Expanded Bed Adsorption Of Bromelain (e.c. 3.4.22.33) From Ananas Comosus Crude Extract

This work focuses on the adsorption of Bromelain in expanded bed conditions, such as the adsorption kinetics parameters. The adsorption kinetics parameters showed that after 40 minutes equilibrium was achieved and maximum adsorption capacity was 6.11 U per resin mL. However, the maximum adsorption capacity was only determined by measuring the adsorption isotherm. Only by the Langmuir model the maximum adsorption capacity, Qm, and dissociation constant, kd, values could be estimated as 9.18 U/mL and 0.591, respectively, at 25°C and 0.1 mol/L phosphate buffer pH 7.5. A column made of glass with an inner diameter of 1 cm was used for the expanded bed adsorption (EBA). The residence time was reduced 10 fold by increasing the expansion degree 2.5 times; nonetheless, the plate number (N) value was reduced only 2 fold. After adsorption, the bromelain was eluted in packed bed mode, with a downward flow. The purification factor was about 13 fold and the total protein was reduced 4 fold. EBA showed to be feasible for purification of bromelain.261149157Amersham Pharmacia Biotech. EBA Handbook: Principles and Methods. Uppsala, ISBN 91-630-5519-8 (1997)Anspach, F.B., Curbelo, D., Hartman, R., Garke, G., Deckwer, W.D., Expanded-bed chromatography in primary protein purification (1999) J. Chromatogr. A, 865, p. 129Bruce, L.J., Chase, H.A., Hydrodynamics and adsorption behaviour within an expanded bed adsorption column studied using in-bed sampling (2001) Chem. Eng. Sci, 56, p. 3149Camprubi, S., Bruguera, M., Canalias, F., Purification of recombinant histidine-tag streptolysin O using immobilized metal affinity expanded bed adsorption (IMA-EBA). International (2006) J. Biol. 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Progr, 17, pp. 1128-1136Gaspani, L., Limiroli, E., Ferrario, P., Bianchi, M., In vivo and in vitro effects of bromelain on PGE(2) and SP concentrations in the inflammatory exudate in rats (2002) Pharmacology, 65, pp. 83-86Haq, S.K., Rasheedi, S., Khan, R.H., Characterization of a partially folded intermediate of stem bromelain at low pH (2002) Eur. J. Biochem, 269, pp. 47-52Harrach, T., Eckert, K., Maurer, H.R., Machleidt, I., Machleidt, W., Nuck, R., Isolation and characterization of two forms of an acidic bromelain stem proteinase (1998) J. Protein Chem, 17, pp. 351-361Hatano, K., Sawano, Y., Tanokura, M., Structure-function relationship of bromelain isoinhibitors from pineapple stem (2002) Biol. Chem, 383, pp. 1151-1156Hatano, K., Tanokura, M., Takahashi, K., The amino acid sequences of isoforms of the bromelain inhibitor from pineapple stem (1998) J. Biochem, 124, pp. 457-461Hjorth, R., Expanded bed adsorption in industrial bioprocessing: Recent developments (1997) Trends Biotechnol, 15, p. 230Hochstrasser, D.F., Patchornik, A., Merril, C.R., Development of Polyacrylamide gels that improve the separation of proteins and their detection by silver staining (1998) Analyt. Biochem, 173, pp. 412-423Hochstrasser, D.F., Merril, C.R., Catalysts' for Polyacrylamide gel polymerization and detection of proteins by silver staining (1998) Appl. Theor. Electrophoresis, 1, pp. 35-40Hebbar, H.U., Sumana, B., Raghavarao, K.S.M.S., Use of reverse micellar systems for the extraction and purification of bromelain term from pineapple wastes (2008) Bioresource Technology, 99, pp. 4896-4902Khan, R.H., Rasheedi, S., Haq, S.K., Effect of pH, temperature and alcohols on the stability of glycosylated and deglycosylated stem bromelain (2003) J. Biosci, 28, pp. 709-714Kim, M. H., Kim, H. K., Lee, J. K., Park, S. Y., Oh, T. K., Thermostable lipase of Bacillus stearothermophilus, high level production, purification and calciumdependent thermostability. Korea Res. Inst. Biosci. Biotechnol. 