Purification and characterization of a unique pectin lyase from aspergillus giganteus able to release unsaturated monogalacturonate during pectin degradation

A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb(2+) and was not significantly affected by Hg(2+). Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca(2+). The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking.

Production, purification, and characterization of an extracellular acid protease from the marine Antarctic yeast Rhodotorula mucilaginosa L7

The production, purification, and characterization of an extracellular protease released by Rhodotorula mucilaginosa L7 were evaluated in this study. This strain was isolated from an Antarctic marine alga and previously selected among others based on the capacity to produce the highest extracellular proteolytic activity in preliminary tests. R. mucilaginosa L7 was grown in Saboraud-dextrose medium at 25 °C, and the cell growth, pH of the medium, extracellular protease production and the glucose and protein consumption were determined as a function of time. The protease was then purified, and the effects of pH, temperature, and salt concentration on the catalytic activity and enzyme stability were determined. Enzyme production started at the beginning of the exponential phase of growth and reached a maximum after 48 h, which was accompanied by a decrease in the pH as well as reductions of the protein and glucose concentrations in the medium. The purified protease presented optimal catalytic activity at pH 5.0 and 50 °C. Finally, the enzyme was stable in the presence of high concentrations of NaCl. These characteristics are of interest for future studies and may lead to potential biotechnological applications that require enzyme activity and stability under acidic conditions and/or high salt concentrations.

Rapid determination of 12 antibiotics and caffeine in sewage and bioreactor effluent by online column-switching liquid chromatography/tandem mass spectrometry

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

Stability, purification, and applications of bromelain: a review

Bromelain is a cysteine protease found in pineapple tissue. Due to its anti-inflammatory and anti-cancer activities, as well as its ability to induce apoptotic cell death, bromelain has proved useful in several therapeutic areas. The market for this protease is growing, and several studies exploring various properties of this molecule have been reported. This review aims to compile this data, and summarize the main findings on bromelain in the literature to date. The physicochemical properties and stability of bromelain under different conditions are discussed. Several studies on the purification of bromelain from crude extracts using a wide range of techniques such as liquid-liquid extractions by aqueous two-phase system, ultrafiltration, precipitation, and chromatography, have been reported. Finally, the various applications of bromelain are presented. This review therefore covers the main properties of bromelain, aiming to provide an up-to-date compilation of the data reported on this enzyme.

Efeitos das lagoas de tratamento de esgoto sobre seu entorno: casos no Pontal do Paranapanema

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

Dextran sulfate/Triton X two-phase micellar systems as an alternative first purification step for clavulanic acid

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A sewage sludge co-composting process using respirometric monitoring method in hermetic rotary reactor

This paper has the objective of monitoring the biological activity of composting process of sewage sludge, sugarcane bagasse and ground coffee in a hermetic rotary reactor using the respirometric method in laboratory scale, in order to obtain parameters and system design for large scale projects. Another particularity of this study is the use of a hermetic reactor with gas purging cycles. Purging was performed when the percentage of oxygen reached less than 5%, thus eliminating the gaseous mixture (with elevated CO2 ratio) and the introduction of environmental air with around 21% of O2, successively until the compost was stabilized. The average purge intervals obtained were 29 h and 2 min with reactor rotation frequency of 15 min. The time of the compost stabilization was optimized in 60% if compared to the 90 days in the traditional method. The results obtained can be used to design the process in industrial scale using a simple O2 gas analyzer.

Lignases Production and Partial Purification of Mnp from Brazilian Fungal Isolate in Submerged Fermentation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

RB30-245 Water Supply and Sewage Disposal Systems for Farm Homes

When the well "goes dry" or when the windmill or pump breaks down, every one in the household immediately appreciates the value fo plenty of water. In other words, "You never miss the water until the well runs dry." Fortunately, in most sections of this state, plenty of pure water may be obtained by sinking wells of moderate depth, yet surprisingly few farm homes are supplied with running water in the kitchen even though the barn yards are equipped with hydrants and tanks. It is the purpose of this bulletin to present a number of water supply and sewage disposal systems which have been used in Nebraska and surrounding states and which add greatly to the comfort and convenience of the farm home.

Distribution of sewage input in marine sediments around a maritime Antarctic research station indicated by molecular geochemical indicators

Sediments from Admiralty Bay, Antarctica were collected during the austral summers of 2002/2003 and 2003/2004 in order to assess the distribution and concentration of sewage indicators originating from Comandante Ferraz Brazilian Antarctic Station. Fecal sterols (coprostanol + epicoprostanol) and linear alkylbenzenes (LABs) ranged from <0.01 to 0.95 mu g g(-1) and <1.0 to 23 ng g(-1) dry weight, respectively. In general, the higher concentrations were found only locally in the vicinity of Ferraz station at Martel Inlet. Baseline values for fecal sterols and coprostanone were calculated as 0.19 and 0.40 mu g g(-1), respectively. According to fecal sterols concentrations, sewage contribution to Martel Inlet has increased more than twice since 1997, as result of the increase in the number of researchers at the station especially during the last decade. A low correlation was found between total LABs and fecal steroids, which could be attributed to the contribution of the natural sources of steroids. (C) 2010 Elsevier B.V. All rights reserved.

Purification and characterization of aprotinin from porcine lungs

Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of similar to 7 kDa. It cross- reacted with polyclonal serum anti- commercial aprotinin. About 1 mu g porcine aprotinin inhibited 6 mu g trypsin whereas 1 mu g commercial soybean inhibitor inhibited only 1 mu g trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.

Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins

Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coil

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa. (C) 2012 Elsevier Inc. All rights reserved.

Purification and characterization of Hb 98-114: A novel hemoglobin-derived antimicrobial peptide from the midgut of Rhipicephalus (Boophilus) microplus

The antimicrobial activity of hemoglobin fragments (hemocidins) has been reported in a variety of models. The cattle tick Rhipicephalus (Boophilus) microplus is a blood sucking arthropod from where the first in vivo-generated hemocidin was characterized (Hb 33-61). In the present work we identified a novel antimicrobial peptide from the midgut of fully engorged R. (B.) microplus females, which comprises the amino acids 98-114 of the alpha subunit of bovine hemoglobin, and was designated Hb 98-114. This peptide was active against several yeast and filamentous fungi, although no activity was detected against bacteria up to 50 mu M of the synthetic peptide. Hb 98-114 was capable of permeabilizing Candida albicans cell membrane and had a fungicidal effect against this yeast. Circulardichroism (CD) and nuclear magnetic resonance (NMR) experiments showed that Hb 98-114 has a random conformation in aqueous solution but switches to an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS). This alpha helix adopts an amphipathic structure which may be the mechanism of cell membrane permeabilization. Importantly, Hb 98-114 may play an important role in defending the tick midgut against fungal pathogens and is the first hemocidin with specific antifungal activity to be characterized. (C) 2012 Elsevier Inc. All rights reserved.