Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast

<p>Abstract</p> <p>Background</p> <p>In the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust high-throughput protocols. The current work describes how to achieve this goal by engineering ligninolytic oxidoreductases (a high-redox potential laccase -HRPL- and a versatile peroxidase, -VP-) functionally expressed in <it>Saccharomyces cerevisiae</it>.</p> <p>Results</p> <p>Taking advantage of the eukaryotic machinery, complex mutant libraries were constructed by different <it>in vivo </it>recombination approaches and explored for improved stabilities and activities. A reliable high-throughput assay based on the analysis of T₅₀was employed for discovering thermostable oxidases from mutant libraries in yeast. Both VP and HRPL libraries contained variants with shifts in the T₅₀values. Stabilizing mutations were found at the surface of the protein establishing new interactions with the surrounding residues.</p> <p>Conclusions</p> <p>The existing tradeoff between activity and stability determined from many point mutations discovered by directed evolution and other protein engineering means can be circumvented combining different tools of <it>in vitro </it>evolution.</p

Kinetics of direct and substrate-mediated electron transfer of versatile peroxidase-modified graphite electrodes

Electron transfer (ET) of versatile peroxidase (VP) was studied in the bioelectrocatalytic reduction reaction of H2O2 at peroxidase- modified graphite electrodes to obtain additional information on the kinetic characteristics of this novel ligninolytic peroxidase. Rotating disk electrodes (RDE) experiments were performed at 0 V (vs. SCE) in two different buffers (tartrate buffer, pH 5.0; and citrate buffer, pH 3.0). From measurements of the mediated and mediatorless currents of H2O2 reduction at the RDE, the percentage of VP molecules involved in direct ET (≈55%) was calculated. The peroxidase-modified electrodes were used for determination of the donor substrates in RDE mode, and the results were interpreted in terms of catalytic efficiencies

Computational Modeling Methods for Understanding the Interaction of Lignin and Its Derivatives with Oxidoreductases as Biocatalysts

This chapter will be presented as follow. First, a brief introduction to structure and characterization of lignin and its derivatives is presented, as well as their importance as chemical scaffolds for obtaining value-added products in chemical, food, pharmaceutical and agriculture industry. Second, an extensive review of different reports using computational modeling methods—like molecular dynamics simulations, quantum mechanics and hybrid calculation methods, among others—in the understanding of enzyme-substrate interaction and biocatalysis will be presented. Third, and as last part of chapter, some hand picked examples from literature will be chosen as successful cases where the interplay between experiment and computation has given as a result protein engineered oxidoreductases with improved catalytic capabilities

MoBiSea: a binary search algorithm for product clustering in Industry 4.0

Proprietary systems used to modernize Industry 4.0 usually involve high financial costs. Consequently, using low-cost devices with the same functionalities, capable of replacing these proprietary systems but at a lower cost, has become an incipient trend. However, these low-cost devices usually come with electromagnetic interference problems as they are encapsulated in electrical panels, sitting alongside electromechanical devices. In this article, we present Mode Binary Search, an algorithm specifically designed for use in a low-cost automated-industrial-productivity-data-collection system. Specifically, productivity data are obtained from the availability and sealing signals of the thermoplastic sealing machines in production lines belonging to the agri-food industry. Mode Binary Search was designed to cluster sealing signals, thus enabling us to identify which products have been made. Furthermore, the algorithm determines when the manufacturing of each product starts and ends, in other words, the exact moment a product change occurs and all this without the need for operator supervision or intervention. Finally, we compared our algorithm, based on binary search, with three clustering mechanisms: k-means, k-rms and x-means. Out of all the cases we analyzed, the maximum error committed by Mode Binary Search is limited to 2.69%, thereby outperforming all others

Multiple implications of an active site phenylalanine in the catalysis of aryl-alcohol oxidase

Aryl-alcohol oxidase (AAO) has demonstrated to be an enzyme with a bright future ahead due to its biotechnological potential in deracemisation of chiral compounds, production of bioplastic precursors and other reactions of interest. Expanding our understanding on the AAO reaction mechanisms, through the investigation of its structure-function relationships, is crucial for its exploitation as an industrial biocatalyst. In this regard, previous computational studies suggested an active role for AAO Phe397 at the active-site entrance. This residue is located in a loop that partially covers the access to the cofactor forming a bottleneck together with two other aromatic residues. Kinetic and affinity spectroscopic studies, complemented with computational simulations using the recently developed adaptive-PELE technology, reveal that the Phe397 residue is important for product release and to help the substrates attain a catalytically relevant position within the active-site cavity. Moreover, removal of aromaticity at the 397 position impairs the oxygen-reduction activity of the enzyme. Experimental and computational findings agree very well in the timing of product release from AAO, and the simulations help to understand the experimental results. This highlights the potential of adaptive-PELE to provide answers to the questions raised by the empirical results in the study of enzyme mechanisms.This work was supported by the EnzOx2 project (H2020-BBI-PPP-2015-720297) of the European Joint Undertaking of Bio-based Industries (http://bbi-europe.eu), the INDOX project (KBBE-2013-7-613549) of the European Seventh Framework Programme, and the NOESIS (BIO2014-56388-R), vMutate (CTQ2016-79138-R) and FLADIMOTEC (BIO2016-75183-P) projects of the Spanish Ministry of Economy and Competitiveness. J.C. acknowledges a FPU fellowship (FPU2012-2041) from the Spanish Ministry of Education, Culture and Sports.Peer ReviewedPostprint (published version

