Partial purification and characterization of a broad-spectrum bacteriocin produced by a Lactobacillus plantarum zrx03 isolated from infant's feces
Lei, Shuang ; Zhao, Ruixiang ; Sun, Junliang ; Ran, Junjian ; Ruan, Xiaoli ; Zhu, Yang - \ 2020
Food Science and Nutrition 8 (2020)5. - ISSN 2048-7177 - p. 2214 - 2222.
antimicrobial stability - bacteriocin - Lactobacillus plantarum zrx03 - purification
Lactobacillus plantarum zrx03 was a bacteriocin-producing strain isolated from infant's feces. The fermentation supernatant produced by this strain could strongly inhibit Escherichia coli JM109 ATCC 67387, Staphylococcus aureus ATCC 25923, and Listeria monocytogenes CICC 21633, in which the diameter of inhibition zone was 12.83 ± 0.62 mm, 15.08 ± 0.31 mm, 6.75 ± 0.20 mm, respectively, compared with lactic acid bacteria N1, N2, M13, M21, M31, and M37. According to amplification of 16S rRNA gene and identification of phylogenetic tree, this strain had a 1,450 bp sequence and 100% identity to the L. plantarum strain. Based on the influence of different protease treatments, such as pepsin, trypsin, papain, and proteinase K on the antimicrobial activity, this antimicrobial substance was considered to be a natural protein. Using bacteriocin produced by this strain as study object of this experiment, it had been extracted from ammonium sulfate precipitation and different organic solvents. The results showed that ethyl acetate was selected as the optimal solution to crude extraction of bacteriocin after comparing ammonium sulfate precipitation method and organic solvent extraction method, such as n-butanol, n-hexane, dichloromethane, trichloromethane, in which the diameter of the inhibition zones was above 28 mm. Results also showed the inhibition spectrum of the obtained bacteriocin had a broad spectrum of inhibition which could inhibit Gram-positive, Gram-negative, yeast. Especially, it could effectively inhibit S. aureus ATCC 25923, Bacillus subtilis CICC 10002, Bacillus anthracis CICC 20443, E. coli JM109 ATCC 67387, and Salmonella CMCC 541, and the zone diameter of inhibition has reached more than 28 mm. Moreover, it had a good thermal stability which antibacterial activity was retained 70.58% after treatment at 121°C for 30 min, and pH-stability was between pH 2.0–9.0. These results suggested bacteriocin produced by L. plantarum zrx03 had potential application prospects in food preservation.
Mobiele waterzuivering glastuinbouw
Ruijven, Jim van; Os, Erik van; Vermeulen, Peter - \ 2017
Bleiswijk : Wageningen University & Research, BU Glastuinbouw (Rapport GTB 1424) - 54
kassen - glastuinbouw - afvalwaterbehandeling - afvalwater - waterverontreiniging - afvoer - zuiveren - waterzuivering - oppervlaktewater - oppervlaktewaterkwaliteit - gewasbescherming - pesticiden - substraten - cultuur zonder grond - mobiele uitrusting - greenhouses - greenhouse horticulture - waste water treatment - waste water - water pollution - discharge - purification - water treatment - surface water - surface water quality - plant protection - pesticides - substrates - soilless culture - mobile equipment
Application of a mobile unit for discharge water purification is one of four options to apply to the purification obligation per 1-1-2018. Depending on the amount of discharge, future water strategy and investment options, mobile purification can be an interesting option. The amount of discharge water varies with crop, irrigation strategy and quality of the irrigation water and is between 122 and 3.340 m3/ha/year for surveyed companies. About 65% of greenhouse companies discharges
Ruijven, J. van; Os, E. van; Beerling, E. ; Staaij, M. van der - \ 2016
Bleiswijk : Wageningen UR Glastuinbouw (Rapport GTB 1419) - 42
kasgewassen - kassen - glastuinbouw - afvoer - zuiveren - gewasbescherming - ozon - greenhouse crops - greenhouses - greenhouse horticulture - discharge - purification - plant protection - ozone
To apply to the Dutch generic obligation to purify discharge water, each horticultural company needs to treat it’s discharge water with a technology that removes 95% of plant protection products. This report shows the process that growers need to go through to make a good choice for a purification technology: mapping of water flows, decrease the amount of discharge water, determine the strategy to apply to the generic obligation and make a choice for a purification technology. For a cucumber production company and a company that combines vegetable plant propagation and growth of potted plants this process is followed. A design for a purification system is developed and built for a semi-practice scale cucumber and sweet pepper production system. In the semi-practice scale and the cucumber production company, an ozone installation (Agrozone) is chosen, either to disinfect drain water and eventually purify discharge water. At the propagation company, an Opticlear Diamond (WaterIQ) is chosen to disinfect the drain water and eventually purify discharge water. For both installations the purification efficacy is measured. In the second part of the project the practical format for the generic obligation for discharge water purification is elaborated.
