Suchergebnisse

The actual problem of experimental medicine is the substantiation of new model organisms that meet modern requirements of bioethics, cost and conditions of detention. The aim of this work was a comparative analysis of the homology degree of proteolytic enzymes in humans and pulmonary freshwater mollusks. The homology of enzymes in nucleotide sequences in humans and pulmonary freshwater mollusks in the analysis of unregulated proteolysis is 66–68 %; regulated proteolysis – 69–76 %; ubiquitin-like modifiers – 78–83 %; extracellular enzymes – 67–76 %; and intracellular enzymes – 65–72 %. The evolutionary conservatism of proteolytic enzymes and the presence of an open blood circulation, which allows the substances under study to be delivered from the hemolymph directly to target cells, make it possible to use these animals as cheap and convenient test organisms. The practical importance of a sufficiently high homology degree of proteolytic enzymes in humans and pulmonary freshwater mollusks justifies the expediency of forming mollusk aquaculture to obtain proteolytic enzyme protein preparations from their tissues within the framework of the tasks of biopharmaceuticals, cosmetics and the food industry.

One: Rearrangement of Prokaryotic Genetic Material and DNA Cloning Vectors -- 1 Genomic Structure and Evolution of Bacillus licheniformis ? and LP52 Phage Family -- 2 Expression of the Synthetic Proenkephalin Gene in E. coli -- 3 Cloning and Expression of Bacillus?-Glucanase Genes -- 4 Regulation of ?-Amylase Synthesis in Bacillus subtilis -- 5 New Variations on an Old Theme: Type I Restriction Enzymes and Their Recognition Sequences -- 6 The EcoDXX1 Restriction and Modification System of Escherichia coli ET7 -- 7 Biological Function of Type I Restriction Enzymes -- 8 The Fosfomycin Resistance Transposon Tn2921 -- 9 Transposition-like Events Mediated by Single-ended Derivatives of Transposon Tn21 -- 10 Cloning and Expression of Determinants Encoding Toxigenicity in Enterobacteria -- 11 Extrachromosomal Replicons in Streptomyces lavendulaegrasserius -- Two: DNA-Dependent RNA Polymerase in Prokaryotes: Structure and Function, Promoter Selection -- 1 Genetic Dissection of E. coli RNA Polymerase -- 2 Non-essential Sequences in the ? Subunit of E. coli RNA Polymerase -- 3 Sequence Analysis of the ? Gene of E. coli RNA Polymerase: Correlation with Structure-Function Studies -- 4 DNA-Dependent RNA Polymerase from Streptomyces granaticolor -- 5 Transcriptional and Translational Signals in Phages PZA and ø29 -- 6 Host-Vector System with the PR' Promoter of Phage Lambda -- 7 On the Difficulties of Defining and Measuring Promoter Strength1 The Case of the Promoters of Bacterial rRNA Genes -- Three: The Role of cAMP in Gene Expression -- 1 Cyclic AMP in Bacteria: Catabolite Repression and Related Effects -- 2 DNA Sequences Involved in Expression and Regulation of deoR-, cytR- and cAMP/CRP-Controlled Genes in Escherichia coli -- 3 Differential Cellular Distribution of Cyclic AMP-Dependent Protein Kinase during Development of Dictyostelium discoideum -- 4 Amplification of the Adenylate Cyclase Gene in Escherichia coli K12 -- 5 Factors Regulating the Activity of Escherichia coli Adenylate Cyclase -- 6 Regulation of Carbohydrate Metabolism by the Bacterial PEP:Sugar Phosphotransferase System -- 7 ATP-Dependent HPr Kinase Involved in Regulation of Carbohydrate Uptake in Gram-positive Bacteria: Inducer Exclusion and Inducer Expulsion -- 8 Regulation of cAMP Synthesis in Streptomyces granaticolor -- 9 Differences in Regulation of a Bacillus megaterium Metalloproteinase during Growth and Sporulation -- Four: Translational Control of Gene Expression -- 1 Ribosomal Protein Sl: "The Messenger RNA-Catching Arm" of Escherichia coli Ribosome -- 2 Initiation of Translation of Genetic Information in Streptomyces aureofaciens -- 3 Bacilli as Hosts for Protein Production -- 4 Structure and Expression of a Chloramphenicol Resistance Gene, cat-86, Cloned from Bacillus pumilus -- 5 An Approach to Controlled Removal of the 3? End of Ribosomal 16S RNA -- 6 Three Ribosomal tRNA-Binding Sites Involved in the Elongation Process -- 7 Structure, Function and Evolution of the 3? Ends of Small Ribosomal Subunit RNA -- 8 Amino Acid Residues In Elongation Factor Tu from Escherichia coli Near the Binding Site for the 3? Terminus of Aminoacyl-tRNA -- 9 Structural and Functional Alterations of Protein-Synthesis Elongation Factor Tu Purified from Streptomyces aureofaciens in an Aggregated State -- 10 Excessive Translational Accuracy Can Inhibit Growth -- 11 Particulate Protein-Synthesis Factors Associated with Translatable mRNA in Mammalian Cells.