Methanol Dehydrogenation on Pt Electrodes : Active Sites and Role of Adsorbed Spectators Revealed through Time-Resolved ATR-SEIRAS

ACKNOWLEDGMENT L.P.-M. acknowledges a doctoral scholarship within the Leverhulme Centre for Doctoral Training in Sustainable Production of Chemicals and Materials (Grant DS-2017-073).Peer reviewedPostprin

Monitoring the influence of marine aquaculture on wild fish communities: benefits and limitations of fatty acid profiles

Fatty acids (FA) have been applied as indicators of the influence of coastal sea-cage fish farming on wild fish communities in several recent scientific publications. Due to the relatively high conservation of FA composition throughout the food web, they are useful for characterizing trophic relationships. The increasing utilization of vegetable or alternative animal oils in the production of aquafeeds results in cultivated fish exhibiting higher levels of terrestrial FAs in their tissues. As previously reported, wild fish ubiquitously aggregate around fish farms as a consequence of the introduction of new habitat and the easy availability of food—fish farms act as enhanced fish aggregation devices (FADs). The influence of food pellets on the composition of wild fish has been detected in recent studies on salmon, sea bass and sea bream aquaculture, with increased levels of linoleic acid (18:2n-6) and a low n-3/n-6 ratio as clear indicators of the consumption of food pellets from the farms. The potential ecological and physiological effects on wild fish are presently unknown. In the present article, guidelines are proposed for the investigation and use of terrestrial FAs to track the effects of coastal aquaculture on wild fish communities and local fisheries, as well as the benefits or limitations of this technique

Prevalence and risk factors of gestational diabetes mellitus: findings from a universal screening feasibility program in Lima, Peru.

BACKGROUND: Gestational diabetes mellitus (GDM) is a global public health concern with potential implications for the health of a mother and her offspring. However, data on the prevalence and risk factors of GDM in Latin America are scarce. The study was designed to estimate the prevalence of GDM and identify maternal risk factors among Peruvian women. METHODS: A cross-sectional study was conducted among 1300 pregnant women attending a prenatal clinic in Lima, Peru. GDM was diagnosed using an Oral Glucose Tolerance Test (OGTT) performed between 24 and 28 gestational weeks using the International Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria. Depression status was assessed using the Patient Health Questionnaire-9. Multivariate logistic regression models were used to identify risk factors of GDM. RESULTS: Approximately 16% of pregnant women were diagnosed with GDM. The prevalence of obesity and depression were 24.4 and 10.6%, respectively. After adjusting for confounders, mid-pregnancy obesity was associated with a 1.64-fold increased odds of GDM (OR: 1.64; 95% CI: 1.03-2.61). Participants with a family history of diabetes had a 1.5-fold increased odds of developing GDM (OR: 1.51, 95% CI: 1.10-2.07) as compared to women without this family history. Depression was associated with a 1.54-fold increased odds of GDM (OR: 1.54; 95% CI:1.09-2.17). CONCLUSIONS: GDM is highly prevalent and was associated with maternal obesity, family history of diabetes and antepartum depression among Peruvian women. Intervention programs aimed at early diagnoses and management of GDM need to take maternal obesity, family history of diabetes and antepartum depression into account

Dioxin Toxicity In Vivo Results from an Increase in the Dioxin-Independent Transcriptional Activity of the Aryl Hydrocarbon Receptor

The Aryl hydrocarbon receptor (Ahr) is the nuclear receptor mediating the toxicity of dioxins -widespread and persistent pollutants whose toxic effects include tumor promotion, teratogenesis, wasting syndrome and chloracne. Elimination of Ahr in mice eliminates dioxin toxicity but also produces adverse effects, some seemingly unrelated to dioxin. Thus the relationship between the toxic and dioxin-independent functions of Ahr is not clear, which hampers understanding and treatment of dioxin toxicity. Here we develop a Drosophila model to show that dioxin actually increases the in vivo dioxin-independent activity of Ahr. This hyperactivation resembles the effects caused by an increase in the amount of its dimerisation partner Ahr nuclear translocator (Arnt) and entails an increased transcriptional potency of Ahr, in addition to the previously described effect on nuclear translocation. Thus the two apparently different functions of Ahr, dioxin-mediated and dioxin-independent, are in fact two different levels (hyperactivated and basal, respectively) of a single function