Extraction of steviol glycosides from fresh Stevia using acidified water; clarification followed by ultrafiltration and nanofiltration
Kootstra, A.M.J. ; Elissen, H.J.H. ; Huurman, Sander - \ 2016
Lelystad : Wageningen UR, PPO/Acrres (Rapport / PPO-AGV 686) - 38
separation technology - stevia - purification - biorefinery - glycosides - ultrafiltration - filtration - scheidingstechnologie - stevia - zuiveren - bioraffinage - glycosiden - ultrafiltratie - filtratie
As part of the PPS Kleinschalige bioraffinage project (WP1b), fresh Stevia material was used in the extraction of steviol glycosides using water acidified through conversion of sugar by microorganisms naturally present on the plant. Two successive harvests from the same plot were used. Previous experiments had resulted in high steviol glycoside extraction rates of 80 % to 90 % but the purity of the final extract was low (15 % to 20 % of steviol glycosides in the dry matter). The first batch of plants was used to test a clarification step by filtration on a small scale. A second batch of plants was used to perform clarification, purification using ultrafiltration, and concentration by nanofiltration on a larger scale.
Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments
Jurak, E. ; Patyshakuliyeva, A. ; Vries, R.P. de; Gruppen, H. ; Kabel, M.A. - \ 2015
PLoS ONE 10 (2015)8. - ISSN 1932-6203
wheat-flour arabinoxylan - h-1-nmr spectroscopy - aspergillus-awamori - enzyme-activities - button mushroom - mode - oligosaccharides - purification
The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, ß-xylosidase and ß-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and a-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments.
High yields of active Thermus thermophilus proline dehydrogenase are obtained using maltose-binding protein as a solubility tag.
Huijbers, M.M.E. ; Berkel, W.J.H. van - \ 2015
Biotechnology Journal 10 (2015)3. - ISSN 1860-6768 - p. 395 - 403.
multifunctional puta flavoprotein - escherichia-coli - purification - domain - stress - biosynthesis - oxidase - crystallization - overexpression - identification
Proline dehydrogenase (ProDH) catalyzes the FAD-dependent oxidation of proline to ¿1-pyrroline-5-carboxylate, the first step of proline catabolism in many organisms. Next to being involved in a number of physiological processes, ProDH is of interest for practical applications because the proline imino acid can serve as a building block for a wide range of peptides and antibiotics. ProDH is a membrane-associated protein and recombinant soluble forms of the enzyme have only been obtained in limited amounts. We here report on the heterologous production of ProDH from Thermus thermophilus (TtProDH) in Escherichia coli. Using maltose-binding protein as solubility tag, high yields of active holoenzyme are obtained. Native TtProDH can be produced from cleaving the purified fusion protein with trypsin. Size-exclusion chromatography shows that fused and clipped TtProDH form oligomers. Thermal stability and co-solvent tolerance indicate the conformational robustness of TtProDH. These properties together with the high yield make TtProDH attractive for industrial applications.
Encapsulation of GFP in complex coacervate core micelles
Nolles, A. ; Westphal, A.H. ; Hoop, J.A. de; Fokkink, R.G. ; Kleijn, J.M. ; Berkel, W.J.H. van; Borst, J.W. - \ 2015
Biomacromolecules 16 (2015)5. - ISSN 1525-7797 - p. 1542 - 1549.
fluorescence correlation spectroscopy - protein - dynamics - behavior - nanocontainers - purification - copolymers - lipase - tag
Protein encapsulation with polymers has a high potential for drug delivery, enzyme protection and stabilization. Formation of such structures can be achieved by the use of polyelectrolytes to generate so-called complex coacervate core micelles (C3Ms). Here, encapsulation of enhanced green fluorescent protein (EGFP) was investigated using a cationic-neutral diblock copolymer of two different sizes: poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205 and poly(2-methyl-vinyl-pyridinium)128-b-poly(ethylene-oxide)477. Dynamic light scattering and fluorescence correlation spectroscopy (FCS) revealed a preferred micellar composition (PMC) with a positive charge composition of 0.65 for both diblock copolymers and micellar hydrodynamic radii of approximately 34 nm. FCS data show that at the PMC, C3Ms are formed above 100 nM EGFP, independent of polymer length. Mixtures of EGFP and nonfluorescent GFP were used to quantify the amount of GFP molecules per C3M, resulting in approximately 450 GFPs encapsulated per micelle. This study shows that FCS can be successfully applied for the characterization of protein-containing C3Ms.
