Mechanism of extracellular silver nanoparticles synthesis by Stereum hirsutum and Fusarium oxysporum

The increasing interest for greener and biological methods of synthesis has led to the development of non-toxic and comparatively more bioactive nanoparticles. Unlike physical and chemical methods of nanoparticle synthesis, microbial synthesis in general and mycosynthesis in particular is cost-effective and environment-friendly. However, different aspects, such as the rate of synthesis, monodispersity and downstream processing, need to be improved. Many fungal-based mechanisms have been proposed for the formation of silver nanoparticles (AgNPs), mainly those involving the presence of nitrate reductase, which has been detected in filtered fungus cell used for AgNPs production. There is a general acceptance that nitrate reductase is the main responsible for the reduction of Ag ions for the formation of AgNPs. However, this generally accepted mechanism for fungal AgNPs production is not totally understood. In order to elucidate the molecules participating in the mechanistic formation of metal nanoparticles, the current study is focused on the enzymes and other organic compounds involved in the biosynthesis of AgNPs. The use of each free fungal mycelium of both Stereum hirsutum and Fusarium oxysporum will be assessed. In order to identify defective mutants on the nitrate reductase structural gene niaD, fungal cultures of S.hirsutum and F.oxysporum will be selected by chlorate resistance. In addition, in order to verify if each compound identified as key-molecule influenced on the production of nanoparticles, an in vitro assay using different nitrogen sources will be developed. Lately, fungal extracellular enzymes will be measured and an in vitro assay will be done. Finally, The nanoparticle formation and its characterization will be evaluated by UV-visible spectroscopy, electron microscopy (TEM), X-ray diffraction analysis (XRD), Fourier transforms infrared spectroscopy (FTIR), and LC-MS/MS.

Protein recovery, separation and purification: selection of optimal techniques using an expert system

The paper discusses the utilization of new techniques ot select processes for protein recovery, separation and purification. It describesa rational approach that uses fundamental databases of proteins molecules to simplify the complex problem of choosing high resolution separation methods for multi component mixtures. It examines the role of modern computer techniques to help solving these questions.

r-Sm14 - pRSETA efficacy in experimental animals

Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system.

Produção de isomaltulose, um substituto da sacarose, utilizando glicosiltransferase microbiana

Isomaltulose, a functional isomer of sucrose, is a non-cariogenic reducing disaccharide; has a low glycemic index; selectively promotes growth of beneficial bifidobacteria in the human intestinal microflora; and has greater stability than sucrose in some foods and beverages. Isomaltulose is a nutritional sugar that is digested more slowly than sucrose, and has health advantages for diabetics and nondiabetics. Immobilization techniques, especially entrapment of the cells, are widely used for conversion of sucrose into isomaltulose. Immobilization offers advantages such as minimum downstream processing, continuous operation and reusability of cells. Isomaltulose is currently considered to be a promising sugar substitute.

