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Background Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts. Results P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive. Conclusion Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts., Microbial Cell Factories, 21 (1), ISSN:1475-2859

This study deals with the genetic and phenotypic heterogeneity of the marine Nocardiopsis alba strains isolated during pre-monsoon, monsoon and post-monsoon seasons. The isolates were characterized for their morphological and biochemical attributes, growth media preferences, antibiotic susceptibility and extracellular enzyme secretion. Nocardiopsis alba strains were assessed against 12 different antibiotics, and the responses were expressed in terms of the multiple antibiotic resistance (MAR) number. The majority of the strains produced multiple extracellular enzymes: proteases, amylases and lipases. Further, the strains were characterized on the basis of 16S rRNA gene sequencing and the majority were identified as Nocardiopsis alba along with few strains of Streptomyces lopnurensis, Nocardiopsis synnemataformans and Nocardiopsis dassonvillei. Neighbor-joining (NJ) phylogenetic tree suggested variation among the genetically similar Nocardiopsis alba species. The study establishes significant heterogeneity with respect to genetic and phenotypic characteristics of the strains of Nocardiopsis alba. Phylogenetic tree and phenogram-based comparison reflect the heterogeneity in terms of different clustering patterns of the strains. Further, the whole genome sequence data available in the literature also confirm the observed heterogeneity. Nocardiopsis alba strains displayed a relatively regressive pattern of dependence on the environmental factors based on the canonical correspondence analysis plot. The study represents cultivation, characterization, phylogenetic analysis and enzymatic potential of the Nocardiopsis alba species of seawater origin.

This review deals with the mechanism of antagonistic action of bacterial and fungal biocontrol agents such as the production of antibiotics, siderophores, enzyme secretion, competition for nutritio...

Consolidated bioprocessing (CBP) of lignocellulosic material into bioethanol has progressed in the past decades; however, several challenges still exist which impede the industrial application of this technology. Identifying the challenges that exist in all unit operations is crucial and needs to be optimised, but only the barriers related to the secretion of recombinant cellulolytic enzymes in Saccharomyces cerevisiae will be addressed in this review. Fundamental principles surrounding CBP as a biomass conversion platform have been established through the successful expression of core cellulolytic enzymes, namely β-glucosidases, endoglucanases, and exoglucanases (cellobiohydrolases) in S. cerevisiae. This review will briefly address the challenges involved in the construction of an efficient cellulolytic yeast, with particular focus on the secretion efficiency of cellulases from this host. Additionally, strategies for studying enhanced cellulolytic enzyme secretion, which include both rational and reverse engineering approaches, will be discussed. One such technique includes bio-engineering within genetically diverse strains, combining the strengths of both natural strain diversity and rational strain development. Furthermore, with the advancement in next-generation sequencing, studies that utilise this method of exploiting intra-strain diversity for industrially relevant traits will be reviewed. Finally, future prospects are discussed for the creation of ideal CBP strains with high enzyme production levels.Key Points• Several challenges are involved in the construction of efficient cellulolytic yeast, in particular, the secretion efficiency of cellulases from the hosts.• Strategies for enhancing cellulolytic enzyme secretion, a core requirement for CBP host microorganism development, include both rational and reverse engineering approaches.• One such technique includes bio-engineering within genetically diverse strains, combining the strengths of both natural strain diversity and rational strain development.

Background Fungal saccharification of lignocellulosic biomass occurs concurrently with the secretion of a diverse collection of proteins, together functioning as a catalytic system to liberate soluble sugars from insoluble composite biomaterials. How different fungi respond to different substrates is of fundamental interest to the developing biomass saccharification industry. Among the cornerstones of fungal enzyme systems are the highly expressed cellulases (endo-β-glucanases and cellobiohydrolases). Recently, a cyclophellitol-derived activity-based probe (ABP-Cel) was shown to be a highly sensitive tool for the detection and identification of cellulases. Results Here we show that ABP-Cel enables endo-β-glucanase profiling in diverse fungal secretomes. In combination with established ABPs for β-xylanases and β-d-glucosidases, we collected multiplexed in-gel fluorescence activity-based protein profiles of 240 secretomes collected over ten days from biological replicates of ten different basidiomycete fungi grown on maltose, wheat straw, or aspen pulp. Our results reveal the remarkable dynamics and unique enzyme fingerprints associated with each species substrate combination. Chemical proteomic analysis identifies significant arsenals of cellulases secreted by each fungal species during growth on lignocellulosic biomass. Recombinant production and characterization of a collection of probe-reactive enzymes from GH5, GH10, and GH12 confirm that ABP-Cel shows broad selectivity towards enzymes with endo-β-glucanase activity. Conclusion Using small-volume samples with minimal sample preparation, the results presented here demonstrate the ready accessibility of sensitive direct evidence for fungal enzyme secretion during early stages of growth on complex lignocellulosic substrates.

