Browsing by Subject "PURIFICATION"

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  • Kjaerbolling, Inge; Vesth, Tammi; Frisvad, Jens C.; Nybo, Jane L.; Theobald, Sebastian; Kildgaard, Sara; Petersen, Thomas Isbrandt; Kuo, Alan; Sato, Atsushi; Lyhne, Ellen K.; Kogle, Martin E.; Wiebenga, Ad; Kun, Roland S.; Lubbers, Ronnie J. M.; Makela, Miia R.; Barry, Kerrie; Chovatia, Mansi; Clum, Alicia; Daum, Chris; Haridas, Sajeet; He, Guifen; LaButti, Kurt; Lipzen, Anna; Mondo, Stephen; Pangilinan, Jasmyn; Riley, Robert; Salamov, Asaf; Simmons, Blake A.; Magnuson, Jon K.; Henrissat, Bernard; Mortensen, Uffe H.; Larsen, Thomas O.; de Vries, Ronald P.; Grigoriev, Igor V.; Machida, Masayuki; Baker, Scott E.; Andersen, Mikael R. (2020)
    Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.
  • Vidilaseris, Keni; Kellosalo, Juho; Goldman, Adrian (2018)
    Membrane-bound pyrophosphatases (mPPases) are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates coupled to the active transport of protons or sodium ions across membranes. They occur in bacteria, archaea, plants, and protist parasites. As they are essential in protist parasites and there are no homologous proteins in animals and humans, these enzymes represent an excellent drug target for treating protistal diseases. Experimental screening to find drug candidates is an important step to discover new hit compounds. For that, a cheap, simple, and robust assay is needed. Here we report the application of the molybdenum blue reaction method for a medium throughput microplate activity assay of the hyperthermophilic bacterium Thermotoga maritima mPPase and the possible application of the assay to screen inhibitors of membrane-bound pyrophosphatases.
  • Cecchetti, Cristina; Strauss, Jannik; Stohrer, Claudia; Naylor, Claire; Pryor, Edward; Hobbs, Jeanette; Tanley, Simon; Goldman, Adrian; Byrne, Bernadette (2021)
    Membrane proteins have a range of crucial biological functions and are the target of about 60% of all prescribed drugs. For most studies, they need to be extracted out of the lipid-bilayer, e.g. by detergent solubilisation, leading to the loss of native lipids, which may disturb important protein-lipid/bilayer interactions and thus functional and structural integrity. Relipidation of membrane proteins has proven extremely successful for studying challenging targets, but the identification of suitable lipids can be expensive and laborious. Therefore, we developed a screen to aid the high-throughput identification of beneficial lipids. The screen covers a large lipid space and was designed to be suitable for a range of stability assessment methods. Here, we demonstrate its use as a tool for identifying stabilising lipids for three membrane proteins: a bacterial pyrophosphatase (Tm-PPase), a fungal purine transporter (UapA) and a human GPCR (A(2A)R). A(2A)R is stabilised by cholesteryl hemisuccinate, a lipid well known to stabilise GPCRs, validating the approach. Additionally, our screen also identified a range of new lipids which stabilised our test proteins, providing a starting point for further investigation and demonstrating its value as a novel tool for membrane protein research. The pre-dispensed screen will be made commercially available to the scientific community in future and has a number of potential applications in the field.
  • Pathak, Lakshmi; Singh, Vineeta; Niwas, Ram; Osama, Khwaja; Khan, Saif; Haque, Shafiul; Tripathi, C. K. M.; Mishra, B. N. (2015)
    Cholesterol oxidase (COD) is a bi-functional FAD-containing oxidoreductase which catalyzes the oxidation of cholesterol into 4-cholesten-3-one. The wider biological functions and clinical applications of COD have urged the screening, isolation and characterization of newer microbes from diverse habitats as a source of COD and optimization and over-production of COD for various uses. The practicability of statistical/artificial intelligence techniques, such as response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) have been tested to optimize the medium composition for the production of COD from novel strain Streptomyces sp. NCIM5500. All experiments were performed according to the five factor central composite design (CCD) and the generated data was analysed using RSM and ANN. GA was employed to optimize the models generated by RSM and ANN. Based upon the predicted COD concentration, the model developed with ANN was found to be superior to the model developed with RSM. The RSM-GA approach predicted maximum of 6.283 U/mL COD production, whereas the ANN-GA approach predicted a maximum of 9.93 U/mL COD concentration. The optimum concentrations of the medium variables predicted through ANN-GA approach were: 1.431 g/50 mL soybean, 1.389 g/50 mL maltose, 0.029 g/50 mL MgSO4, 0.45 g/50 mL NaCl and 2.235 ml/50 mL glycerol. The experimental COD concentration was concurrent with the GA predicted yield and led to 9.75 U/mL COD production, which was nearly two times higher than the yield (4.2 U/mL) obtained with the un-optimized medium. This is the very first time we are reporting the statistical versus artificial intelligence based modeling and optimization of COD production by Streptomyces sp. NCIM 5500.