DNA repair inhibitors are one of the latest additions to cancer chemotherapy. In general, chemotherapy produces DNA damage but tumoral cells may become resistant if enzymes involved in DNA repair are overexpressed and are able to reverse DNA damage. One of the most successful drugs based on modulating DNA repair are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Several PARP1 inhibitors have been recently developed and approved for clinical treatments. We envisaged that PARP inhibition could be potentiated by simultaneously modulating the expression of PARP 1 and the enzyme activity, by a two-pronged strategy. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter has been recently identified. In this study, we explored the potential binding of clinically approved PARP1 inhibitors to the G-quadruplex structure found at the gene promoter region. The results obtained by NMR, CD, and fluorescence titration confirmed by molecular modeling demonstrated that two out the four PARP1 inhibitors studied are capable of forming defined complexes with the PARP1 G-quadruplex. These results open the possibility of exploring the development of better G-quadruplex binders that, in turn, may also inhibit the enzyme. ; This research was supported by PIANO DI SOSTEGNO ALLA RICERCA 2020—Linea 2 azione B (DEFENS). ICTS Nanbiosis Oligonucleotide Synthesis Platform (CIBER-BBN). Funding from the Spanish Ministerio de Ciencia e Innovación (PID2019-107158GB-I00) and recognition from the Autonomous Catalan government (2017SGR114) are acknowledged. ; Peer reviewed

Nitramines are key constituents of most of the explosives currently in use and consequently contaminate soil and groundwater at many military facilities around the world. Toxicity from nitramine contamination poses a health risk to plants and animals. Thus, understanding how nitramines are biodegraded is critical to environmental remediation. The biodegradation of synthetic nitramine compounds such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been studied for decades, but little is known about the catabolism of naturally produced nitramine compounds. In this study, we report the isolation of a soil bacterium, Variovorax sp. strain JS1663, that degrades N-nitroglycine (NNG), a naturally produced nitramine, and the key enzyme involved in its catabolism. Variovorax sp. JS1663 is a Gram-negative, non-spore-forming motile bacterium isolated from activated sludge based on its ability to use NNG as a sole growth substrate under aerobic conditions. A single gene (nnlA) encodes an iron-dependent enzyme that releases nitrite from NNG through a proposed β-elimination reaction. Bioinformatics analysis of the amino acid sequence of NNG lyase identified a PAS (Per-Arnt-Sim) domain. PAS domains can be associated with heme cofactors and function as signal sensors in signaling proteins. This is the first instance of a PAS domain present in a denitration enzyme. The NNG biodegradation pathway should provide the basis for the identification of other enzymes that cleave the N—N bond and facilitate the development of enzymes to cleave similar bonds in RDX, nitroguanidine, and other nitramine explosives.