Many challenges are faced in the conversion of biomass into advanced biofuels, one of which is finding the correct organism for the job. The filamentous fungus Aspergillus niger has been chosen as a biocatalyst for cellulose, hemicellulose, and lignin degradation because it can secrete numerous hydrolytic enzymes, such as lignin-degrading enzymes and, in particular, laccase enzymes (1). However, low transformation efficiency has hindered efforts to unlock the full potential of this organism. This thesis presents a novel method to efficiently edit the genome of A. niger that overcomes several of the current challenges encountered when using CRISPR/Cas9 (2). We designed a genetic construct that is efficient and precise concerning knockout efficiency and phenotype control and demonstrated its utility for genetic knockouts, integrations, and alterations. Using the new CRISPR/Cas9 toolbox, we developed a high throughput platform to transform A. niger using robotics. We then transformed a library of 81 laccases that could be potentially secreted by A. niger in two different strain backgrounds and obtained six new laccases secreted by A. niger. We engineered a strain that increased total protein secretion fourfold and extracellular laccase activity threefold. We are currently applying for a patent for this discovery. We characterized each secreted enzyme by downscaling in micro-culture to study their optimum efficiency (e.g., media, pH, and fermentation time). These discoveries bring us significantly closer to generating strains that can produce a cocktail of laccases to unlock recalcitrant biomass for downstream processing. Additionally, the methods we developed will enable the rapid building and testing of genetic variants in A. niger for metabolic engineering, synthetic biology, and many other applications.

Trypanosoma brucei are protozoan parasites infecting mammals in Sub-Saharan Africa causing the disease known as Sleeping sickness. The parasites are transmitted between the mammal hosts through bites of an insect vector. The transmission from insect to mammal is one out of many steps in a complex life cycle causing numbers of metabolic and physiological changes. While the majority of a population is preparing to reenter the insect vector, a fraction of the population continuously adapt to the immune system of the host, showing a heterogeneous pattern within the population. Though numerous analytical methods for studying single cells are continuously developed, little is still know of T. bruceion a single-cell level. Droplet-based microfluidicsis a high-throughput tool for single cell analysis emerging within the last decades. We here developed an analytical method for quantitative measurements at the single cell level based on encapsulation and cultivation of T. brucei in microcompartment droplets. We described a microfluidic system for single-cell encapsulation and analysis based on fluorescence measurements. We first showed the survival of T. brucei for several hours to days in droplets with influence on both survival and growth by the droplet size. We unraveled various growth patterns within a population, and found that droplet cultivation of trypanosomes revealed highly dividing cell variants with10-fold higher cell densities compared to standard cultivation techniques. We further combined the encapsulation of T. brucei with an enzymatic detection assay to study protease secretion. Using a fluorescence based substrate allowed detection of protease activity secreted from single cells. Lastly, we tested the setup in two additional applications both used on single-cell T. brucei. Conclusively, droplet-based microfluidics have shown promising as an analytical tool for trypanosome cultivation and analysis with a further potential for high-throughput single-cell analysis., Trypanosoma brucei est un parasite protozoaire infectant les mammifères en Afrique subsaharienne et provoquant la maladie connue sous le nom de maladie du sommeil. Les parasites sont transmis entre les hôtes mammifères par les piqûres d’un insecte vecteur. La transmission de l’insecte au mammifère est l’une des nombreuses étapes d’un cycle de vie complexe provoquant de nombreux changements métaboliques et physiologiques. Alors que la majorité d’une population se prépare à réintégrer l’insecte vecteur, une fraction de la population s’adapte en permanence au système immunitaire de l’hôte, montrant un schéma hétérogène au sein de la population. Bien que de nombreuses méthodes analytiques soient continuellement développées pour étudier les cellules individuelles, on sait encore peu de choses sur T. brucei au niveau cellulaire. La microfluidique en gouttes est un outil à haut débit pour l’analyse de cellules uniques qui a émergé au cours des dernières décennies. Nous avons développé ici une méthode analytique pour les mesures quantitatives au niveau de la cellule unique basées sur l’encapsulation et la culture de T. brucei dans des gouttes agissant comme des microcompartiments. Nous avons décrit un système microfluidique pour l’encapsulation et l’analyse de cellules individuelles basées sur des mesures de fluorescence. Nous avons d’abord montré la survie de T. brucei pendant plusieurs heures à plusieurs jours dans des gouttelettes avec une influence à la fois sur la survie et la croissance par la taille des gouttelettes. Nous avons observé divers modèles de croissance au sein d’une population et avons découvert que la culture par gouttes de trypanosomes révélait des variants cellulaires à division élevée avec des densités cellulaires 10 fois plus élevées que les techniques de culture standard. Nous avons en outre combiné l’encapsulation de T.brucei avec un test de détection enzymatique pour étudier la sécrétion de protéase. L’utilisation d’un substrat basé sur la fluorescence a permis la détection de l’activité protéase sécrétée par des cellules individuelles. Enfin, nous avons testé la configuration dans deux applications supplémentaires, toutes deux utilisées sur T. brucei unicellulaire. En conclusion, la microfluidique en gouttes s’est révélée prometteuse en tant qu’outil analytique pour la culture et l’analyse des trypanosomes avec un potentiel supplémentaire pour l’analyse unicellulaire à haut débit.