  • Kögler, Martin; Itkonen, Jaakko; Viitala, Tapani; Casteleijn, Marco G. (2020)
    Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time in order to boost the Raman signal, while simultaneously suppressing background signals. We evaluated the expression of hCNTF, hHspA1, and hHsp27 in complex media using the batch fermentation mode. HCNTF was also cultivated using EnBase in a fed-batch like mode. HspA1 expressed poorly due to aggregation problems within the cell, while hCNTF expressed in batch mode was correctly folded and protein instabilities were identified in the EnBase cultivation. Time-gated Raman spectroscopy showed to be a powerful tool to evaluate protein production and correct folding within living E. coli cells during the cultivation.
  • Rizzello, Carlo G.; Coda, Rossana; Wang, Yaqin; Verni, Michela; Kajala, Ilkka; Katina, Kati; Laitila, Arja (2019)
    The interest towards legumes in food applications has risen over the past decades. However, the presence of antinutritional factors (ANF) and the poor technological performances still restricts their application in food fortification. In this study, four lactic acid bacteria (LAB) isolated from faba bean were applied as starter cultures for faba bean bioprocessing. None of the strains employed produced exopolysaccharides from raffinose, on the contrary, they did with sucrose as substrate. The fermented doughs were characterized and the strains were compared for their adaptation capacity and metabolic performance including the formation of dextrans, the degradation of ANF and the ability to improve antioxidant activity and in vitro protein digestibility (IVPD). A contribution to the proteolysis was given by the presence of endogenous enzymes, responsible for the increase of peptides and amino acids in dough from irradiated flour. However, the LAB strains further enhanced proteolysis. Weissella cibaria VTT E-153485 led to the highest peptide release and consequentially to the highest IVPD. In doughs fermented with Pediococcus pentosaceus VTT E-153483 and Leuconostoc kimchi VTT E-153484, phytic acid was reduced to more than half the initial concentration. Inoculated doughs had significantly lower content of oligosaccharides after 24 h of incubation compared to the controls. The most efficient raffinose consumption was found for Leuc. kimchi and W. cibaria. Doughs inoculated with weissellas contained > 1% of dextrans. Weissella confusa VTT E-143403 induced a significant increment in viscosity (ca. 7 times higher than the controls). This study revealed that well-characterized, indigenous LAB provided beneficial biotechnological features in faba bean dough processing and contributed to its implementation in the food production.
  • Nivala, Outi; Faccio, Greta; Arvas, Mikko; Permi, Perttu; Buchert, Johanna; Kruus, Kristiina; Mattinen, Maija-Liisa (2017)
    Background: Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear. Results: The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initial activity was retained after incubation at 50 degrees C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide-and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant. Conclusions: AtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides.
  • Ran, Li; Tan, Xiaoqian; Li, Shuhong; Jin, Yu; Li, Song; Li, Shulei; Takala, Timo; Saris, Per (2021)
    Cystatins are proteins, which inhibit cysteine proteases, such as papain. In this study, the 336-bp cystatin C gene (family II, HmCysC) of silver carp (Hypophthalmichthys molitrix) was cloned and expressed in Escherichia coli BL21 (DE3). HmCysC encodes the mature peptide of cystatin C (HmCystatin C), with 111 amino acids. A typical QXXXG motif was found in HmCystatin C and it formed a cluster with Cyprinus carpio and Danio rerio cystatin C in the phylogenetic tree. Quantitative real-time polymerase chain reaction analysis indicated that HmCysC was transcribed at different levels in five tested tissues of silver carp. Following purification with Ni2+– nitrilotriacetic acid agarose affinity chromatography, HmCystatin C displayed a molecular weight of 20 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified HmCystatin C had strong inhibitory effects toward the proteolytic activity of papain. Immunochemical staining with anti-HmCystatin C antibody showed that HmCystatin C was widely distributed in silver carp tissues. These results collectively demonstrated the properties of HmCystatin C, providing information for further studies of cystatins from fish organisms.
