Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom

An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65 kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAC-cDNA of 1548 bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58 kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Elp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents. (C) 2009 Elsevier Masson SAS. All rights reserved.

Lipase Production by Endophytic Fungus Cercospora kikuchii: Stability of Enzymatic Activity after Spray Drying in the Presence of Carbohydrates

The present work deals with improving the production and stabilization of lipases from Cercospora kikuchii. Maximum enzyme production (9.384 U/ml) was obtained after 6 days in a medium supplemented with 2% soybean oil. The lipases were spray dried with different adjuvants, and their stability was studied. The residual enzyme activity after drying with 10% (w/v) of lactose, b- cyclodextrin, maltodextrin, mannitol, gum arabic, and trehalose ranged from 63 to 100%. The enzyme activity was lost in the absence of adjuvants. Most of the adjuvants used kept up at least 50% of the enzymatic activity at 5 degrees C and 40% at 25 degrees C after 8 months. The lipase dried with 10% of beta-cyclodextrin retained 72% of activity at 5 degrees C. Lipases were separated by butyl-sepharose column into 4 pools, and pool 4 was partially purified (33.1%; 269.5 U/mg protein). This pool was also spray dried in maltodextrin DE10, and it maintained 100% of activity.

Evidence of thermostable amylolytic activity from Rhizopus microsporus var. rhizopodiformis using wheat bran and corncob as alternative carbon source

Rhizopus microsporus var. rhizopodiformis produced high levels of alpha-amylase and glucoamylase under solid state fermentation, with several agricultural residues, such as wheat bran, cassava flour, sugar cane bagasse, rice straw, corncob and crushed corncob as carbon sources. These materials were humidified with distilled water, tap water, or saline solutions-Segato Rizzatti (SR), Khanna or Vogel. The best substrate for amylase production was wheat bran with SR saline solution (1:2 v/v). Amylolytic activity was still improved (14.3%) with a mixture of wheat bran, corncob, starch and SR saline solution (1:1:0.3:4.6 w/w/w/v). The optimized culture conditions were initial pH 5, at 45 degrees C during 6 days and relative humidity around 76%. The crude extract exhibited temperature and pH optima around 65 degrees C and 4-5, respectively. Amylase activity was fully stable for 1 h at temperatures up to 75 degrees C, and at pH values between 2.5 and 7.5.

Regulation of xylanase in Aspergillus phoenicis: a physiological and molecular approach

Microbial xylanolytic enzymes have a promising biotechnological potential, and are extensively applied in industries. In this study, induction of xylanolytic activity was examined in Aspergillus phoenicis. Xylanase activity induced by xylan, xylose or beta-methylxyloside was predominantly extracellular (93-97%). Addition of 1% glucose to media supplemented with xylan or xylose repressed xylanase production. Glucose repression was alleviated by addition of cAMP or dibutyryl-cAMP. These physiological observations were supported by a Northern analysis using part of the xylanase gene ApXLN as a probe. Gene transcription was shown to be induced by xylan, xylose, and beta-methylxyloside, and was repressed by the addition of 1% glucose. Glucose repression was partially relieved by addition of cAMP or dibutyryl cAMP.

Production and properties of xylanases from Aspergillus terricola Marchal and Aspergillus ochraceus and their use in cellulose pulp bleaching

Aspergillus terricola and Aspergillus ochraceus, isolated from Brazilian soil, were cultivated in Vogel and Adams media supplemented with 20 different carbon sources, at 30 A degrees C, under static conditions, for 120 and 144 h, respectively. High levels of cellulase-free xylanase were produced in birchwood or oat spelt xylan-media. Wheat bran was the most favorable agricultural residue for xylanase production. Maximum activity was obtained at 60 A degrees C and pH 6.5 for A. terricola, and 65 A degrees C and pH 5.0 for A. ochraceus. A. terricola xylanase was stable for 1 h at 60 A degrees C and retained 50% activity after 80 min, while A. ochraceus xylanase presented a t (50) of 10 min. The xylanases were stable in an alkali pH range. Biobleaching of 10 U/g dry cellulose pulp resulted in 14.3% delignification (A. terricola) and 36.4% (A. ochraceus). The brightness was 2.4-3.4% ISO higher than the control. Analysis in SEM showed defibrillation of the microfibrils. Arabinase traces and beta-xylosidase were detected which might act synergistically with xylanase.