64:280-286 (2000)Kordel, M., Hofmann, B., Schomburg, D., Schimid, R.D., Extracelluar lipase of Pseudomonas sp. strain ATCC 21808: Purification, characterization, crystallization and preliminary X-ray diffraction data (1991) J. Bacteriol, 173, pp. 4836-4841Kunitz, M., Crystalline soybean trypsin inhibitor: II general properties (1974) J. Gen. Physiol, 30, pp. 291-310Lali, A.M., Khare, A.S., Joshi, J.B., Behaviour of solid particles in viscous non-newtonian solutions: Settling velocity, wall effects and bed expansion in solid-liquid fluidized beds (1989) Powder Tech, 57, pp. 39-50Maurer, H.R., Bromelain: Biochemistry, pharmacology and medical use (2001) Cell. Mol. 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Biochem, 71, pp. 817-830Rasheedi, S., Haq, S.K., Khan, R.H., Guanidine hydrochloride denaturation of glycosylated and deglycosylated stem bromelain (2003) Biochemistry, 68, pp. 1097-1100Roy, I., Pai, A., Lali, A., Gupta, M.N., Comparison of batch, packed bed and expanded bed purification of A. niger cellulose beads (1999) Bioseparation, 8, pp. 317-326Santos, E.S., Guirardello, R., Franco, T.T., Distributor Effect on Expanded Bed Adsorption (2000) International Conference IEX 2000 (Ion Exchange at the Millennium), , Cambridge/UKSantos, E.S., Guirardello, R., Franco, T.T., Preparative chromatography of xylanase using expanded bed adsorption (2002) J. Chrom. A, 944, pp. 217-224Silveira, E., (2007) Purificação e caracterização de bromelina a partir do extrato bruto de Ananas comosus por adsorção em leito expandido, , Dissertação de Mestrado. Faculdade de Engenharia Química: UnicampSilverstein, R.M., Kezdy, F.J., Characterization of the pineapple stem proteases (bromelain) (1975) Arch. 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B, 846, pp. 51-56Trinh, L., Phue, J.-N., Jaluria, P., Tsai, C.W., Narum, D.L., Shiloach, J., Screen-less expanded bed column: New approach for the recovery and purification of a malaria transmission blocking vaccine candidate from Pichia pastoris (2006) Biotechnol. Lett, 28, pp. 951-958Walter, H. E., Proteinases: methods with hemoglobin, casein, and azocoll as substrates. Pp. 270-277 in Methods of Enzymatic Analysis, 5, H.U. Bergmeyer, ed. Verlag Chemie, Weinheim, Germany (1984)Wang, S.-L., Peng, J.-H., Liang, T.-W., Liu, K.-C., Purification and characterization of a chitosanase from Serratia marcescens TKU011 (2008) Carbohydrate Research, 343, pp. 1316-1323Wharton, C.W., The structure and mechanism of stem bromelain. Evaluation of the homogeneity of purified stem bromelain, determination of the molecular weight and kinetic analysis of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-L-phenylalanyl-L-serine methyl ester (1974) Biochem. 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Can artisanal “Coalho” cheese from Northeastern Brazil be used as a functional food?

