Purification, characterization and crystallization of Jararacussin-I, a fibrinogen-clotting enzyme isolated from the venom of Bothrops jararacussu

A fibrinogen-clotting enzyme, Jararacussin-I, was purified from the venom of Bothrops jararacussu by a combination of ion exchange chromatography using Resource 15S resin and affinity chromatography using Benzamidine Sepharose 6B resin. Jararacussin-I displays a molecular mass of 28 kDa as estimated by sodium dodecyl sulphate-PAGE and possesses an isoetectric point of 5.0. The coagulant specific activity of the enzyme was determined to be 45.8 NIH U/mg using bovine fibrinogen as the substrate and the esterase specific activity was determined to be 258.7 U/mg. The protease inhibitors, benzamidine and DTT inhibited the esterase specific activity by 72.4 and 69.7%, respectively. The optimal temperature and pH for the degradation of both chains of fibrinogen and esterase specific activity were determined to be 37 degreesC and 7.4-8.0, respectively. The enzyme was inactivated at both 4 and 75 T. Single crystals of Jararacussin-I were obtained and complete three-dimensional X-ray diffraction data was collected at the Brazilian National Synchrotron Source (LNLS) to a resolution of 2.4 Angstrom. (C) 2002 Published by Elsevier B.V. Ltd.

Production, purification and partial characterization of a novel protease from marine Engyodontium album BTMFS10 under solid state fermentation

Engyodontium album isolated from marine sediment produced protease, which was active at pH 11. Process parameters influencing the production of alkaline protease by marine E. album was optimized. Particle size of <425 mm, 60% initial moisture content and incubation at 25 8C for 120 h were optimal for protease production under solid state fermentation (SSF) using wheat bran. The organism has two optimal pH (5 and 10) for maximal enzyme production. Sucrose as carbon source, ammonium hydrogen carbonate as additional inorganic nitrogen source and amino acid leucine enhanced enzyme production during SSF. The protease was purified and partially characterized. A 16-fold purified enzyme was obtained after ammonium sulphate precipitation and ion-exchange chromatography. Molecular weight of the purified enzyme protein was recorded approximately 38 kDa by SDS-PAGE. The enzyme showed maximum activity at pH 11 and 60 8C. Activity at high temperature and high alkaline pH suggests suitability of the enzyme for its application in detergent industry

Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus

The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106 kDa (gel filtration) and 55 kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, A beta NA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74 mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3. Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species. (c) 2010 Elsevier Inc. All rights reserved.

Leishmania amazonensis: Partial purification and study of the biochemical properties of the telomerase reverse transcriptase activity from promastigote-stage

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

Purification and properties of an alkaline protease of Aspergillus clavatus

An extracellular alkaline serine protease has been purified from a strain of Aspergillus clavatus, to apparent homogeneity, by ammonium sulfate precipitation and chromatography on Sephadex G-75. Its molar mass, estimated by SDS-PAGE, was 35 kDa. Maximum protease activity was observed at pH 9.5 and 40 degrees C. The enzyme was active between pH 6.0 and 11.0 and was found to be unstable up to 50 degrees C. Calcium at 5 mM increased its thermal stability. The protease was strongly inhibited by PMSF and chymostatin as well as by SDS, Tween 80 and carbonate ion. Substrate specificity was observed with N-p-Tos-Gly-Pro-Arg-p-nitroanilide and N-Suc-Ala-Ala-Ala-p-nitroanilide being active substates. Parts of the amino acid sequence were up to 81% homologous with those of several fungal alkaline serine proteases.

Production, purification and characterization of a minor form of xylanase from Aspergillus versicolor

A strain of Aspergillus versicolor produces a xylanolytic complex containing two components, the minor component being designated xylanase II. The highest production of xylanase II was observed in cultures grown for 5 days in 1% wheat bran as carbon source, at pH 6.5. Xylanase II was purified 28-fold by DEAE-Sephadex and HPLC GF-5 10 gel filtration. Xylanase II was a monomeric glycoprotein, exhibiting a molecular mass of 32 kDa with 14.1% of carbohydrate content. Optimal pH and temperature values for the enzyme activity were about 6.0-7.0 and 55 degreesC, respectively. Xylanase II thermoinactivation at 50degreesC showed a biphasic curve. The ions Hg2+, Cu2+ and the detergent SDS were strong inhibitors, while Mn2+ ions and dithiothreitol were stimulators of the enzyme activity. The enzyme was specific for xylans, showing higher specific activity on birchwood xylan. The Michaelis-Menten constant (K-m) for birchwood xylan was estimated to be 2.3 mg ml(-1) while maximal velocity (V-max) was 233.1 mumol mg(-1) min(-1) of protein. The hydrolysis of oat spell xylan released only xylooligosaccharides. Published by Elsevier Ltd.

Purification and some properties of an extracellular acid protease from Aspergillus clavatus

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

Purification and Characterization of Two Extracellular Xylanases from Penicillium sclerotiorum: A Novel Acidophilic Xylanase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Purification and characterization of the exopolygalacturonase produced by Aspergillus giganteus in submerged cultures

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

Purification and properties of an acid beta-xylosidase from Penicillium sclerotiorum

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Production and purification of an Endo–1,4-beta-Xylanase from Humicola grisea var. thermoidea by electroelution

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Purification, characterization, and specificity determination of a new serine protease secreted by penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl2, KCl, and BaCl, and partially inhibited by CuCl2, CoCl2 and totally inhibited by AlCl3 and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k cat/K m of 10,666 mM-1 s-1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k cat/K m of 7,500 mM -1 s-1. Basic and acidic side chain-containing amino acids performed best at subsite S1. Subsites S2, S3, S′ 2, and S′ 1, S ′ 3 showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k cat/K m were observed for the subsites S2, S3, and S′ 2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level. © 2012 Springer Science+Business Media New York.

Production, purification, and characterization of a major penicillium glabrum xylanase using brewer's spent grain as substrate

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost. © 2013 Adriana Knob et al.

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.