New analytical approaches for faster or greener phytochemical analyses
Shen, Y. - \ 2015
Wageningen University. Promotor(en): Han Zuilhof; B. Chen, co-promotor(en): Teris van Beek. - Wageningen : Wageningen University - ISBN 9789462573307 - 206
giftige planten - chemische samenstelling - massaspectrometrie - niet-destructief testen - bemonsteren - zuiveren - oplosmiddelen - illicium - poisonous plants - chemical composition - mass spectrometry - nondestructive testing - sampling - purification - solvents - illicium
Chapter 1 provides a short introduction into the constraints of phytochemical analysis. In order to make them faster, less laborious and greener, there is a clear scope for miniaturized and simplified sample preparation, solvent-free extractions and the use of cleaner solvents in preparative HPLC. Possible modern techniques to achieve this, such as microfluidic chips, ambient mass spectrometry, selective magnetic nanoparticles, and use of less toxic but equally efficient solvents are discussed. Clear aims were formulated and research towards fulfilling these aims in the field of phytochemical analysis is carried out in this thesis.
A first version of a 3-phase liquid-liquid extraction (LLE) chip for the miniaturized sample pretreatment of alkaloids was introduced by our group in 2009. In Chapter 2 more biodegradable and less-toxic solvents for the transport phase and a more suitable pH for the feed phase were evaluated. The extraction efficiency improved. On-line hyphenation of the 3-phase chip to nanoLC-UV/MS was also investigated. This combination saved a lot of time and solvent in comparison with traditional methods for the purification of alkaloids from plant materials.
A novel Induced Phase Separation Extraction (IPSE) chip was introduced in Chapter 3 for efficient sample pretreatment. The acetonitrile – water (1:1) sample solutions were separated in organic and aqueous phases in this IPSE chip based on their affinity for both phases. In turn this could be correlated with the log D values of the analytes. Some optimization regarding design, operation, flows and solvents was carried out. Extraction efficiencies of several model compounds were determined. A real sample application with a plant used in Traditional Chinese Medicines (TCMs) was carried out to show the usefulness of the IPSE chip in dealing with complex matrixes.
Chapter 4 presented an unambiguous distinction between toxic Japanese star anise and non-toxic Chinese star anise fruits within seconds without any sample pretreatment by DART-orbitrap MS technology. Both positive and negative mode gave the same result, although the latter mode is preferred because of its higher sensitivity and cleaner spectra. Not only raw plant materials but also a herbal tea containing both Chinese and Japanese star anise could be quickly and accurately distinguished by DART-HRMS.
In Chapter 5, direct plant spray in combination with orbitrap HRMS allowed, like DART-HRMS, for an unambiguous distinction between toxic Japanese star anise and non-toxic Chinese star anise fruits within seconds without any sample pretreatment in both positive and negative mode. Direct plant spray ionization has the advantage of low cost, simplicity, room temperature and low standard deviations. Neither the DART nor the direct spray method is very suitable for quantitative measurements of solid samples like star anise fruits.
Chapter 6 describes the purification of eight ginkgolic acids (GAs) from raw plant material (Ginkgo biloba) by using only three steps, namely (1) extraction; (2) selective purification by cheap Fe3O4 magnetic nanoparticles (MNPs); (3) preparative HPLC on a C8 column. The three main constituents occurring at concentrations of 0.15% - 0.60% were enriched to >95% absolute purity without using tedious (gravity) column chromatography with halogenated solvents.
Preparative RP-HPLC is an efficient but not very green technique for the final purification of fine chemicals and natural products as large volumes of acetonitrile, methanol and tetrahydrofuran (THF) are consumed. In Chapter 7 it was investigated whether less toxic organic solvents could replace them. As a test case the preparative separation of Ginkgo terpene trilactones (TTLs) was selected. By a two-step chromatographic optimization procedure a 30 min gradient using only water, ethanol, acetone and ethyl acetate was developed, which gave a baseline separation of 480 mg of an injected TTLs mixture. All five individual TTLs were > 95% pure.