AbstractBrazilian artisanal “Coalho” cheeses from six Northeast towns were investigated as a functional food based on their peptide profiles and antioxidant, zinc-binding and antimicrobial activities. The peptides (WSP) from “Coalho” cheese showed high antioxidant activity, the best value of TEAC being 2223±10.10μM, which means 91.1±0.43% oxidative inhibition and peptide concentration for IC50 of 7mg/mL (21μg of peptides) for sample from the town of Correntes. The smallest TEAC value (1896±17μM), which means 75.9±0.7% oxidative inhibition and IC50 of 10.5mg/mL (31.5μg of peptide), was obtained for samples from the town of São Bento do Una. The zinc-binding activities were: Arcoverde (72.21±0.24%) Cachoeirinha (75.02±0.02%), Capoeiras (61.78±0.65%), Correntes (75.47±0.5%), São Bento do Una (75.41±0.15%), and Venturosa (74.36±0.04%). The WSP extracts showed antimicrobial activity against Enterococcus faecalis, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. All the results obtained suggest that “Coalho” cheese has potential as a functional food

Screening, production and biochemical characterization of a new fibrinolytic enzyme produced by Streptomyces sp. (Streptomycetaceae) isolated from Amazonian lichens

Thrombosis is a pathophysiological disorder caused by accumulation of fibrin in the blood. Fibrinolytic proteases with potent thrombolytic activity have been produced by diverse microbial sources. Considering the microbial biodiversity of the Amazon region, this study aimed at the screening, production and biochemical characterization of a fibrinolytic enzyme produced by Streptomyces sp. isolated from Amazonian lichens. The strain Streptomyces DPUA1576 showed the highest fibrinolytic activity, which was 283 mm2. Three variables at two levels were used to assess their effects on the fibrinolytic production. The parameters studied were agitation (0.28 - 1.12 g), temperature (28 - 36 ºC) and pH (6.0 - 8.0); all of them had significant effects on the fibrinolytic production. The maximum fibrinolytic activity (304 mm2) was observed at 1.12 g, 28 ºC, and pH of 8.0. The crude extract of the fermentation broth was used to assess the biochemical properties of the enzyme. Protease and fibrinolytic activities were stable during 6 h, at a pH ranging from 6.8 to 8.4 and 5.8 to 9.2, respectively. Optimum temperature for protease activity ranged between 35 and 55 °C, while the highest fibrinolytic activity was observed at 45 ºC. Proteolytic activity was inhibited by Cu2+ and Co2+ ions, phenylmethylsulfonyl fluoride (PMSF) and pepstatin A, which suggests that the enzyme is a serine protease. Enzymatic extract cleaved fibrinogen at the subunits A-chain, A-chain, and -chain. The results indicated that Streptomyces sp. DPUA 1576 produces enzymes with fibrinolytic and fibrinogenolytic activity, enzymes with an important application in the pharmaceutical industry.The authors grateful acknowledge the financial support of Fundação de Amparo a Pesquisa do Estado de Pernambuco (FACEPE, Pernambuco, Brazil, N. 0158-2.12/11), CNPq/ RENORBIO (National Counsel of Technological and Scientific Development, N.55146/2010-3) and National Council for the Improvement of Higher Education (CAPES, Brazil) for the scholarship. The author thanks editor and reviewers for their review and comments.info:eu-repo/semantics/publishedVersio

Produção de biocompostos com atividade antimicrobiana de Streptomyces sp. ante isolados de mastite caprina

Actinomicetos são um dos principais produtores de enzimas, vitaminas e metabólitos secundários, destacando-se o gênero Streptomyces, o qual tem uma ampla capacidade de produção de antibióticos eficazes no combate a diferentes microrganismos, entre eles o Staphylococcus sp. Em virtude dessa eficiência no combate a patógenos, o objetivo deste trabalho foi avaliar a produção de metabólitos com atividade antimicrobiana produzidos por 67 Streptomyces isolados de liquens da região amazônica, ante isolados de mastite caprina (Staphylococcus aureus) do estado de Pernambuco, Brasil. Foi utilizado um planejamento fatorial para avaliar a influência das fontes de carbono (glicose) 0%, 0,5% e 1% e de nitrogênio (farinha de soja) 1%, 2,5% e 4% na produção dos antimicrobianos, bem como das variáveis pH, biomassa e atividade antimicrobiana. Dos Streptomyces estudados, o DPUA 1566 foi o que se destacou por formação de halos de inibição entre 18 e 26mm ante os isolados de mastite caprina. Foi possível verificar que a fonte de carbono inibiu a produção de antimicrobianos quando submetidos a uma concentração de glicose de 1%; com a retirada desta, os Streptomyces apresentaram uma elevada capacidade de produção de metabólitos com atividade antimicrobiana tendo potencial para o tratamento de mastite caprina

Investiga\ue7\ue3o da cin\ue9tica e termodin\ue2mica na atividade e estabilidade da ascorbato oxidase do extrato de Cucurbita maxima

The objective of this work was to estimate the main kinetic and thermodynamic parameters of the activity and stability of ascorbate oxidase in an extract of Cucurbita maxima

Extractive Cultivation Of Xylanase By Penicillium Janthinellum In A Poly(ethylene Glycol)/cashew-nut Tree Gum Aqueous Two-phase System