To fight against antibiotic‐resistant bacteria adhering and developing on medical devices, which is a growing problem worldwide, researchers are currently developing new “smart” materials and coatings. They consist in delivery of antimicrobial agents in an intelligent way, i.e., only when bacteria are present. This requires the use of new and sophisticated tools combining antimicrobial agents with lipids or polymers, synthetic and/or natural. In this review, three classes of innovative materials are described: hydrogels, nanomaterials, and thin films. Moreover, smart antibacterial materials can be classified into two groups depending on the origin of the stimulus used: those that respond to a nonbiological stimulus (light, temperature, electric and magnetic fields) and those that respond to a biological stimulus related to the presence of bacteria, such as changes in pH or bacterial enzyme secretion. The bacteria presence can induce a pH change that constitutes a first potential biological trigger allowing the system to become active. A second biological trigger signal consists in enzymes produced by bacteria themselves. A complete panel of recent studies will be given focusing on the design of such innovative smart materials that are sensitive to biological triggers.

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, β-glucosidase, cellobiohydrolase I and II, xylanase, β-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks. ispartof: METABOLIC ENGINEERING vol:59 pages:131-141 ispartof: location:Belgium status: published

The pancreas is a glandular organ responsible for diverse homeostatic functions, including hormone production from the endocrine islet cells to regulate blood sugar levels and enzyme secretion from the exocrine acinar cells to facilitate food digestion. These pancreatic functions are essential for life; therefore, preserving pancreatic function is of utmost importance. Pancreas dysfunction can arise either from developmental disorders or adult onset disease, both of which are caused by defects in shared molecular pathways. In this chapter, we discuss what is known about the molecular mechanisms controlling pancreas development, how disruption of these mechanisms can lead to developmental defects and disease, and how essential pancreas functions can be modeled using human pluripotent stem cells. At the core of understanding of these molecular processes are animal model studies that continue to be essential for elucidating the mechanisms underlying human pancreatic functions and diseases.