Chromatographic recovery of chemicals from acidic biomass hydrolysates

Lignocellulosic biomasses (e.g., wood and straws) are a potential renewable source for the production of a wide variety of chemicals that could be used to replace those currently produced by petrochemical industry. This would lead to lower greenhouse gas emissions and waste amounts, and to economical savings. There are many possible pathways available for the manufacturing of chemicals from lignocellulosic biomasses. One option is to hydrolyze the cellulose and hemicelluloses of these biomasses into monosaccharides using concentrated sulfuric acid as catalyst. This process is an efficient method for producing monosaccharides which are valuable platforn chemicals. Also other valuable products are formed in the hydrolysis. Unfortunately, the concentrated acid hydrolysis has been deemed unfeasible mainly due to high chemical consumption resulting from the need to remove sulfuric acid from the obtained hydrolysates prior to the downstream processing of the monosaccharides. Traditionally, this has been done by neutralization with lime. This, however, results in high chemical consumption. In addition, the by-products formed in the hydrolysis are not removed and may, thus, hinder the monosaccharide processing. In order to improve the feasibility of the concentrated acid hydrolysis, the chemical consumption should be decreased by recycling of sulfuric acid without neutralization. Furthermore, the monosaccharides and the other products formed in the hydrolysis should be recovered selectively for efficient downstream processing. The selective recovery of the hydrolysis by-products would have additional economical benefits on the process due to their high value. In this work, the use of chromatographic fractionation for the recycling of sulfuric acid and the selective recovery of the main components from the hydrolysates formed in the concentrated acid hydrolysis was investigated. Chromatographic fractionation based on the electrolyte exclusion with gel type strong acid cation exchange resins in acid (H+) form as a stationary phase was studied. A systematic experimental and model-based study regarding the separation task at hand was conducted. The phenomena affecting the separation were determined and their effects elucidated. Mathematical models that take accurately into account these phenomena were derived and used in the simulation of the fractionation process. The main components of the concentrated acid hydrolysates (sulfuric acid, monosaccharides, and acetic acid) were included into this model. Performance of the fractionation process was investigated experimentally and by simulations. Use of different process options was also studied. Sulfuric acid was found to have a significant co-operative effect on the sorption of the other components. This brings about interesting and beneficial effects in the column operations. It is especially beneficial for the separation of sulfuric acid and the monosaccharides. Two different approaches for the modelling of the sorption equilibria were investigated in this work: a simple empirical approach and a thermodynamically consistent approach (the Adsorbed Solution theory). Accurate modelling of the phenomena observed in this work was found to be possible using the simple empirical models. The use of the Adsorbed Solution theory is complicated by the nature of the theory and the complexity of the studied system. In addition to the sorption models, a dynamic column model that takes into account the volume changes of the gel type resins as changing resin bed porosity was also derived. Using the chromatography, all the main components of the hydrolysates can be recovered selectively, and the sulfuric acid consumption of the hydrolysis process can be lowered considerably. Investigation of the performance of the chromatographic fractionation showed that the highest separation efficiency in this separation task is obtained with a gel type resin with a high crosslinking degree (8 wt. %); especially when the hydrolysates contain high amounts of acetic acid. In addition, the concentrated acid hydrolysis should be done with as low sulfuric acid concentration as possible to obtain good separation performance. The column loading and flow rate also have large effects on the performance. In this work, it was demonstrated that when recycling of the fractions obtained in the chromatographic fractionation are recycled to preceding unit operations these unit operations should included in the performance evaluation of the fractionation. When this was done, the separation performance and the feasibility of the concentrated acid hydrolysis process were found to improve considerably. Use of multi-column chromatographic fractionation processes, the Japan Organo process and the Multi-Column Recycling Chromatography process, was also investigated. In the studied case, neither of these processes could compete with the single-column batch process in the productivity. However, due to internal recycling steps, the Multi-Column Recycling Chromatography was found to be superior to the batch process when the product yield and the eluent consumption were taken into account.

Recuperação e purificação de proteínas do soro de queijo tipo coalho usando cromatografia de troca iônica e interação hidrofóbica em leito na forma expandida

Expanded Bed Adsorption plays an important role in the downstream processing mainly for reducing costs as well as steps besides could handling cells homogenates or fermentation broth. In this work Expanded Bed Adsorption was used to recover and purify whey proteins from coalho cheese manufacture using Streamline DEAE and Streamline SP both ionic resins as well as a hydrophobic resin Streamline Phenyl. A column of 2.6 cm inner diameter with 30 cm in height was coupled to a peristaltic pump. Hydrodynamics study was carried out with the three resins using Tris-HCl buffer in concentration of 30, 50 and 70 mM, with pH ranging from 7.0 to 8.0. In this case, assays of the expansion degree as well as Residence Time Distribution (RTD) were carried out. For the recovery and purification steps, a whey sample of 200 mL, was submitted to a column with 25mL of resin previously equilibrated with Tris/HCl (50 mM, pH 7.0) using a expanded bed. After washing, elution was carried out according the technique used. For ionic adsorption elution was carried out using 100 mL of Tris/HCl (50 mM, pH 7.0 in 1M NaCl). For Hydrophobyc interaction elution was carried out using Tris/HCl (50 mM, pH 7.0). Adsorption runs were carried out using the three resins as well as theirs combination. Results showed that for hydrodynamics studies a linear fit was observed for the three resins with a correlation coefficient (R2) about 0.9. In this case, Streamline Phenyl showed highest expansion degree reaching an expansion degree (H0/H) of 2.2. Bed porosity was of 0.7 when both resins Streamline DEAE and Streamline SP were used with StremLine Phenyl showing the highest bed porosity about 0.75. The number of theorical plates were 109, 41.5 and 17.8 and the axial dipersion coefficient (Daxial) were 0.5, 1.4 and 3.7 x 10-6 m2/s, for Streamline DEAE, Streamline SP and Streamline Phenyl, respectively. Whey proteins were adsorved fastly for the three resins with equilibrium reached in 10 minutes. Breakthrough curves showed that most of proteins stays in flowthrough as well as washing steps with 84, 77 and 96%, for Streamline DEAE, Streamline SP and Streamline Phenyl, respectively. It was observed protein peaks during elution for the three resins used. According to these peaks were identified 6 protein bands that could probably be albumin (69 KDa), lactoferrin (76 KDa), lactoperoxidase (89 KDa), β-lactoglobulin (18,3 KDa) e α-lactoalbumin (14 KDa), as well as the dimer of beta-lactoglobulin. The combined system compound for the elution of Streamline DEAE applied to the Streamline SP showed the best purification of whey proteins, mainly of the α-lactoalbumina