Traditional Chinese Medicines (TCMs) are one of the oldest and most used traditional drugs in the world. Many plants are used for their preparation. An overview of HPLC-related methods such as: multicomponent quantitation, fingerprinting, bioaffinity chromatography and on-flow assays for screening and quality control of TCMs was presented and discussed in Chapter 8.
The final Chapter 9 discusses the major findings of this work and gives further perspectives.
Quantitative trait locus mapping for bruising sensitivity and cap color of Agaricus bisporus (button mushrooms)
Gao, W. ; Weijn, A. ; Baars, J.J.P. ; Mes, J.J. ; Visser, R.G.F. ; Sonnenberg, A.S.M. - \ 2015
Fungal Genetics and Biology 77 (2015). - ISSN 1087-1845 - p. 69 - 81.
melanin biosynthesis pathway - latent isoform ppo4 - pseudomonas-tolaasii - genetic-analysis - tyrosinase - purification - behavior - strains - genome
White button mushrooms discolor after mechanical damage of the cap skin. This hampers the development of a mechanical harvest system for the fresh market. To unravel the genetic basis for bruising sensitivity, two haploid populations (single spore cultures) were generated derived from crosses between parental lines differing in discoloration after mechanical damage (bruising sensitivity). The haploids were crossed with different homokaryotic tester lines to generate mushrooms and allow assessment of the bruising sensitivity in different genetic backgrounds. Bruising sensitivity appears to be a polygenic highly heritable trait (H2: 0.88-0.96) and a significant interaction between genotypes and tester lines and genotypes and flushes was found. Using SNP markers evenly spread over all chromosomes, a very low recombination was found between markers allowing only assignment of QTL for bruising sensitivity to chromosomes and not to sub-regions of chromosomes. The cap color of the two parental lines of population 1 is white and brown respectively. A major QTL for bruising sensitivity was assigned to chromosome 8 in population 1 that also harbors the main determinant for cap color (brown versus white). Splitting offspring in white and non-white mushrooms made minor QTL for bruising sensitivity on other chromosomes (e.g. 3 and 10) more prominent. The one on chromosome 10 explained 31% phenotypic variation of bruising sensitivity in flush 2 in the subpopulations of population 1. The two parental lines of population 2 are both white. Major QTL of bruising sensitivity were detected on chromosome 1 and 2, contributing totally more than 44% variation of the bruising sensitivity in flush 1 and 54% variation of that in flush 2. A considerable consistency was found in QTL for bruising sensitivity in the different populations studied across tester lines and flushes indicating that this study will provide a base for breeding cultivars that are less sensitive for bruising allowing the use of mechanical harvest and automatic postharvest handling for produce for the fresh market. The low recombination between homologous chromosomes, however, underlines the need to introduce a normal recombination pattern found in a subspecies of the button mushroom.
Lignin solubilisation and gentle fractionation in liquid ammonia
Strassberger, Z. ; Prinsen, P. ; Klis, F. van der; Es, D.S. van; Tanase, S. ; Rothenberg, G. - \ 2015
Green Chemistry 17 (2015). - ISSN 1463-9262 - p. 325 - 334.
technical lignins - renewable chemicals - catalysts - extraction - conversion - fuels - kraft - wood - purification - valorization
We present a simple method for solubilising lignin using liquid ammonia. Unlike water, which requires harsh conditions, ammonia can solubilise technical lignins, in particular kraft lignin. A commercial pine wood Kraft lignin (Indulin AT) was solubilized instantaneously at room temperature and 7–11 bars autogeneous pressure, while a commercial mixed wheat straw/Sarkanda grass soda lignin (Protobind™ 1000) was solubilized within 3 h at ambient temperature, and 30 min at. 85 °C. Hydroxide salts were not required. Wheat straw, poplar and spruce organosolv lignins, as well as elephant grass native lignin (MWL) were also solubilized, albeit at lower values. Different sequences of solubilisation and extraction were tested on the Protobind™ 1000 lignin. The remaining lignin residues were characterized by FTIR, size exclusion chromatography (SEC), elemental analysis (ICP), 2D-NMR and 31P NMR. Liquid ammonia is not an innocent solvent, as some nitrogen was incorporated in the residual lignin which then rearranged to higher molecular weight fractions. Nevertheless, the mild solubilisation conditions make liquid ammonia an attractive candidate as a solvent for lignin in future biorefinery processes.
Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor
Raven, N. ; Rasche, F. ; Kuehn, C. ; Anderlei, T. ; Klöckner, W. ; Schuster, F. ; Henquet, M.G.L. ; Bosch, H.J. ; Büchs, J. ; Fischer, R. ; Schillberg, S. - \ 2015
Biotechnology and Bioengineering 112 (2015)2. - ISSN 0006-3592 - p. 308 - 321.
expanded-bed adsorption - enzyme replacement therapy - high-yield production - full-size antibody - pharmaceutical proteins - monoclonal-antibody - suspension-cultures - foreign proteins - cho-cells - purification
Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387¿g/L and M12 yields of ~20¿mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium.
Expression studies in the embryo and in the micropylar endosperm of germinating coffee (Coffea arabica cv. Rubi) seeds
Farias, E.T. de; Amaral da Silva, E.A. ; Toorop, P.E. ; Bewley, J.D. ; Hilhorst, H.W.M. - \ 2015
Plant Growth Regulation 75 (2015)2. - ISSN 0167-6903 - p. 575 - 581.
beta-mannanase - growth - purification - cloning
Germination of coffee (Coffea arabica L.) seed is slow and uneven. Its germination is the net result of events that occur simultaneously in the embryo and endosperm and which are controlled by abscisic acid (ABA). The aim of the study was to monitor the expression of genes related to the cell cycle and to cell wall modifications, including an actin (ACT), a cyclin-dependent kinase (CDK2a) and a-expansin (a-EXP) in the embryo, and a-galactosidase (a-GAL), ß-mannosidase (LeMSIDE2), endo-ß-mannanase (MANA) in the micropylar endosperm. The first seed germinated after 5 days of imbibition and 50 % germination was reached after 10 days. The embryo grew inside the seed prior to radicle protrusion and ABA inhibited both embryo growth and radicle protrusion. The expression of the genes associated with the embryo growth increased during germination and ABA partially inhibited expression. The expression of ß-mannosidase and endo-ß-mannanase increased during imbibition and ABA completely inhibited expression of these genes. However, a-galactosidase displayed a more constitutive expression and was less affected by ABA. ABA plays a dual role in the regulation of coffee seed germination; it concomitantly controls both endosperm weakening and embryo growth.
Mild disintegration of the green microalgae Chlorella vulgaris using bead milling
Postma, P.R. ; Miron, T.L. ; Olivieri, G. ; Barbosa, M.J. ; Wijffels, R.H. ; Eppink, M.H.M. - \ 2015
Bioresource Technology 184 (2015). - ISSN 0960-8524 - p. 297 - 304.
protein aggregation - cell disruption - microbial-cells - release - food - biomass - purification - extraction - economics - products
In this work, the mild disintegration of the microalgae Chlorella vulgaris for the release of intracellular products has been studied. By means of bead milling the microalgae suspensions were successfully disintegrated at different biomass concentrations (25–145 gDW kg-1) over a range of agitator speeds (6–12 m s-1). In all cases over 97% of cell disintegration was achieved resulting in a release of water soluble proteins. A clear optimum rate of disintegration and protein release was observed at an agitator speed of 9–10 m s-1 regardless of the biomass concentration. Selective extraction of water soluble proteins was observed as proteins released sooner than cell disintegration took place. Proteins could be released at 85% lower energy input than for cell disintegration resulting in specific energy consumptions well below 2.5 kWh kgDW-1.
Biocatalytic asymmetric phosphorylation of mevalonate
Matsumi, R. ; Hellriegel, C. ; Schoenenberger, B. ; Milesi, T. ; Oost, J. van der; Wohlgemuth, R. - \ 2014
RSC Advances : An international journal to further the chemical sciences 4 (2014)25. - ISSN 2046-2069 - p. 12989 - 12994.