Properties of a purified thermostable glucoamylase from Aspergillus niveus

A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40A degrees C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0-5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0-9.5. The temperature optimum was 65A degrees C and retained 100% activity after 240 min at 60A degrees C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-d-glucan glucohydrolase). The K (m) was calculated as 0.32 mg ml(-1). Circular dichroism spectroscopy estimated a secondary structure content of 33% alpha-helix, 17% beta-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.

Heterologous expression of an Aspergillus niveus xylanase GH11 in Aspergillus nidulans and its characterization and application

A xylanase was cloned from Aspergillus niveus and successfully expressed in Aspergillus nidulans (XAN). The full-length gene consisted of 890 bp and encoded 275 mature amino acids with a calculated mass of 31.3 kDa. The deduced amino acid sequence was highly homologous with the xylanase belonging to family 11 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by anion-exchange chromatography and gel filtration. The optima of pH and temperature for the recombinant enzyme were 5.0 and 65 degrees C, respectively. The thermal stability of the recombinant xylanase was extremely improved by covalent immobilization on glyoxyl agarose with 91.4% of residual activity after 180 min at 60 degrees C, on the other hand, the free xylanase showed a half-life of 9.9 min at the same temperature. Affinity chromatography on Concanavalin A- and Jacalin-agarose columns followed by SDS-PAGE analyses showed that the XAN has O- and N-glycans. XAN promotes hydrolysis of xylan resulting in xylobiose, xylotriose and xylotetraose. Intermediate degradation of xylan resulting in xylo-oligomers is appealing for functional foods as the beneficial effect of oligosaccharides on gastrointestinal micro flora includes preventing proliferation of pathogenic intestinal bacteria and facilitates digestion and absorption of nutrients. (C) 2011 Elsevier Ltd. All rights reserved.

Effect of glycosylation on the biochemical properties of beta-xylosidases from Aspergillus versicolor

Aspergillus versicolor grown on xylan or xylose produces two beta-xylosidases with differences in biochemical properties and degree of glycosylation. We investigated the alterations in the biochemical properties of these beta-xylosidases after deglycosylation with Endo-H or PNGase F. After deglycosylation, both enzymes migrated faster in PAGE or SDS-PAGE exhibiting the same R(f). Temperature optimum of xylan-induced and xylose-induced beta-xylosidases was 45A degrees C and 40A degrees C, respectively, and 35A degrees C after deglycosylation. The xylan-induced enzyme was more active at acidic pH. After deglycosylation, both enzymes had the same pH optimum of 6.0. Thermal resistance at 55A degrees C showed half-life of 15 min and 9 min for xylose- and xylan-induced enzymes, respectively. After deglycosylation, both enzymes exhibited half-lives of 7.5 min. Native enzymes exhibited different responses to ions, while deglycosylated enzymes exhibited identical responses. Limited proteolysis yielded similar polypeptide profiles for the deglycosylated enzymes, suggesting a common polypeptide core with differential glycosylation apparently responsible for their biochemical and biophysical differences.

Production of beta-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of beta-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of beta-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular beta-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 degrees C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 degrees C. beta-Fructofuranosidase activity was slightly activated by Cu(2+), Mn(2+), Mg(2+), and Na(+) at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K(d) values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively. (C) 2008 Elsevier Ltd. All rights reserved.

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4-5 days in liquid medium at 40A degrees C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60-70A degrees C. The enzymes were stable for 30 min at 60A degrees C, maintaining 95-98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5-5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0-8.0, while the enzyme from A. niveus was more stable at pH 4.5-6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.

Removal from the Membrane Affects the Interaction of Rat Osseous Plate Ecto-Nucleosidetriphosphate Diphosphohydrolase-1 with Substrates and Ions

We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M(r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always < 2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.