Recuperação e purificação de ramnolipídeos produzidos por pseudomonas aeruginosa P029-GVIIA utilizando melaço de cana como substrato

Biosurfactants are molecules produced by microorganisms mainly bacteria as Pseudomonas and Bacillus. Among the biosurfactants, rhamnolipids play an important role due to their tensoactive as well as emulsifying properties. Besides can be produced in a well consolidated way the production costs of biosurfactants are quite expansive mainly if downstream processing is goning to be considered. Actually, attention has been given to identification of biosurfactants as well as optimization of its fermentative processes including downstream ones. This work deals with the development of strategies to recovery and purification of rhamnolipids produced by Pseudomonas aeruginosa P029-GVIIA using sugar-cane molasses as substrate. Broth free of cells was used in order to investigate the best strategies to recovery and purification produced by this system. Between the studied acids (HCl and H2SO4) for the acid precipitation step, HCl was the best one as has been showed by the experimental design 24. Extraction has been carried out using petroleum ether and quantification has been done using the thioglycolic acid method. Adsorption studies were carried out with activated carbon in a batch mode using a 24 experimental design as well as combined with an hydrophobic resin Streamline Phenyl aiming to separate the produced biosurfactant. Biosurfactant partial identification was carried out using High Performance Liquid Chromatography (HPLC). Experiments in batch mode showed that adsorption has been controlled mainly by pH and temperature. It was observed a reduction of 41.4% for the liquid phase and the solid phase it was possible to adsorb up to 15 mg of rhamnolipd/g of activated carbon. The kinetics of adsorption has been well fitted to a pseudo-first order reaction with velocity constant (k1) of 1.93 x 10-2 min-1. Experiments in packed bed ranging concentration on eluent (acetone) has been shown the highest recovery factor of 98% when pure acetone has been used. The combined effect if using activated carbon with an hydrophobic resin Streamline Phenyl has been shown successful for the rhamnolipids purification. It has been possible to purify a fraction of the crude broth with 98% of purity when the eluted of activated carbon packed bed was used with pure acetone

Rhamnolipids as biosurfactants from renewable resources: Concepts for next-generation rhamnolipid production

Several microorganisms are known to produce a wide variety of surface-active substances, which are referred to as biosurfactants. Interesting examples for biosurfactants are rhamnolipids, glycolipids mainly known from Pseudomonas aeruginosa produced during cultivation on different substrates like vegetable oils, sugars, glycerol or hydrocarbons. However, besides costs for downstream processing of rhamnolipids, relatively high raw-material prices and low productivities currently inhibit potential economical production of rhamnolipids on an industrial scale. This review focuses on cost-effective and sustainable production of rhamnolipids by introducing new possibilities and strategies regarding renewable substrates. Additionally, past and recent production strategies using alternative substrates such as agro-industrial byproducts or wastes are summarized. Requirements and concepts for next-generation rhamnolipid producing strains are discussed and potential targets for strain-engineering are presented. The discussion of potential new strategies is supported by an analysis of the metabolism of different Pseudomonas species. According to calculations of theoretical substrate-to-product conversion yields and current world-market price analysis, different renewable substrates are compared and discussed from an economical point of view. A next-generation rhamnolipid producing strain, as proposed within this review, may be engineered towards reduced formation of byproducts, increased metabolic spectrum, broadened substrate spectrum and controlled regulation for the induction of rhamnolipid synthesis. (C) 2012 Elsevier Ltd. All rights reserved.

Kinetic studies on clavulanic acid degradation

Clavulanic acid (CA), a potent beta-lactamase inhibitor, is very sensitive to pH and temperature. It is produced by Streptomyces clavuligerus and to optimize both the fermentation step and the downstream process, the expression of the hydrolysis kinetics has to be determined. In the present work the CA degradation rate from various sources was investigated at temperatures of 10, 20, 25, 30 and 40degreesC and PH values of 6.2 and 7.0. The results showed that first-order kinetics explained very well the hydrolysis kinetics and the Arrhenius equation could be applied to establish a relationship between the degradation rate constant and temperature, at both pHs. It has been observed that CA from fermentation medium was much more unstable than that from standard solution and from a commercially available medicine. Also, it was observed that CA was more stable at PH 6.2 than at pH 7.0, irrespective of the CA source. (C) 2004 Elsevier B.V. All rights reserved.

