Directed Evolution of Fungal Laccases

Fungal laccases are generalists biocatalysts with potential applications that range from bioremediation to novel green processes. Fuelled by molecular oxygen, these enzymes can act on dozens of molecules of different chemical nature, and with the help of redox mediators, their spectrum of oxidizable substrates is further pushed towards xenobiotic compounds (pesticides, industrial dyes, PAHs), biopolymers (lignin, starch, cellulose) and other complex molecules. In recent years, extraordinary efforts have been made to engineer fungal laccases by directed evolution and semi-rational approaches to improve their functional expression or stability. All these studies have taken advantage of Saccharomyces cerevisiae as a heterologous host, not only to secrete the enzyme but also, to emulate the introduction of genetic diversity through in vivo DNA recombination. Here, we discuss all these endeavours to convert fungal laccases into valuable biomolecular platforms on which new functions can be tailored by directed evolution

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

Enzyme-mediated surface functionalisation of stimuli-responsive microgels

Aqueous microgels based on N-Vinylcaprolactam (VCL) gained much attention in the biomedical research due to their temperature-sensitive behavior and their high biocompatibility.[1] However, the post-modification of PVCL-microgels with proteins is still challenging. The so-called sortase-mediated ligation is one possible method for the conjugation of biomolecules to polymers.[2] Sortases are bacterial enzymes with transpeptidase activity, responsible for the attachment of proteins to the cell wall of gram-positive bacteria. Sortases of class A (SrtA) recognize a LPXTG-sorting motif (X being any amino acid) at the C-terminus of the targeted protein, cleave it between the threonine and the glycine and ligate it to a second protein via an oligoglycine nucleophile.[3] In this work, we present the use of sortase-mediated ligation for the conjugation of different proteins to PVCL-microgels with the aim to incorporate special functionalities. For this purpose, microgels based on PVCL containing 5 mol% glycidyl methacrylate (GMA) as comonomer in the particles shell were synthesized and modified with the specific recognition peptide sequence LPETG for SrtA. The coupling of the LPETG sequence was analyzed via UV-Vis spectroscopy and Raman spectroscopy. To perform Sortase-mediated ligation, oligoglycin-tagged enhanced Green Fluorescent Protein (GGG‑eGFP) was used as a model protein. The conjugation of the eGFP to the microgel was investigated qualitatively via confocal microscopy and quantitatively via fluorescence intensity measurements. It was shown that the fluorescence intensity increased linearly with increasing eGFP-concentration and exponentially with increasing reaction time up to seven hours. Additionally, we were able to show that also oligoglycin-tagged CueO-laccase can be conjugated to the PVCL-microgel using sortase-mediated ligation. To this end, the protein activity of the microgels was measured using ABTS as laccase substrate and the amount of conjugated protein was analyzed via BCA assay. These results indicate that sortase-mediated ligation is a very promising and powerful tool for the modification of microgels with biomacromolecules for applications in drug delivery, biointerface coatings and sensors. Aqueous microgels based on N-Vinylcaprolactam (VCL) gained much attention in the biomedical research due to their temperature-sensitive behavior and their high biocompatibility.[1] However, the post-modification of PVCL-microgels with proteins is still challenging. The so-called sortase-mediated ligation is one possible method for the conjugation of biomolecules to polymers.[2] Sortases are bacterial enzymes with transpeptidase activity, responsible for the attachment of proteins to the cell wall of gram-positive bacteria. Sortases of class A (SrtA) recognize a LPXTG-sorting motif (X being any amino acid) at the C-terminus of the targeted protein, cleave it between the threonine and the glycine and ligate it to a second protein via an oligoglycine nucleophile.[3] In this work, we present the use of sortase-mediated ligation for the conjugation of different proteins to PVCL-microgels with the aim to incorporate special functionalities. For this purpose, microgels based on PVCL containing 5 mol% glycidyl methacrylate (GMA) as comonomer in the particles shell were synthesized and modified with the specific recognition peptide sequence LPETG for SrtA. The coupling of the LPETG sequence was analyzed via UV-Vis spectroscopy and Raman spectroscopy. To perform Sortase-mediated ligation, oligoglycin-tagged enhanced Green Fluorescent Protein (GGG‑eGFP) was used as a model protein. The conjugation of the eGFP to the microgel was investigated qualitatively via confocal microscopy and quantitatively via fluorescence intensity measurements. It was shown that the fluorescence intensity increased linearly with increasing eGFP-concentration and exponentially with increasing reaction time up to seven hours. Additionally, we were able to show that also oligoglycin-tagged CueO-laccase can be conjugated to the PVCL-microgel using sortase-mediated ligation. To this end, the protein activity of the microgels was measured using ABTS as laccase substrate and the amount of conjugated protein was analyzed via BCA assay. These results indicate that sortase-mediated ligation is a very promising and powerful tool for the modification of microgels with biomacromolecules for applications in drug delivery, biointerface coatings and sensors. Please click Additional Files below to see the full abstract