liver phosphomevalonate kinase - substrate-specificity - zur biosynthese - pig-liver - isopentenyl pyrophosphate - quantitative nmr - acid - cholesterol - purification - terpene
The excellent selectivity of the mevalonate kinase-catalyzed phosphorylation of mevalonate simplifies lengthy multi-step routes to (R)-mevalonate-5-phosphate to a one-step biocatalytic reaction, because the phosphate group can be transferred directly and without any additional reaction steps involving introduction and removal of protecting groups. By adjusting the required reaction time for complete conversion, the kinetic resolution of racemic mevalolactone can be easily used for the preparation of (S)-mevalonate and (R)-mevalonate-5-phosphate. A new recombinant mevalonate kinase has been prepared by the expression of the mevalonate kinase gene from the hyperthermophilic archaeon Thermococcus kodakarensis in Escherichia coli and by the subsequent purification. Direct quantitative 31P-NMR kinetic analysis has been utilized to characterize the enantioselectivity of the mevalonate kinase. This method is useful for determining the biocatalyst's utility for the synthesis of enantiomerically pure (R)-mevalonate-5-phosphate as well as for biocatalytic process development.
Lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system
Desguin, B. ; Goffin, P. ; Viaene, E. ; Kleerebezem, M. ; Martin-Diaconescu, V. ; Maroney, M.J. ; Declercq, J.P. ; Soumillion, P. ; Hols, P. - \ 2014
Nature Communications 5 (2014). - ISSN 2041-1723
lactobacillus-plantarum - lactic-acid - racemization - binding - dehydrogenase - purification - proteins - bacteria - growth - model
Racemases catalyse the inversion of stereochemistry in biological molecules, giving the organism the ability to use both isomers. Among them, lactate racemase remains unexplored due to its intrinsic instability and lack of molecular characterization. Here we determine the genetic basis of lactate racemization in Lactobacillus plantarum. We show that, unexpectedly, the racemase is a nickel-dependent enzyme with a novel a/ß fold. In addition, we decipher the process leading to an active enzyme, which involves the activation of the apo-enzyme by a single nickel-containing maturation protein that requires preactivation by two other accessory proteins. Genomic investigations reveal the wide distribution of the lactate racemase system among prokaryotes, showing the high significance of both lactate enantiomers in carbon metabolism. The even broader distribution of the nickel-based maturation system suggests a function beyond activation of the lactate racemase and possibly linked with other undiscovered nickel-dependent enzymes.
Separation of Whey Proteins using Cascaded Ultrafiltration
Patil, N.V. ; Janssen, A.E.M. ; Boom, R.M. - \ 2014
Separation Science and Technology 49 (2014)15. - ISSN 0149-6395 - p. 2280 - 2288.
tangential flow filtration - membrane cascades - nanofiltration cascades - monoclonal-antibody - bed chromatography - beta-lactoglobulin - fractionation - purification - oligosaccharides - configuration
Whey protein isolate, containing a-Lactalbumin and ß-Lactoglobulin, was separated by using a continuous three-stage ultrafiltration cascade system. Single-stage experiments were optimized to enable good and stable cascade operation. Three different cascade configurations, a non-constrained ideal system (Configuration A), and adapted version (Configuration B), and a countercurrent cascade (Configuration C) were experimentally tested and compared. The countercurrent cascade system showed the traditional trade-off between yield and purity. Both the adapted cascade system and the non-constrained ideal cascade gave better performance in terms of recovery and purity and show potential for application, albeit for different purposes.
Integration of growth and patterning during vascular tissue formation in Arabidopsis
Rybel, B. De; Adibi, M. ; Breda, A.S. ; Wendrich, J.R. ; Smit, M.E. ; Novák, O. ; Yamaguchi, N. ; Yoshida, S. ; Isterdael, G. van; Palovaara, J. ; Nijsse, B. ; Boekschoten, M.V. ; Hooiveld, G.J.E.J. ; Beeckman, T. ; Wagner, D. ; Ljung, K. ; Fleck, C. ; Weijers, D. - \ 2014
Science 345 (2014)6197. - ISSN 0036-8075 - 9 p.
dependent auxin gradients - solid-phase extraction - shoot apical meristem - transcription factor - root - cytokinins - purification - expression - transport - genes
Coordination of cell division and pattern formation is central to tissue and organ development, particularly in plants where walls prevent cell migration. Auxin and cytokinin are both critical for division and patterning, but it is unknown how these hormones converge upon tissue development. We identify a genetic network that reinforces an early embryonic bias in auxin distribution to create a local, nonresponding cytokinin source within the root vascular tissue. Experimental and theoretical evidence shows that these cells act as a tissue organizer by positioning the domain of oriented cell divisions. We further demonstrate that the auxin-cytokinin interaction acts as a spatial incoherent feed-forward loop, which is essential to generate distinct hormonal response zones, thus establishing a stable pattern within a growing vascular tissue.