Evidence for involvement of Saccharomyces cerevisiae protein kinase C in glucose induction of HXT genes and derepression of SUC2

The PKC1 gene in the yeast Saccharomyces cerevisiae encodes protein kinase C that is known to control a mitogen-activated protein (MAP) kinase cascade consisting of Bck1, Mkk1 and Mkk2, and Mpk1. This cascade affects the cell wall integrity but the phenotype of Pkc1 mutants suggests additional targets which have not yet been identified. We show that a pkc1Δ mutant, as opposed to mutants in the MAP kinase cascade, displays two major defects in the control of carbon metabolism. It shows a delay in the initiation of fermentation upon addition of glucose and a defect in derepression of SUC2 gene after exhaustion of glucose from the medium. After addition of glucose the production of both ethanol and glycerol started very slowly. The V max of glucose transport dropped considerably and Northern blot analysis showed that induction of the HXT1, HXT2 and HXT4 genes was strongly reduced. Growth of the pkc1Δ mutant was absent on glycerol and poor on galactose and raffinose. Oxygen uptake was barely present. Derepression of invertase activity and SUC2 transcription upon transfer of cells from glucose to raffinose was deficient in the pkc1Δ mutant as opposed to the wild-type. Our results suggest an involvement of Pkc1p in the control of carbon metabolism which is not shared by the downstream MAP kinase cascade. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

BIOMOLECULES EXTRACTED BY ATPS: PRACTICAL EXAMPLES

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biomolecules extracted by ATPs: practical examples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production of Fructooligosaccharides by Aspergillus phoenicis Biofilm on Polyethylene as Inert Support

Aspergillus phoenicis biofilms on polyethylene as inert support were used to produce fructooligosaccharides (FOS) in media containing 25% (m/V) of sucrose as a carbon source. The maximum production of total FOS (122 mg/mL), with 68% of 1-kestose and 32% of nystose, was obtained in Khanna medium maintained at 30 degrees C for 48 h under orbital agitation (100 rpm). At high concentrations of sucrose (30%, m/V), the recovery of FOS was higher than that observed at a low concentration (5%, m/V). High levels of FOS (242 mg/mL) were also recovered when using the biofilm in sodium acetate buffer with high sucrose concentration (50%, m/V) for 10 h. When the dried biofilm was reused in a fresh culture medium, there was a recovery of approx. 13.7% of total FOS after 72 h of cultivation at 30 C, and 10% corresponded to 1-kestose. The biofilm morphology, analyzed by scanning electron microscope, revealed a noncompact mycelium structure, with unfilled spaces and channels present among the hyphae. The results obtained in this study show that A. phoenicis biofilms may find application for FOS production in a single-step fermentation process, which is cost-effective in terms of reusability, downstream processing and efficiency.

Purification of bromelain from pineapple wastes by ethanol precipitation

Bromelain is an aqueous extract of pineapple that contains a complex mixture of proteases and non-protease components. These enzymes perform an important role in proteolytic modulation of the cellular matrix in numerous physiologic processes, including anti-inflammatory, anti-thrombotic and fibrinolytic functions. Due to the scale of global production of pineapple (Ananas comosus L.), and the high percentage of waste generated in their cultivation and processing, several studies have been conducted on the recovery of bromelain. The aim of this study was to purify bromelain from pineapple wastes using an easy-to-scale-up process of precipitation by ethanol. The results showed that bromelain was recovered by using ethanol at concentrations of 30% and 70%, in which a purification factor of 2.28 fold was achieved, and yielded more than 98% of the total enzymatic activity. This enzyme proved to be susceptible to denaturation after the lyophilization process. However, by using 10% (w/v) glucose as a cryoprotector, it was possible to preserve 90% of the original enzymatic activity. The efficiency of the purification process was confirmed by SDS-PAGE, and native-PAGE electrophoresis, fluorimetry, circular dichroism and FTIR analyzes, showing that this method could be used to obtain highly purified and structurally stable bromelain. (C) 2012 Elsevier B.V. All rights reserved.