Bakteri yang hidup pada tanah mangrove bersifat halofilik yaitu mampu hidup pada lingkungan dengan kondisi kadar garam yang tinggi. Bakteri yang hidup pada zona yang ekstrim memerlukan protein yang tinggi untuk mempertahankan hidup dan dapat melakukan metabolisme, salah satunya dengan membentuk enzim terutama enzim ekstra seluler (enzim amilase, protease, selulase dan lipase). Tujuannya yaitu untuk mengisolasi dan menyeleksi bakteri penghasil enzim ekstraseluler. Metode yang digunakan adalah survey dan eksperimental terhadap isolasi bakteri dari tanah mangrove yang dikarakterisasi berdasarkan morfologi, pewarnaan gram, dan uji aktivitas enzim amilase dengan media amilum, protease dengan media skim milk, selulase dengan media Carboxy Methyl Cellulosed an lipase dengan media tween 80. Analisis data secara deskriptif kuantitatif. Terdapat tiga parameter yang diukur yaitu suhu, salinitas dan pH. Berdasarkan data penelitian ditemukan bahwa tanah mangrove yang ada di pantai Noelbaki menghasilkan 70 isolat bakteri penghasil enzim ekstra seluler. 33 isolat bakteri memiliki aktifitas enzim amilase, 20 isolat memiliki aktifitas enzim proteolitik, 56 isolat memiliki aktifitas selulolitik,10 isolat memiliki aktifitas lipolitik, sehingga total isolat yang memproduksi enzim ekstraseluler adalah 119 isolat, hal ini menjelaskan bahwa beberapa isolat memiliki kemampuan menghasilkan enzim multi-aktifitas atau mampu menghasilkan beberapa enzim.ISOLATION AND SELECTION OF BACTERIA PRODUCING EXTRACELLULAR ENZYMES ON MANGROVE LAND OF NOELBAKI BEACH. Bacteria living in the halofilic mangrove land are able to live in environments that are high salinity. Bacteria that live in the extreme zone require high proteins to sustain life and can metabolize, one by forming enzymes especially extra-cellular enzymes (amilase, protease, selulase and lipase). The aim was to isolate and select extra-cellular enzyme bacteria. This method was a survey and experimental against the insulation of mangrove land controlled by morphology, dyeing grams, and testing for amilase enzyme activity for amilum media, protease with skim milk, selulase with medium carboxy methyl cellulose and lipase in tween media. Quantitative descriptive analysis of data. Three parameters, measured temperature, salinity and ph. Based on research data, were found that mangrove soils located on the coast of noelbaki produced 70 insulations of extreme enzyme yielding bacteria. 33 bacterial isolates have activity in amilase enzyme, 20 isolates have protein activity, 56 isolates had activity cellulase enzyme, 10 isolates had activities lipase enzyme, that include 119 isolates which produced extra-cellular enzymes, which made it clear that some isolates had the ability to create a multi-activity enzyme.

Military personnel and athletes exposed to traumatic brain injury may develop chronic traumatic encephalopathy (CTE). Brain pathology in CTE includes intracellular accumulation of abnormally phosphorylated tau proteins (p-tau), the main constituent of neurofibrillary tangles (NFTs). Recently, we found that cholinergic basal forebrain (CBF) neurons within the nucleus basalis of Meynert (nbM), which provide the major cholinergic innervation to the cortex, display an increased number of NFTs across the pathological stages of CTE. However, molecular mechanisms underlying nbM neurodegeneration in the context of CTE pathology remain unknown. Here, we assessed the genetic signature of nbM neurons containing the p-tau pretangle maker pS422 from CTE subjects who came to autopsy and received a neuropathological CTE staging assessment (Stages II, III, and IV) using laser capture microdissection and custom-designed microarray analysis. Quantitative analysis revealed dysregulation of key genes in several gene ontology groups between CTE stages. Specifically, downregulation of the nicotinic cholinergic receptor subunit β-2 gene (CHRNB2), monoaminergic enzymes catechol-O-methyltransferase (COMT) and dopa decarboxylase (DDC), chloride channels CLCN4 and CLCN5, scaffolding protein caveolin 1 (CAV1), cortical development/cytoskeleton element lissencephaly 1 (LIS1), and intracellular signaling cascade member adenylate cyclase 3 (ADCY3) was observed in pS422-immunreactive nbM neurons in CTE patients. By contrast, upregulation of calpain 2 (CAPN2) and microtubule-associated protein 2 (MAP2) transcript levels was found in Stage IV CTE patients. These single-population data in vulnerable neurons indicate alterations in gene expression associated with neurotransmission, signal transduction, the cytoskeleton, cell survival/death signaling, and microtubule dynamics, suggesting novel molecular pathways to target for drug discovery in CTE.