Clinkering of calcium sulfoaluminate clinkers: polymorphism of ye'elimite

The manufacture of CSA cements is more environmentally friendly than that of OPC as it releases less CO2. This reduction depends on CSA composition and is due to three factors: i) less emissions from decarbonation in the kilns; ii) lower clinkering temperature, consequently less fuel is needed, and iii) it is easier to grind, implying a depletion in indirect emissions. CSA cements are prepared by mixing the clinker with different amounts of calcium sulfate as a set regulator. Their main performances are fast setting time (followed by a rapid hardening), good chemical resistance and, depending on the amount of the added sulfate source they can work as shrinkage controllers. CSA cements present a wide range of phase assemblages, but all of them contain over 50 wt% of ye'elimite (C4A3s) jointly with belite (C2S), tetracalcium aluminoferrite (C4AF) and other minor components such as CA, Cs, CsH2 and so on [1]. Ye'elimite is also included (~25 wt%) in BYF (Belite- Ye'elimite-Ferrite) or BAY (Belite-Alite-Ye'elimite) clinkers. Ye'elimite has a sodalite type structure with general composition, M4[T6O12]X. Stoichiometric ye'elimite crystal structure at room temperature will be described in detailed. The role of different amounts of minor elements on the synthetic procedure and crystal structures will be also presented [2,3]. This keynote will be also focused on a revision of the effect of raw materials on the mineralogical composition of CSA, BYF and BAY. Specifically, the role of main elements contents in the ye'elimite formation in these systems will be described. Moreover, the effect of minor elements on the polymorphism of both ye'elimite and belite, especially on BYF and BAY clinkers, will be presented [4,5,6].Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Spanish MINECO and FEDER [BIA2017-82391-R] research project and I3 [IEDI-2016-0079] program

Lacasa de alto potencial redox funcional en sangre mediante evolución dirigida método de obtención y sus aplicaciones

La presente invención describe una lacasa de alto potencial redox obtenida mediante evolución molecular dirigida que es activa en condiciones electrofisiológicas, que resiste elevadas concentraciones de haluros, que tiene una actividad significativa a pHs neutros/alcalinos y que es activa en sangre y plasma humano. La presente invención se refiere a la secuencia aminoacídica de dicha lacasa, a la secuencia nucleotídica que codifica para dicha lacasa y células que permiten su obtención. La lacasa de la invención presenta aplicaciones en diversos sectores: nano-biotecnología, biomedicina, procesos de biorremediación, industria papelera y química fina.Peer reviewedConsejop Superior de Investigaciones CientíficasA1 Solicitud de patente con informe sobre el estado de la técnic

Lacasa de alto potencial redox funcional en sangre mediante evolución dirigida método de obtención y sus aplicaciones