Dissertation, RWTH Aachen University, 2020; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2021). = Dissertation, RWTH Aachen University, 2020, Enzymes are biological catalysts that are produced by living organisms. Their properties make them indispensable in different fields of industry and research. Obtaining enzymes in sufficient quantities is important for their application, as well as for their scientific analysis. Heterologous enzyme production is a common method to achieve high enzyme titers and employing secretion can improve enzyme yield as well as reduce downstream processing. The goal of this thesis was to establish a platform for enzyme production that can be used for simple and straightforward optimization of enzyme expression and secretion. The main objective was the generation of a signal peptide library fusion platform that can be used to screen for suitable signal peptides. To this end, a cloning strategy was established for simple and fast library generation. Subsequently, a signal peptide library was built with signal peptides from B. subtilis. This library served as starting point for subcloning into different vectors and for generating fusion libraries of different target genes. Two different vectors were used for subcloning: a C. glutamicum vector to enable enzyme production in the gram positive bacterium and a vector containing the fluorescent reporter protein Pp1FbFP to enable monitoring the target protein secretion during the expression process. A cutinase, a phytase and a diguanylate cyclase were cloned into the library to test the library’s applicability. For cutinase, the signal peptide screening resulted in a threefold increase in activity, which was achieved by the signal peptide YobB. The best signal peptide for the phytase AppA showed 3.5-fold improvement compared to the starting variant. For the diguanylate cyclase tDGCsh, secretion could not be observed. Fusion with signal peptides does not only enable enzyme secretion, it can also be used for the secretion of peptides. Secretory production of antimicrobial peptides in C. glutamicum was tested for the four antimicrobial peptides AFP1, LCI, Psoriasin and VarvF. With the signal peptide NprE, LCI and Psoriasin were secreted at high levels. Titers of up to 130 mg/L have been reached for LCI while Psoriasin concentration was estimated to be 54 mg/L. Directed evolution was performed to improve different signal peptides towards more efficient secretion. For this purpose, the signal peptides PhrF and YbdN were chosen. Two rounds of directed evolution for PhrF resulted in a 2.4-fold increase in activity. YbdN was evolved in one round of mutagenesis, resulting in a 1.36-fold improvement. Coexpression of enzymes with additional unfused signal peptides resulted in significantly improved enzyme secretion. The effect of coexpressed signal peptides on secretion was strongly depended on the expression conditions and the used signal peptide. To further improve enzyme production, vector backbone evolution with the epMEGAWHOP method was performed for the compact E. coli vector pALXtreme-1a and the E. coli-C. glutamicum shuttle vector pEKEx2. The activity of the model enzyme cutinase was increased up to 2.5-fold after two evolution rounds for pEKEx2 and up to ninefold after three rounds of directed evolution for pALXtreme-1a., Published by RWTH Aachen University, Aachen

Yeasts occur in all environments and have been described as potent antagonists of various plant pathogens. Due to their antagonistic ability, undemanding cultivation requirements, and limited biosafety concerns, many of these unicellular fungi have been considered for biocontrol applications. Here, we review the fundamental research on the mechanisms (e.g., competition, enzyme secretion, toxin production, volatiles, mycoparasitism, induction of resistance) by which biocontrol yeasts exert their activity as plant protection agents. In a second part, we focus on five yeast species (Candida oleophila, Aureobasidium pullulans, Metschnikowia fructicola, Cryptococcus albidus, Saccharomyces cerevisiae) that are or have been registered for the application as biocontrol products. These examples demonstrate the potential of yeasts for commercial biocontrol usage, but this review also highlights the scarcity of fundamental studies on yeast biocontrol mechanisms and of registered yeast-based biocontrol products. Yeast biocontrol mechanisms thus represent a largely unexplored field of research and plentiful opportunities for the development of commercial, yeast-based applications for plant protection exist. Electronic supplementary material The online version of this article (10.1007/s11274-019-2728-4) contains supplementary material, which is available to authorized users.

SummaryDuring the degradation of biological materials such as biopolymers, extracellular enzymes liberate oligosaccharides that act as common goods and become available for all cells in the local neighborhood. This phenomenon can lead to cooperative growth, whereby cell-cell aggregation increases both the per-capita availability of resources and the per cell growth rate. However, aggregation can also have detrimental consequences for growth, as gradients form within aggregates limiting the resource accessibility. We used a computational model to show that high bacterial densities and high enzyme secretion rates restrict cooperation in aggregates larger than 10μm, due to the emergence of polymer and oligomer counter-gradients. We compared these predictions against experiments performed with two well-studied alginate degrading Vibrios, one of which displayed a strong density dependent growth. We observed that both strains can form large aggregates (

Made available in DSpace on 2020-12-12T02:23:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-11-14 Background: β-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. Results: In this study, BxlB-a highly secreted GH3 β-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants' catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. Conclusions: This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes. Department of Biochemistry and Tissue Biology Institute of Biology University of Campinas (UNICAMP), Rua Monteiro Lobato, 255 Cidade Universitária Zeferino Vaz Department of Physics Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP) Department of Medical Science Faculty of Medicine University of Campinas (UNICAMP) Department of Biochemistry and Immunology Ribeirão Preto Medical School University of São Paulo (USP) Institute of Chemistry and Center for Computing in Engineering and Sciences University of Campinas (UNICAMP) Department of Physics Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP)