Cultivation of the fungus Penicillium janthinellum for xylanase production was studied in a poly(ethylene glycol)/cashew-nut tree gum aqueous two-phase system, using a two-level fractional factorial design. The parameters studied were initial pH, cultivation time, type of agro-industrial residue (oat husk or corn cob), agitation, temperature, and phase-forming polymers. The xylanase produced during fermentation partitioned into the top phase. The agitation and temperature (negative), cultivation time and initial pH (positive) effects proved statistically significant for xylanase production. The highest percentage yield of the xylanase in the top and its production in the top phase, about 97% and 160.7 U/mL, were obtained in cultures of 120 h, 40 rpm, 25°C, and pH 5.0.20618801884Albertsson, P.A., (1986) Partition of Cell Particles and Macromolecules, 3rd Ed., , Wiley Interscience: New YorkSinha, J., Dey, P.K., Panda, T., Aqueous two-phase: The system of choice for extractive fermentation (2000) Appl. Microbiol. Biotechnol., 54, pp. 476-486Persson, I., Tjerneld, F., Hahn-Hagerdal, B., Production of β-glucosidase by Aspergillus phoenicis QM329 in aqueous two-phase systems (1989) Biotechnol. Tech., 3, pp. 265-270Hotha, S., Banik, R.M., Production of alkaline protease by Bacillus thurigiensis H14 in aqueous two-phase systems (1997) J. Chem. Technol. Biotechnol., 69, pp. 5-10Kuboi, R., Umakoshi, H., Komasawa, I., Extractive cultivation of Escherichia coli using poly(ethylene glycol)/phosphate aqueous two-phase system to produce intracellular β-galactosidase (1995) Biotechnol. Prog., 11, pp. 202-207Chen, J.P., Lee, M.S., Enhanced production of Serratia marcescens chitinase in PEG/dextran aqueous two-phase systems (1995) Enzyme Microb. Technol., 17, pp. 1021-1027Antov, M.G., Pericin, D.M., Dimic, G.R., Cultivation of Polyporus squamosus for pectinase production in aqueous two-phase system containing sugar beet extraction waste (2001) J. Biotechnol., 91, pp. 83-87Christian, T.J., Manley-Harris, M., Richards, G.N., A preliminary study of the use of larch arabinogalactan in aqueous two-phase systems (1998) Carbohydr. Polym., 35, pp. 7-12Skuse, D.R., Norris-Jones, R., Yalpani, M., Brooks, D.E., Hydroxypropyl cellulose/poly(ethylene glycol)-co-poly(propylene glycol) aqueous two-phase systems: System characterization and partition of cells and proteins (1992) Enzyme Microb. Technol., 14, pp. 785-790Venâncio, A., Teixeira, J.A., Mota, M., Evaluation of crude hydroxypropyl starch as a bioseparation aqueous two-phase-forming polymer (1993) Biotechnol. Prog., 9, pp. 635-638Oliveira, L.A., Sarubbo, L.A., Porto, A.L.F., Campos-Takaki, G.M., Tambourgi, E.B., Partition of trypsin in aqueous two-phase systems of poly(ethylene glycol) and cashew-nut tree gum (2002) Process Biochem., 38, pp. 693-699Sarubbo, L.A., Oliveira, L.A., Porto, A.L.F., Duarte, H.S., Carneiro-Leão, A.M.A., Lima-Filho, J.L., Campos-Takaki, G.M., Tambourgi, E.B., New aqueous two-phase system based on cashew-nut tree gum and poly(ethylene glycol) (2000) J. Chromatogr. B, 743, pp. 79-84Paula, R.C.M., Rodrigues, J.F., Composition and rheological properties of cashew tree gum, the exudate polysaccharide from Anacardium occidentale L. (1995) Carbohydr. Polym., 26, pp. 177-181Rodrigues, J.F., Paula, R.C.M., Costa, S.M., (1993) Polim. Cienc. Tecnol., 3, pp. 31-36Milagres, A.M.F., (1988) Alguns Aspectos Da Regulação de β-Xilanases Extracelulares de Penicillium Janthinellum, , Viçosa: Tese de Mestrado, UFVVogel, H.J., A convenient growth medium for Neurospora crassa (Medium N) (1956) Microb. Gen. Bull., 13, pp. 42-43Bailey, M.J., Biely, P., Poutanen, K., Inter-laboratory testing of methods for assay of xylanase activity (1992) J. Biotechnol., 23, pp. 257-270Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing sugar (1959) Anal. Chem., 31, pp. 426-428Sedmak, J.J., Grossberg, S.E., A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250 (1977) Anal. Biochem., 79, pp. 544-1522Oliveira, L.A., Sarubbo, L.A., Porto, A.L.F., Lima-Filho, J.L., Campos-Takaki, G.M., Tambourgi, E.B., Physical and rheological characterisation of poly(ethylene glycol)-cashew-nut tree gum aqueous two-phase systems (2002) J. Chromatogr. B, 766, pp. 27-36Barros Neto, B., Scarminio, I.S., Bruns, R.E., (2001) Como Fazer Experimentos: Pesquisa e Desenvolvimento Na Ciência e Na Indústria, 1st Ed., , UNICAMP: BrazilHaltrich, D., Nidetzky, B., Kulbe, K.D., Steiner, W., Zupancic, S., Production of fungal xylanases (1996) Bioresour. Technol., 58, pp. 137-161Parajó, J.C., Domínguez, H., Dominguez, J.M., Biotechnological production of xylitol. Part 3: Operation in culture media made from lignocellulose hydrolysates (1998) Bioresour. Technol., 66, pp. 25-40Costa, S.A., Pessoa Jr., A., Roberto, I.C., Partitioning of xylanolytic complex from Penicillium janthinellum by an aqueous two-phase system (2000) J. Chromatogr. B, 743, pp. 339-348Bim, M.A., Franco, T.T., Extraction in aqueous two-phase systems of alkaline xylanase produced by Bacillus pumilus and its application in kraft pulp bleaching (2000) J. Chromatogr. B, 743, pp. 349-356Kulkarni, N., Vaidya, A., Rao, M., Extractive cultivation of recombinant Escherichia coli using aqueous two phase systems for production and separation of extracellular xylanase (1999) Biochem. Biophys. Res. Commun., 255, pp. 274-27