1 Introduction -- Nomenclature of multiple forms of enzymes. Numbering of isoenzymes -- 2 Techniques for the Separation of Isoenzymes -- Preparation of materials for isoenzyme separation. Electrophoretic techniques: general considerations; paper electrophoresis; starch-block electrophoresis; cellulose acetate electrophoresis; agar-gel electrophoresis; starch-gel electrophoresis; polyacrylamide-gel electrophoresis; column electrophoresis; iso-electric focusing. Chromatographic techniques:ion-exchange celluloses. Gel filtration -- 3 Detection and Determination of Isoenzymes -- Elution techniques: NAD- and NADP-dependent dehydrogenases; esterases; peroxidases. Detection of isoenzymes in electrophoretic media: 'enzymoelectrophoresis'; tetrazoliumstaining techniques; 'nothing dehydrogenase' effect; staining techniques for esterases -- 4 The Chemical Nature of Isoenzymes -- Sub-unit structure: lactate dehydrogenase; creatine kinase;aldolase; glucose 6-phosphate dehydrogenase; tryptophan synthetase; catalase; caeruloplasmin. Presence of charged groups in isoenzyme molecules: alkaline phosphatase. Polymerization as the source of enzyme heterogeneity: cholinesterases; amylase. Conformational isomerism: malate dehydrogenase. Differences in amino-acid sequence -- 5 Enzyme Multiplicity in the Glycolytic Pathway and the Pentose—Phosphate Cycle -- Hexokinase: detection of hexokinase after zone electrophoresis; hexokinase isoenzymes in the erythrocyte; hexokinase in other tissues. Phosphoglucomutase. Glucose 6-phosphate dehydrogenase: demonstration of glucose 6-phosphate dehydrogenase heterogeneity; chemical structure and properties of glucose 6-phosphate dehydrogenase; genetic variants of human erythrocytic glucose 6-phosphate dehydrogenase; glucose 6-phosphate dehydrogenase heterogeneity in other tissues. 6-Phosphogluconate dehydrogenase: heterogeneity in human erythrocytes; 6-phosphogluconate dehydrogenase isoenzymes in other species; structure of 6-phosphogluconate dehydrogenase isoenzymes. Phosphoglucose isomerase. Aldolase: classification of the aldolases;electrophoretic studies; fructose 1-phosphate aldolase. Glyceraldehyde 3-phosphate dehydrogenase. ?-Glycerophosphate dehydrogenase. Phosphopyruvate hydratase (enolase). Pyruvate kinase -- 6 Lactate Dehydrogenase Isoenzymes -- Distribution. Chemical and kinetic properties: substrate specificities and affinities; effect of pH variation; effects of inhibitors; reactions with coenzyme analogues; effect of temperature. Lactate dehydrogenase isoenzymes in developing tissues. Genetic variants of lactate dehydrogenase isoenzymes. Diagnostic applications of lactate dehydrogenase isoenzymes:myocardial infarction; liver diseases; malignant diseases;diseases of muscle; diseases of joints: anaemia; pregnancy;renal diseases; non-electrophoretic diagnostic