  • Sah-Teli, Shiv K.; Hynönen, Mikko J.; Schmitz, Werner; Geraets, James A.; Seitsonen, Jani; Pedersen, Jan Skov; Butcher, Sarah J.; Wierenga, Rik K.; Venkatesan, Rajaram (2019)
    The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid beta-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.
  • Yegutkin, Gennady G.; Auvinen, Kaisa; Karikoski, Marika; Rantakari, Pia; Gerke, Heidi; Elima, Kati; Maksimow, Mikael; Quintero, Ileana B.; Vihko, Pirkko; Salmi, Marko; Jalkanen, Sirpa (2014)
  • Megta, Abhin Kumar; Pratap, Shivendra; Kant, Abhiruchi; Palva, Airi; von Ossowski, Ingemar; Krishnan, Vengadesan (2020)
    To successfully colonize a host or environment, certain genera and species of Gram-positive bacteria have evolved to utilize the so-called sortase-dependent pilus, a long multi-subunit and non-flagellar surface adhesin. One example of this is Lactobacillus rhamnosus GG, a gut-adapted probiotic strain that produces SpaCBA pili. These structures are covalent hetero-oligomers built from three types of pilin subunit, each with a specific location and function (i.e., backbone SpaA for length, tip SpaC for adhesion, and basal SpaB for anchoring). Functionally, the SpaCBA pilus exhibits a promiscuous affinity for components on intestinal surfaces (e.g., mucus, collagen, and epithelial cells), which is largely attributed to the SpaC subunit. Then again, the basal SpaB pilin, in addition to acting as the terminal subunit during pilus assembly, displays an out of character mucoadhesive function. To address the structural basis of this unusual dual functionality, we reveal the 2.39 A resolution crystal structure of SpaB. SpaB consists of one immunoglobulin-like CnaB domain and contains a putative intermolecular isopeptide bond-linking lysine and internal isopeptide bond-asparagine in an FPKN pilin motif within the C-terminal end. Remarkably, we found that a C-terminal stretch of positively charged lysine and arginine residues likely accounts for the atypical mucoadhesiveness of SpaB. Although harboring an autocatalytic triad of residues for a potential internal isopeptide interaction, the SpaB crystal structure lacked the visible electron density for intact bond formation, yet its presence was subsequently confirmed by mass spectral analysis. Finally, we propose a structural model that captures the exclusive basal positioning of SpaB in the SpaCBA pilus.
  • Mishra, Arjun K.; Megta, Abhin Kumar; Palva, Airi; von Ossowski, Ingemar; Krishnan, Vengadesan (2017)
    SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensal Lactobacillus rhamnosus GG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria, L. rhamnosus GG SpaE for crystallization was produced by recombinant expression in Escherichia coli. Although several attempts to crystallize the SpaE protein were unsuccessful, trigonal crystals that diffracted to a resolution of 3.1 angstrom were eventually produced using PEG 3350 as a precipitant and high protein concentrations. Further optimization with a combination of additives led to the generation of SpaE crystals in an orthorhombic form that diffracted to a higher resolution of 1.5 angstrom. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 angstrom resolution.
  • Jiang, Guangyang; He, Juan; Gan, Longzhan; Li, Xiaoguang; Xu, Zhe; Yang, Li; Li, Ran; Tian, Yongqiang (2022)
    The novel exopolysaccharide EPS-E8, secreted by Pediococcus pentosaceus E8, was obtained by anion-exchange and gel filtration chromatography. Structural analyses identified EPS-E8 as a heteropolysaccharide containing mannose, glucose, and galactose. Its major backbone consists of -> 2)-alpha-D-Manp-(1 -> 2,6)-alpha-D-Glcp-(1 -> 6)-alpha-D-Manp-(1 ->, and its molecular weight is 5.02 x 10(4) g/mol. Using atomic force microscopy and scanning electron microscopy, many spherical and irregular reticular-like shapes were observed in the microstructure of EPS-E8. EPS-E8 has outstanding thermal stability (305.7 degrees C). Both the zeta potential absolute value and average particle diameter increased gradually with increasing concentration. Moreover, at a concentration of 10 mg/ml, the antioxidant capacities of, 1-Diphenyl-2-picrylhydrazyl (DPPH), ABTS and hydroxyl radical were 50.62 +/- 0.5%, 52.17 +/- 1.4%, and 58.91 +/- 0.7%, respectively. EPS-E8 possesses excellent emulsifying properties against several food-grade oils, and its activity is retained under various conditions (temperature, pH, and ionic strength). Finally, we found that EPS-E8 as a polysaccharide-based coating could reduce the weight loss and malondialdehyde (MDA) content of strawberry, as well as preserving the vitamin C and soluble solid content during storage at 20 degrees C. Together, the results support the potential application of EPS-E8 as an emulsifier, and a polysaccharide-based coating in fruit preservation.