Biochemical properties of an extracellular beta-D-fructofuranosidase II produced by Aspergillus phoenicis under Solid-Sate Fermentation using soy bran as substrate

The filamentous fungus A. phoenicis produced high levels of beta-D-fructofuranosidase (FFase) when grown for 72 hrs under Solid-State Fermentation (SSF), using soy bran moistened with tap water (1:0.5 w/v) as substrate/carbon source. Two isoforms (I and II) were obtained, and FFase II was purified 18-fold to apparent homogeneity with 14% recovery. The native molecular mass of the glycoprotein (12% of carbohydrate content) was 158.5 kDa with two subunits of 85 kDa estimated by SDS-PAGE. Optima of temperature and pH were 55 degrees C and 4.5. The enzyme was stable for more than 1 hr at 50 degrees C and was also stable in a pH range from 7.0 to 8.0. FFase II retained 80% of activity after storage at 4 degrees C by 200 hrs. Dichroism analysis showed the presence of random and beta-sheet structure. A. phoenicis FFase II was activated by Mn(2+), Mg(2+) and Co(2+), and inhibited by Cu(2+), Hg(2+) and EDTA. The enzyme hydrolyzed sucrose, inulin and raffinose. K(d) and V(max) values were 18 mM and 189 U/mg protein using sucrose as substrate.

Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed

Solid-state fermentation obtained from different and low-cost carbon sources was evaluated to endocellulases and endoxylanases production by Aspergillus japonicus C03. Regarding the enzymatic production the highest levels were observed at 30 degrees C, using soy bran added to crushed corncob or wheat bran added to sugarcane bagasse, humidified with salt solutions, and incubated for 3 days (xylanase) or 6 days (cellulase) with 70% relative humidity. Peptone improved the xylanase and cellulase activities in 12 and 29%, respectively. The optimum temperature corresponded to 60 degrees C and 50-55 degrees C for xylanase and cellulase, respectively, both having 4.0 as optimum pH. Xylanase was fully stable up to 40 degrees C, which is close to the rumen temperature. The enzymes were stable in pH 4.0-7.0. Cu(++) and Mn(++) increased xylanase and cellulase activities by 10 and 64%, respectively. A. japonicus C03 xylanase was greatly stable in goat rumen fluid for 4 h during in vivo and in vitro experiments.

Proteomic characterization of the multiple forms of the PLAs from the venom of the social wasp Polybia paulista

The phospholipases A(1) (PLA(1)s) from the venom of the social wasp Polybia paulista occur as a mixture of different molecular forms. To characterize the molecular origin of these structural differences, an experimental strategy was planned combining the isolation of the pool of PLAs from the wasp venom with proteomic approaches by using 2-D, MALDI-TOF-TOF MS and classical protocols of protein chemistry, which included N- and C-terminal sequencing. The existence of an intact form of PLA(1) and seven truncated forms was identified, apparently originating from controlled proteolysis of the intact protein; in addition to this, four of these truncated forms also presented carbohydrates attached to their molecules. Some of these forms are immunoreactive to specific-IgE, while others are not. These observations permit to raise the hypothesis that naturally occurring proteolysis of PLA(1), combined with protein glycosylation may create a series of different molecular forms of these proteins, with different levels of allergenicity. Two forms of PLA(2)s, apparently related to each other, were also identified; however, it was not possible to determine the molecular origin of the differences between both forms, except that one of them was glycosylated. None of these forms were immunoreactive to human specific IgE.

Peritoneal Dialysis in Acute Kidney Injury: Lessons Learned and Applied

Peritoneal dialysis (PD) is a simple, safe, gentle, and efficient renal replacement therapy (RRT) method. It is able to correct acute kidney injury (AKI)-induced metabolic, electrolytic, and acid-base disorders and volume overload both in and out the intensive care unit setting. Some PD modalities, such as high-volume PD and continuous flow PD, can provide RRT doses and efficiency comparable to extracorporeal blood purification methods. PD is particularly suitable for children, patients with refractory heart failure or hemodynamically instable, conditions where systemic anticoagulation should be avoided, patients with difficulty for vascular access and hypo- and hyperthermia conditions. In the following manuscript, PD technical aspects and the possible advantages and limitations of this RRT method will be discussed, and the more recent literature on clinical experience with PD for treatment of AKI will be reviewed.