techniques:serum 2-hydroxybutyrate dehydrogenase in diagnosis; relative heat-stability test; solvent-precipitation techniques; selective absorption techniques. Summary -- 7 Multiple Molecular Forms of Other Oxidoreductases -- Malate dehydrogenase: mitochondrial and supernatant malate dehydrogenase isoenzymes; biological significance of malate dehydrogenase isoenzymes; malate dehydrogenase isoenzymes in mammalian tissues; malate dehydrogenase isoenzymes in insect development; malate dehydrogenase isoenzymes of the chicken embryo. Isocitrate dehydrogenase: isocitrate dehydrogenase isoenzymes in heart and liver diseases. Glutamate dehydrogenase. Alcohol dehydrogenase: alcohol dehydrogenases of insects; avian alcohol dehydrogenases; alcohol dehydrogenases in mammalian liver; alcohol dehydrogenase polymorphism in plants. Xanthine dehydrogenase. Miscellaneous dehydrogenases -- 8 Aminotransferase (Transaminase) Isoenzymes -- Separation of aspartate aminotransferase isoenzymes. Properties of aspartate aminotransferase isoenzymes. Clinical applications of aspartate aminotransferase isoenzymes. Aspartate aminotransferase isoenzymes in developing tissues. Alanine aminotransferase -- 9 Phosphatase Isoenzymes -- Alkaline phosphatase: distribution, paper electrophoresis; starch-block electrophoresis; agar-gel electrophoresis; starchgel electrophoresis; acrylamide-gel electrophoresis; electrophoresis on Sephadex G-200; cellulose acetate electrophoresis; ion-exchange chromatography; properties of alkaline phosphatases; substrate affinities; effects of inhibitors; heat inactivation; effect of neuraminidase; summary of catalytic properties of tissue alkaline phosphatases; immunochemical reactions; genetic studies; alkaline phosphatase isoenzymes in bacteria. Acid phosphatase; electrophoretic separation; chromatographic investigations; genetic variants of human acid phosphatases -- 10 Multiple Forms of Esterases -- Aliesterases: genetic variations of non-specific esterases; ontogenetic observations. Cholinesterases: plasma cholinesterases; genetic polymorphism of serum cholinesterases; Cholinesterase isoenzymes in other species. Arylesterases:serum arylesterases; tissue esterases. Esterases and isoenzymes -- 11 Miscellaneous Enzymes -- Arylamidase: arylamidase isoenzymes in man; arylamidases in other species. Alanine aminopeptidase. Creatine kinase: creatine kinase isoenzymes. Arginine kinase. Caeruloplasmin (copper oxidase). Catalase. Carbonic anhydrase. Amylase: serum and tissue amylases; urinary amylase; macroamylasaemia. Other enzymes occurring in multiple forms: glycogen Phosphorylase; adenylate kinase; adenosine deaminase; tyrosinase; ribonuclease; deoxyribonucleic acid (DNA) polymerase; peroxidase; hyaluronate lyase (hyaluronidase); ?-glycosidases -- 12 The Biological Significance of Isoenzymes -- Author Index.