Faox enzymes inhibited Maillard reaction development during storage both in protein glucose model system and low lactose UHT milk
Troise, A.D. ; Dathan, N.A. ; Fiore, A. ; Roviello, G. ; Fiore, A. Di; Caira, S. ; Cuollo, M. ; Simone, G. De; Fogliano, V. ; Monti, S.M. - \ 2014
Amino Acids 46 (2014)2. - ISSN 0939-4451 - p. 279 - 288.
glycation end-products - aspergillus-fumigatus - liquid-chromatography - amadoriase-i - ec 1.5.3 - oxidoreductase - complications - purification - metabolites - dysfunction
Fructosamines, also known asAmadori products, are formed by the condensation of glucose with the amino group of amino acids or proteins. These compounds are precursors of advanced glycation end products (AGEs) that can be formed either endogenously during aging and diabetes, and exogenously in heat-processed food. The negative effects of dietary AGEs on human health as well as their negative impact on the quality of dairy products have been widely described, therefore specific tools able to prevent the formation of glycation products are needed. Two fructosamine oxidase enzymes isolated fromAspergillus sp. namely, Faox I and Faox II catalyze the oxidative deglycation of Amadori products representing a potential tool for inhibiting the Maillard reaction in dairy products. In this paper, the ability of recombinant Faox I and II in limiting the formation of carboxy-methyl lysine (CML) and protein-bound hydroxymethyl furfurol (b-HMF) in a commercial UHT low lactose milk and a beta-lactoglobulin (b-LG) glucose model system was investigated. Results show a consistent reduction of CML and b-HMF under all conditions. Faox effects were particularly evident on b-HMF formation in low lactose commercial milk. Peptide analysis of the b-LG glucose system identified some peptides, derived from cyanogen bromide hydrolysis, as suitable candidates to monitor Faox action in milk-based products. All in all data suggested that non-enzymatic reactions in dairy products might be strongly reduced by implementing Faox enzymes.
Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the international space station: a challenge for rapid and easy molecular detection of virulent B. anthracis.
Tongeren, S.P. van; Roest, H.I.J. ; Degener, J.E. ; Harmsen, H.J.M. - \ 2014
PLoS ONE 9 (2014)9. - ISSN 1932-6203
polymerase-chain-reaction - toxin genes - identification - purification - sequence - strains - gyrb
For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.
Microorganisms hydrolyse amide bonds; knowledge enabling read-across of biodegradability of fatty acid amides
Geerts, R. ; Kuijer, P. ; Ginkel, C.G. van; Plugge, C.M. - \ 2014
Biodegradation 25 (2014)4. - ISSN 0923-9820 - p. 605 - 614.
pseudomonas aeruginosa - purification - metabolism
To get insight in the biodegradation and potential read-across of fatty acid amides, N-[3-(dimethylamino)propyl] cocoamide and N-(1-ethylpiperazine) tall oil amide were used as model compounds. Two bacteria, Pseudomonas aeruginosa PK1 and Pseudomonas putida PK2 were isolated with N-[3-(dimethylamino)propyl] cocoamide and its hydrolysis product N,N-dimethyl-1,3-propanediamine, respectively. In mixed culture, both strains accomplished complete mineralization of N-[3-(dimethylamino)propyl] cocoamide. Aeromonashydrophila PK3 was enriched with N-(1-ethylpiperazine) tall oil amide and subsequently isolated using agar plates containing dodecanoate. N-(2-Aminoethyl)piperazine, the hydrolysis product of N-(1-ethylpiperazine) tall oil amide, was not degraded. The aerobic biodegradation pathway for primary and secondary fatty acid amides of P. aeruginosa and A. hydrophila involved initial hydrolysis of the amide bond producing ammonium, or amines, where the fatty acids formed were immediately metabolized. Complete mineralization of secondary fatty acid amides depended on the biodegradability of the released amine. Tertiary fatty acid amides were not transformed by P. aeruginosa or A. hydrophila. These strains were able to utilize all tested primary and secondary fatty acid amides independent of the amine structure and fatty acid. Read-across of previous reported ready biodegradability results of primary and secondary fatty acid amides is justified based on the broad substrate specificity and the initial hydrolytic attack of the two isolates PK1 and PK3.