La presente invención describe una lacasa de alto potencial redox obtenida mediante evolución molecular dirigida que es activa en condiciones electrofisiológicas, que resiste elevadas concentraciones de haluros, que tiene una actividad significativa a pHs neutros/alcalinos y que es activa en sangre y plasma humano. La presente invención se refiere a la secuencia aminoacídica de dicha lacasa, a la secuencia nucleotídica que codifica para dicha lacasa y células que permiten su obtención. La lacasa de la invención presenta aplicaciones en diversos sectores: nano-biotecnología, biomedicina, procesos de biorremediación, industria papelera y química fina.Peer reviewedConsejop Superior de Investigaciones CientíficasB1 Patente sin examen previ

Tailored setting times with high compressive strengths in bassanite calcium sulfoaluminate eco-cements

This work deals with the hydration of a calcium sulfoaluminate (CSA) eco-cement prepared with bassanite and different additives (type and content) at a fixed water/CSA ratio of 0.5. Pastes prepared with bassanite show high water demands, high viscosity values and short initial setting times which are related to the fast dissolution rate of bassanite and the subsequent precipitation of gypsum. These facts have a dramatic effect onto the mechanical strength values, and make necessary the addition of additives. Here, the addition of different amounts of specific retarders (polycarboxylate, tartaric acid and phosphonic acid) not only improved the workability of pastes and mortars, but also delayed the setting time, by modifying the dissolution rates of the phase(s), and improved mechanical strengths. Finally, mortars with high compressive strengths (46 and 84 MPa at 1 and 7 days of hydration, respectively) and, chiefly, tailored setting times with high strengths have been prepared.Funding from Junta de Andalucía (P11-FQM-7517 and P12-FQM-1656), FEDER/University of Málaga (FC14-MAT-23), MINECO (BIA2014-57658-C2-1-R and BIA2014-57658-C2-2-R, the latter co-funded by FEDER) are acknowledged

X-ray diffraction, cements and environment, three worlds in one

This keynote lecture will be focused on the strategies for reducing CO2 emissions in the cement production. Concretely, the production of ecocements with optimised formulations that yield reductions in CO2 emissions of up to 25%, when compared to OPC production. Phase assemblage has to be carefully optimised to be competitive and these new ecocements should develop compressive strengths of at least 50 MPa at 28 days of hydration. Optimised compositions of several ecocements will be discussed, but all of them are ye'elimite or calcium sulphoaluminate containing ones: belite-ye'elimite-ferrite (BYF), belite-alite-ye'elimite (BAY) and ye'elimite rich ones (CSA). The clinkering temperature of BYF and BAY has to be established to obtain the targeted phase assemblages. Moreover, the stabilisation of alpha-forms of belite is needed to develop high mechanical strengths at early ages. The benefits of the use of waste materials (such as fly ash or slag) as additions to ecocements are three-fold: lower CO2 emissions due to clinker replacement; valorisation of “useless” products that need a lot of landscape and the consequent efficient consumption of raw materials; and to enhance mechanical properties of the corresponding mortars. The design of appropriate CSA, BYF and BAY mortars, with the final aim of knowing and controlling the hydration mechanisms, will be presented. Particularly, the role of i) type and amount of set regulator (gypsum, anhydrite, etc.), ii) water/cement ratio (w/c); iii) superplasticiser; and iv) pozzolanic additions will be discussed. The role of these parameters in the microstructure and hydraulic behaviour has been investigated through traditional techniques as well as advanced synchrotron characterisation. The formers include laboratory/synchrotron X-ray powder diffraction combined with Rietveld methodology (to obtain phase assemblage), electron microscopy techniques for paste microstructure determination, rheological studies (to control the effect of the different additives, w/c ratio and setting time retarders) and mechanical tests (setting times, compressive strengths and dimensional stability). The latters comprise a group of techniques available at synchrotrons such as: i) high temperature x-ray diffraction for clinkering studies and ii) total scattering data to be analysed by pair distribution function, PDF.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. PhD D. Londono-Zuluaga thanks Colciencias and Enlaza Mundos program PhD grant. Spanish MINECO (BIA2014-57658-C2-2-R, which is co-funded by FEDER, BIA2014-57658-C2-1-R and I3 (IEDI-2016-0079) grants) are acknowledged

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Diseño de lacasas fúngicas activas en sangre mediante evolución molecular dirigida

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-06-201