Untersuchungen zur Chemosensitivität von testikulären Tumorzellen gegenüber Cisplatin

Metastasierender Krebs ist bei Erwachsenen in der Regel nicht heilbar. Eine Ausnahme stellen testikuläre Keimzelltumoren (TKZT) dar, da über 75 % der Patienten mit fortgeschrittenen metastasierenden TKZT mit einer auf Cisplatin basierenden Kombinations-Chemotherapie geheilt werden können. Zelllinien, die aus TKZT isoliert wurden, behalten diese Cisplatin-Sensitivität in vitro bei. Somit spiegeln Testistumorzelllinien die klinische Situation wider und sind deswegen ein gutes Modellsystem um zu untersuchen, welche Faktoren der Cisplatin-Sensitivität zugrunde liegen. Die Ursachen der Cisplatin-Sensitivität in Testistumoren sind nicht bekannt. Es wurde bereits gezeigt, dass Testistumorzellen eine geringe Kapazität für die Entfernung von Cisplatin-induzierten DNA-Platinierungen aufweisen. Dieser Defekt in der DNA-Reparatur könnte ein Faktor für die beobachtete Cisplatin-Sensitivität sein. Cisplatin induziert sowohl Intrastrang-Vernetzungen als auch Interstrang-Vernetzungen (ICLs). Die Bildung und Reparatur der Cisplatin-induzierten Intrastrang-Vernetzungen wurde mittels DNA-Slot-Blot, die Bildung und Entfernung von Interstrang-Vernetzungen wurde mithilfe des Comet-Assays untersucht. In der vorliegenden Arbeit wurde gezeigt, dass die Reparatur von Intrastrang-Vernetzungen in Testis- und Blasentumorzelllinien vergleichbar ist. Somit sind Testistumorzellen in diesem Reparaturweg nicht beeinträchtigt. Im Unterschied dazu zeigte sich, dass Testistumorzellen die ICLs nicht oder nur mit einer reduzierten Kapazität entfernen können.Da die ICL-Reparatur über die Bildung von DNA-Doppelstrangbrüchen (DSB) mit anschließender DSB-Reparatur verläuft, wurde die Kinetik der DSB-Reparatur anhand der Immundetektion der Histon-Variante γH2AX, die zur Visualisierung von DSB verwendet wird, verfolgt. γH2AX Foci wurden nach Behandlung mit Cisplatin in Testistumorzellen und Blasentumorzellen gebildet. Anders als in Blasentumorzellen blieb der Prozentsatz an γH2AX-positiven Zellen in Testistumorzellen bestehen. Offensichtlich konnten die Testistumorzellen die Cisplatin-induzierten ICLs nicht korrekt prozessieren, was dazu führte, dass γH2AX Foci persistierten. Da unreparierte DNA-Läsionen eine DNA-schadensabhängige Antwort einleiten können, wurde die Aktivierung der Hauptfaktoren dieser Signalwege untersucht. In den Testistumorzellen zeigte sich eine Erhöhung der p53 Proteinmenge nach Cisplatin-Behandlung. Des Weiteren wurde die durch Cisplatin induzierte Aktivierung von ATM/ATR, Chk1/Chk2, Bax und Noxa in Testis- und Blasentumorzellen vergleichend untersucht. Es wurde bereits gezeigt, dass der Reparaturfaktor ERCC1-XPF in Testistumorzelllinien reduziert vorliegt. Um eine mögliche Rolle von ERCC1-XPF für die Reparatur-Defizienz der ICLs und Cisplatin-Sensitivität in Testistumorzellen zu analysieren, wurde ERCC1-XPF in der Testistumorenzelllinie 833K mithilfe eines Expressionsvektors überexprimiert, und der Einfluss von ERCC1-XPF auf ICL-Reparatur sowie Cisplatin-Sensitivität wurde ermittelt. Überexpression von ERCC1-XPF führte zur Reparatur der ICLs in 833K-Zellen und verminderte die Cisplatinsensitivität. Somit scheint die Cisplatinsensitivität der Testistumorzellen, zumindest zum Teil, auf einer verminderten ICL-Reparatur zu beruhen. Des Weiteren wurde in „proof of principle“ Experimenten ERCC1-XPF in der Cisplatin-resistenten Blasentumorzelllinie MGH-U1 mittels siRNA herunterreguliert, und die Auswirkung der Herunterregulation auf die ICL-Reparatur und die Cisplatinsensitivität wurde geprüft. RNA-Interferenz-vermittelte Herunterregulierung von ERCC1-XPF reduzierte die Prozessierung der Cisplatin-induzierten ICLs und verstärkte die Cisplatinsensitivität in MGH-U1 Zellen. Somit wurde in dieser Arbeit zum ersten Mal gezeigt, dass die Testistumorzellen in Vergleich zu Blasentumorzellen in der Reparatur von ICLs defizient sind, wobei die verminderte ICL-Reparatur auf die geringe Expression von ERCC1-XPF zurückgeführt werden konnte. Diese ICL-Reparatur-Defizienz könnte, zumindest zu einem Teil, für die Sensitivität der Testistumoren gegenüber Cisplatin verantwortlich sein.