Produção e caracterização de protease fibrinolítica de Streptomyces parvulus DPUA 1573

RESUMO As proteases fibrinolíticas são capazes de degradar coágulos de fibrina formados dentro dos vasos sanguíneos, evitando a trombose intravascular. Em animais, a tromboflebite, que acomete frequentemente os equinos, ocasiona, em seus casos graves, a obstrução jugular e também um edema de laringe, derivando a obstrução das vias aéreas, o que possibilita um edema cerebral, ocorrendo o óbito do animal. Devido ao fato de o tratamento ser de custo elevado, faz-se necessária a investigação de outras fontesde proteases fibrinolíticas com custos menores e com menos efeitos colaterais. Diante disso, este estudo tem como objetivo produzir e caracterizar proteases fibrinolíticas obtidas de Streptomyces parvulus DPUA 1573. Para produção da enzima, foi utilizado um planejamento fatorial 24 avaliando a concentração da farinha de soja (0,5, 1,0 e 1,5%) e da glicose (0, 0,5 e 1,0g/L), temperatura (28, 32 e 37ºC) e agitação (150, 200 e 250rpm) sobre a biomassa e a atividade fibrinolítica. Pode-se verificar que a protease fibrinolítica apresentou atividade máxima (835U/mL) nas condições de concentração de 1,5% de soja, 1g/L de glicose, 28°C e 150rpm com 48 horas de fermentação. A protease fibrinolítica obtida teve temperatura e pH ótimos de 55°C e pH 9,0, respectivamente. A atividade enzimática foi inibida pelo EDTA, pelo íon Fe2+ e pelo SDS, o que indicou a enzima ser uma metaloprotease. A linhagem Streptomyces parvulus DPUA 1573 foi capaz de produzir protease fibrinolítica, possuindo características bioquímicas favoráveis à aplicação na medicina veterinária e possivelmente humana

Clavulanic acid purification by extractive fermentation using aqueous two-phase system

A factorial design was employed to stady the influence of PEG molar mass, PEG concentration, phosphate salt concentration and agitation intensity on the extractive clavulanic acid fermentation by Streptomyces DAUFPE 3060