  • Dilokpimol, Adiphol; Mäkelä, Miia R.; Mansouri, Sadegh; Belova, Olga; Waterstraat, Martin; Bunzel, Mirko; de Vries, Ronald P.; Hilden, Kristiina S. (2017)
    A feruloyl esterase (FAE) from Aspergillus niger N402, FaeC was heterologously produced in Pichia pastoris X-33 in a yield of 10 mg/L. FaeC was most active at pH 7.0 and 50 degrees C, and showed broad substrate specificity and catalyzed the hydrolysis of methyl 3,4-dimethoxycinnamate, ethyl ferulate, methyl ferulate, methyl p-coumarate, ethyl coumarate, methyl sinapate, and methyl caffeate. The enzyme released both ferulic acid and p-coumaric acid from wheat arabinoxylan and sugar beet pectin (up to 3 mg/g polysaccharide), and acted synergistically with a commercial xylanase increasing the release of ferulic acid up to six-fold. The expression of faeC increased over time in the presence of feruloylated polysaccharides. Cinnamic, syringic, caffeic, vanillic and ferulic acid induced the expression of faeC. Overall expression of faeC was very low in all tested conditions, compared to two other A. niger FAE encoding genes, faeA and faeB. Our data showed that the fae genes responded differently towards the feruloylated polysaccharides and tested monomeric phenolic compounds suggesting that the corresponding FAE isoenzymes may target different substrates in a complementary manner. This may increase the efficiency of the degradation of diverse plant biomass. (C) 2017 Elsevier B.V. All rights reserved.
  • Dilokpimol, Adiphol; Mäkelä, Miia Riitta; Varriale, Simona; Zhou, Miaomiao; Cerullo, Gabriella; Gidijala, Loknath; Hinkka, Harri Tapio; Brás, Joana L.A.; Jütten, Peter; Piechot, Alexander; Verhaert, Raymond; Hilden, Sari Kristiina; Faraco, Vincenza; de Vries, Ronald (2018)
    Feruloyl esterases (FAEs) are a diverse group of enzymes that specifically catalyze the hydrolysis of ester bonds between a hydroxycinnamic (e.g. ferulic) acid and plant poly- or oligosaccharides. FAEs as auxiliary enzymes significantly assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass saccharification for biofuel and biochemical production. A limited number of FAEs have been functionally characterized compared to over 1000 putative fungal FAEs that were recently predicted by similarity-based genome mining, which divided phylogenetically into different subfamilies (SFs). In this study, 27 putative and six characterized FAEs from both ascomycete and basidiomycete fungi were selected and heterologously expressed in Pichia pastoris and the recombinant proteins biochemically characterized to validate the previous genome mining and phylogenetical grouping and to expand the information on activity of fungal FAEs. As a result, 20 enzymes were shown to possess FAE activity, being active towards pNP-ferulate and/or methyl hydroxycinnamate substrates, and covering 11 subfamilies. Most of the new FAEs showed activities comparable to those of previously characterized fungal FAEs.