Background: The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris.[br/] Results: Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either β-D-xylosidase (EC 3.2.1.37) or α-L-arabinofuranosidase (EC 3.2.1.55) activity, while others displayed the ability to degrade AZCL-xylan or AZCL-β-(1,3)-β-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens.[br/] Conclusions: This study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of biomass within the termite mound, although further investigation will be needed to clarify the complex synergies that might exist between the different microbiomes that constitute the termitosphere of fungus-growing termites. This study exemplifies the power of functional metagenomics for the discovery of biomass-active enzymes and has provided a collection of potentially interesting biocatalysts for further study.

The review describes the history of the development of research on carbohydrasеs conducted at the Department of Chemical Enzymology from the mid-1970s to the present. The results concerning the study of the mechanism and kinetics of the processes of enzymatic conversion of cellulose and renewable plant raw materials under the action of a multi-enzyme cellulases complexes; the role of individual components of these complexes - basic (endoglucanases and cellobiohydrolases) and auxiliary enzymes (polysaccharide monooxygenase, β-glucosidase, xylanase) and their synergistic interaction. The features of using reactors of various designs for bioconversion of plant raw materials are described: periodic type, continuous column type, reactor for hydrolysis in a constant electric field, reactor with intensive mixing by ferromagnetic particles in magnetic field. The possibilities of increasing the reactivity of plant raw materials using various pretreatment methods, as well as the influence of the structural and physico-chemical properties of cellulose on the efficiency of its enzymatic conversion are discussed. Data on the creation of highly active strains of microscopic fungi-producers of cellulases and other carbohydrases using methods of induced mutagenesis - Trichoderma (Hypocrea), Penicillium (Talaromyces), Aspergillus, Chrysosporium (Myceliophtora) spp., as well as data on the composition of the enzyme complexes produced by them and the properties of the enzymes forming them are presented. It describes the creation of expression systems based on P. canescens and P. verruculosum and the production of recombinant producer strains with their help, which made it possible to obtain enzyme preparations (EP) that ensure high efficiency of bioconversion processes of plant raw materials, as well as to create producers of a wide range of carbohydrases for practical use in various fields of industry and agriculture. A number of industrially important EP obtained using the P. verruculosum expression system are currently being produced at the Agroferment LLC plant.

Chantier qualité GA ; Background: The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris.[br/] Results: Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either β-D-xylosidase (EC 3.2.1.37) or α-L-arabinofuranosidase (EC 3.2.1.55) activity, while others displayed the ability to degrade AZCL-xylan or AZCL-β-(1,3)-β-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens.[br/] Conclusions: This study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of ...

Chantier qualité GA ; Background: The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris.[br/] Results: Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either β-D-xylosidase (EC 3.2.1.37) or α-L-arabinofuranosidase (EC 3.2.1.55) activity, while others displayed the ability to degrade AZCL-xylan or AZCL-β-(1,3)-β-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens.[br/] Conclusions: This study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of ...

The aldol reaction is one of the most fundamental stereocontrolled carbon-carbon bond‐forming reactions and is mainly catalyzed by aldolases in nature. Despite the fact that the aldol reaction has been widely proposed to be involved in fungal secondary metabolite biosynthesis, a dedicated aldolase that catalyzes stereoselective aldol reactions has only rarely been reported in fungi. Herein, we activated a cryptic polyketide biosynthetic gene cluster that was upregulated in the fungal wheat pathogen Parastagonospora nodorum during plant infection; this resulted in the production of the phytotoxic stemphyloxin II (1). Through heterologous reconstruction of the biosynthetic pathway and in vitro assay by using cell‐free lysate from Aspergillus nidulans, we demonstrated that a berberine bridge enzyme (BBE)‐like protein SthB catalyzes an intramolecular aldol reaction to establish the bridged tricyclo[6.2.2.02,7]dodecane skeleton in the post‐assembly tailoring step. The characterization of SthB as an aldolase enriches the catalytic toolbox of classic reactions and the functional diversities of the BBE superfamily of enzymes. ; This study was supported by an Australian Research Council (ARC) Discovery Project grant (DP170100228). Y.-H.C. is supported by an ARC Future Fellowship (FT160100233). H.L. and J.H. are supported by an Australian Government International Postgraduate Research Scholarship. NMR and X-ray diffraction data were collected at the CMCA.

This work was funded by The Novo Nordisk Foundation grant to the Center for Biosustainability (NNF10CC1016517). P.I.N. was funded by grants from The Novo Nordisk Foundation (NNF20CC0035580, and LiFe, NNF18OC0034818), the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 814418 (SinFonia) and the Danish Council for Independent Research (SWEET, DFF-Research Project 8021-00039B). T.K. and M.N.D. were funded by fellowships from the European Union's Horizon 2020 research and innovation program under a Marie Skłodowska Curie project under grant agreement No. 713683 (COFUNDfellowsDTU). ; The fluorinase enzyme represents the only biological mechanism capable of forming stable C–F bonds characterized in nature thus far, offering a biotechnological route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis constant (KM) for S-adenosyl-l-methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-l-methionine affinity points to a long-range effect of hexamerization on substrate binding – likely decreasing substrate dissociation and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system. ; Publisher PDF ; Peer reviewed