Agradecimentos: We thank the LNBio Mass Spectrometry staff for their assistance with LC–MS/MS and Professor Hernandes Faustino de Carvalho Laboratory for his assistance with the immunohistochemistry methodology. We would like to thank Paul Whitford for the simulation and analysis program codes. The simulations were performed at CENAPAD-SP (Centro Nacional de Processamento de Alto Desempenho em São Paulo), under the UNICAMP/FINEP-MCT project. Research was supported by LNBR-Brazilian Biorenewables National Laboratory (CNPEM/MCTIC) using the Characterization of Macromolecules (MAC) open-access facility. The authors thank Espaço da Escrita-Pró-Reitoria de Pesquisa, UNICAMP for the language services provided Abstract: ß-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. In this study, BxlB - a highly secreted GH3 ß-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites - was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants’ catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Fechado

The aim of the present study was to assess the ability of different white-rot fungi to tolerate polychlorinated biphenyls (PCBs) using predictive mycology, by relating fungal growth inhibition to ligninolityc enzyme secretion. Fungal strains were grown in the presence of PCBs in solid media and their radial growth values were modelled through the Dantigny-logistic like function in order to estimate the time required by the fungal colonies to attain half their maximum diameter. The principal component analysis (PCA) revealed an inverse correlation between strain tolerance to PCBs and the laccase secretion over time, being laccase production closely associated with fungal growth capacity. Finally, a PCA was run to regroup and split between resistant and sensitive fungi. Simultaneously, a function associated with a model predicting the tolerance to PCBs was developed. Some of the assayed isolates showed a promising capacity to be applied in PCB bioremediation. Abbreviations: Polychlorinated biphenyls (PCBs), white-rot fungi (WRF). Fil: Sadañoski, Marcela Alejandra. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Velázquez, Juan Ernesto. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Fonseca, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina Fil: Zapata, Pedro Dario. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Levin, Laura Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Villalba, Laura. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina

Deinococcus radiodurans is one of the most yet discovered extremophilic microbe, the isolation of which from the various habitats of Kotumsar cave is always a matter of enticement to discover its ecological economics. In the present work we studied the intra versus extracellular alkaline protease and glucose isomerase secretion capabilities of Deinococcus radiodurans; KCB21, KCB50, KCB93 isolated from three distinct subterranean niches of Kotumsar cave. The selected niches/zones were the entrance zone, transient zone and the deep inner zone from where the soil sediments were collected to isolate the bacterial strains. The results revealed high extracellular alkaline protease activity from the Deinococcus radiodurans strain which was isolated from the deeper zones of the cave, whereas no such phenomenon was revealed for glucose isomerase. The possible reason for the obtained results has been discussed.

Melatonin is an indoleamine produced from the amino acid l-tryptophan, whereas metabolites of melatonin are known as kynuramines. One of the best-known kynuramines is N1-acetyl-N1-formyl-5-methoxykynuramine (AFMK). Melatonin has attracted scientific attention as a potent antioxidant and protector of tissue against oxidative stress. l-Tryptophan and kynuramines share common beneficial features with melatonin. Melatonin was originally discovered as a pineal product, has been detected in the gastrointestinal tract, and its receptors have been identified in the pancreas. The role of melatonin in the pancreatic gland is not explained, however several arguments support the opinion that melatonin is probably implicated in the physiology and pathophysiology of the pancreas. (1) Melatonin stimulates pancreatic enzyme secretion through the activation of entero-pancreatic reflex and cholecystokinin (CCK) release. l-Tryptophan and AFMK are less effective than melatonin in the stimulation of pancreatic exocrine function; (2) Melatonin is a successful pancreatic protector, which prevents the pancreas from developing of acute pancreatitis and reduces pancreatic damage. This effect is related to its direct and indirect antioxidant action, to the strengthening of immune defense, and to the modulation of apoptosis. Like melatonin, its precursor and AFMK are able to mimic its protective effect, and it is commonly accepted that all these substances create an antioxidant cascade to intensify the pancreatic protection and acinar cells viability; (3) In pancreatic cancer cells, melatonin and AFMK activated a signal transduction pathway for apoptosis and stimulated heat shock proteins. The role of melatonin and AFMK in pancreatic tumorigenesis remains to be elucidated.

Low back pain is a common disorder. Pathological innervation and intervertebral disc degeneration are two major factors associated with this disease. Semaphorin 3A, originally known for its potent inhibiting effect on axonal outgrowth, is recently found to correlate with disease activity and histological features in some skeletal disorders. Based on its effects on innervation and vascularization, as well as enzyme secretion, we presume that semaphorin 3A may act as a potential target for low back pain.