  • Sun, Peicheng; Li, Xinxin; Dilokpimol, Adiphol; Henrissat, Bernard; de Vries, Ronald P.; Kabel, Mirjam A.; Mäkelä, Miia R. (2022)
    Xyloglucan is a prominent matrix heteropolysaccharide binding to cellulose microfibrils in primary plant cellwalls. Hence, the hydrolysis of xyloglucan facilitates the overall lignocellulosic biomass degradation. Xyloglucanases (XEGs) are key enzymes classified in several glycoside hydrolase (GH) families. So far, family GH44 has been shown to contain bacterial XEGs only. Detailed genome analysis revealed GH44 members in fungal species from the phylum Basidiomycota, but not in other fungi, which we hypothesized to also be XEGs. Two GH44 enzymes from Dichomitus squalens and Pleurotus ostreatus were heterologously produced and characterized. They exhibited XEG activity and displayed a hydrolytic cleavage pattern different fromthat observed in fungal XEGs from other GH families. Specifically, the fungal GH44 XEGs were not hindered by substitution of neighboring glucosyl units and generated various," "XXXG- type,'' "GXXX(G)-type,'' and "XXX-type'' oligosaccharides. Overall, these fungal GH44 XEGs represent a novel class of enzymes for plant biomass conversion and valorization.
  • Urquhart, Andrew; Mondo, Stephen; Mäkelä, Miia Riitta; Hane, James; Wiebenga, Ad; He, Guifen; Mihaltcheva, Sirma; Pangilinan, Jasmyn; Lipzen, Anna; Barry, Kerrie; de Vries, Ronald; Grigoriev, Igor V.; Idnurm, Alexander (2018)
    Species in the genus Paecilomyces, a member of the fungal order Eurotiales, are ubiquitous in nature and impact a variety of human endeavors. Here, the biology of one common species, Paecilomyces variotii, was explored using genomics and functional genetics. Sequencing the genome of two isolates revealed key genome and gene features in this species. A striking feature of the genome was the two-part nature, featuring large stretches of DNA with normal GC content separated by AT-rich regions, a hallmark of many plant-pathogenic fungal genomes. These AT-rich regions appeared to have been mutated by repeat-induced point (RIP) mutations. We developed methods for genetic transformation of P. variotii, including forward and reverse genetics as well as crossing techniques. Using transformation and crossing, RIP activity was identified, demonstrating for the first time that RIP is an active process within the order Eurotiales. A consequence of RIP is likely reflected by a reduction in numbers of genes within gene families, such as in cell wall degradation, and reflected by growth limitations on P. variotii on diverse carbon sources. Furthermore, using these transformation tools we characterized a conserved protein containing a domain of unknown function (DUF1212) and discovered it is involved in pigmentation.
  • Adams, Michael P.; Atanasova, Nina S.; Sofieva, Svetlana; Ravantti, Janne; Heikkinen, Aino; Brasseur, Zoe; Duplissy, Jonathan; Bamford, Dennis H.; Murray, Benjamin J. (2021)
    In order to effectively predict the formation of ice in clouds we need to know which subsets of aerosol particles are effective at nucleating ice, how they are distributed and where they are from. A large proportion of ice-nucleating particles (INPs) in many locations are likely of biological origin, and some INPs are extremely small, being just tens of nanometres in size. The identity and sources of such INPs are not well characterized. Here, we show that several different types of virus particles can nucleate ice, with up to about 1 in 20 million virus particles able to nucleate ice at -20 degrees C. In terms of the impact on cloud glaciation, the ice-nucleating ability (the fraction which are ice nucleation active as a function of temperature) taken together with typical virus particle concentrations in the atmosphere leads to the conclusion that virus particles make a minor contribution to the atmospheric ice-nucleating particle population in the terrestrial-influenced atmosphere. However, they cannot be ruled out as being important in the remote marine atmosphere. It is striking that virus particles have an ice-nucleating activity, and further work should be done to explore other types of viruses for both their ice-nucleating potential and to understand the mechanism by which viruses nucleate ice.
  • Pinto de Magalhães, Joana; Franko, Nina; Raboni, Samanta; Annunziato, Giannamaria; Tammela, Päivi; Bruno, Agostino; Bettati, Stefano; Mozzarelli, Andrea; Pieroni, Marco; Cambanini, Barbara; Costantino, Gabriele (2020)
    In Upsilon-proteobacteria and Actinomycetales, cysteine biosynthetic enzymes are indispensable during persistence and become dispensable during growth or acute infection. The biosynthetic machinery required to convert inorganic sulfur into cysteine is absent in mammals; therefore, it is a suitable drug target. We searched for inhibitors of Salmonella serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The virtual screening of three ChemDiv focused libraries containing 91 243 compounds was performed to identify potential SAT inhibitors. Scaffold similarity and the analysis of the overall physicochemical properties allowed the selection of 73 compounds that were purchased and evaluated on the recombinant enzyme. Six compounds displaying an IC50