Discovery of a New Enzyme: Round Trip from Theory to Practice

NADP+-dependent formate dehydrogenases (FDHs) are biotechnologically relevant enzymes for cofactors regeneration in industrial processes employing redox biocatalysts. Their effective applicability is however hampered by the low cofactor and substrate affinities of the few enzymes described so far. After different efforts to ameliorate the previously studied GraFDH from the acidobacterium Granulicella mallensis MP5ACTX8, an enzyme having double (NAD+ and NADP+) cofactor specificity, we started over our search with the advantage of hindsight. We identified and characterized GraFDH2, a novel highly active FDH, which proved to be a good NAD+-dependent catalyst. A rational engineering approach permitted to switch its cofactor specificity, producing an enzyme variant that displays a 10-fold activity improvement over the wild-type enzyme with NADP+. Such variant resulted to be one of the best performing enzyme among the NADP+-dependent FDHs reported so far in terms of catalytic performance

POLYPYRROLE AND POLYPYRROLE/WOOD-DERIVED MATERIALS CONDUCTING COMPOSITES: A REVIEW

Wood and cellulose derivatives, in both fibrous and water-soluble macromolecular form, are emerging as outstanding candidates for organic electronics applications due to their large-scale availability, low cost, and easy processability. Paper and wood fibre-based derivatives are considered to be materials of choice as supports for communication world-wide. The interest in producing inexpensive and universally available conducting polymer/cellulose fibres substrates resides in the possibility of creating new materials that can be used for a broad range of advanced applications. For instance, PPy/cellulose fibres composites can be used for the preparation of energy storage devices thanks to the conjugation of the high specific area of cellulose fibres and the electrochemical properties of PPy. Other possible applications of such composites are in the area of the antistatic materials, sensors, electromagnetic interference shielding materials, smart packaging, and tissues. Concerning the woody polymers, some of them (i.e. cellulose derivatives) also exhibit biocompatibility, as well as film-forming properties and transparency. In combination with the electrical properties of PPy, these features make PPy/macromolecular cellulose composites suitable for applications as displays, lighting, and photovoltaics. Due to their chemical structure, macromolecular wood derivatives have been proposed with success as enhancing conductivity additives in Py polymerisation. The aim of the present review is to provide an overview of PPy chemistry and of the most relevant advances attained in the production of PPy/wood derived materials conducting composites

Enzymatic Baeyer-Villiger oxidation of steroids with cyclopentadecanone monooxygenase

Peer reviewed: YesNRC publication: Ye

Biochemical and structural characterization of a novel thermophilic esterase EstD11 provide catalytic insights for the HSL family

[Abstract]: A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60 C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester. The tridimensional structures of EstD11 and the inactive mutant have been determined at atomic resolutions. Structural and bioinformatic analysis, confirm that EstD11 belongs to the family IV, the hormone-sensitive lipase (HSL) family, from the α/β-hydrolase superfamily. The canonical a/b hydrolase domain is completed by a cap domain, composed by two subdomains that can unmask of the active site to allow the substrate to enter. Eight crystallographic complexes were solved with different substrates and reaction products that allowed identification of the hot-spots in the active site underlying the specificity of the protein. Crystallization and/or incubation of EstD11 at high temperature provided unique information on cap dynamics and a first glimpse of enzymatic activity in vivo. Very interestingly, we have discovered a unique Met zipper lining the active site and the cap domains that could be essential in pivotal aspects as thermo-stability and substrate promiscuity in EstD11Ministerio de Ciencia e Innovación; BFU2017-90030-

Novel foods, food enzymes, and food additives derived from food by-products of plant or animal origin: principles and overview of the EFSA safety assessment

The European Union (EU) is committed to transitioning toward a circular economy model, with food waste being one of the areas to be targeted. To close the loop of food waste generated during food processing and discarded at the retail or consumption phases, research and innovation parties proposed to valorize agro-food by-products to produce novel foods and food improvement agents (food additives, food enzymes, and food flavorings). In the EU, the authorization of such novel foods and food improvement agents is governed by different regulatory frameworks. A centralized safety assessment by the European Food Safety Authority (EFSA) is the prerequisite for their authorization through the so-called Union Lists. Up to December 2023, EFSA published 45 scientific opinions on the safety of novel foods, food enzymes, and food additives derived from by-products of plant and animal origin. The current study illustrates examples of these by-products for the production of novel foods or food improvement agents and the data requirements behind their respective safety assessments conducted by EFSA. In this review, applications on novel foods, food enzymes, and food additives received by EFSA were screened and analyzed to find the common scientific requirements and differences in terms of the safety evaluation of such products. Various by-products (i.e., corncobs, coffee husks, spent grains of barley and rice, grape pomace, pumpkin peels, bovine whey, eggshells, shrimp heads, and animal organs or tissues) were described in the applications as being processed (extraction, physical treatments, and chemical and enzymatic reactions) to obtain novel foods and food improvement agents. The heterogeneity and complexity of these products emphasize the challenge of their safety assessment, depending on the characteristics of each product. However, as this study shows, the scientific requirements underpinning their safety do not differ substantially in the different regulated product areas considered, with similar information needed to assess their safety in terms of identity, production process, compositional characterization, proposed/intended uses and exposure assessment, toxicological information, and allergenicity data. Additional nutritional information and data on the history of use are required in the case of novel foods