scholarly journals Partial purification and characterization of xylanase produced by Penicillium expansum

2006 ◽  
Vol 49 (3) ◽  
pp. 475-480 ◽  
Author(s):  
André Luiz de Souza Querido ◽  
Jorge Luiz Cavalcante Coelho ◽  
Elza Fernandes de Araújo ◽  
Virgínia Maria Chaves-Alves
Keyword(s):  
Enzymatic Activity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Penicillium Expansum ◽  
Partial Purification ◽  
Major Protein ◽  
Purification And Characterization ◽  
Exclusion Chromatography ◽  
Molecular Exclusion Chromatography

An extracellular xylanase was found to be the major protein in the filtrate culture of Penicillium expansum when grown on 0.3 % wheat bran, which showed no xylanase multiplicity. The enzime was partial purified by.ammonium sulfate fractioning, molecular exclusion chromatography, ultrafiltration and anion exchange chromatography. The protein eluation profile showed only one form of xylanase that was partially characterized. The activity of purified xylanase was optimal at pH 5.5 and 40 ºC. The enzyme was stable at pH between 5.5 and 6.5 and temperatures between 20-40 ºC. The enzyme showed a Km of 3.03 mM and Vmax of 0.027 mumol min-1 mug -1 of protein. The enzymatic activity was increased 31 % by Mg2+ and 28 % by Al3+.

scholarly journals Purification and characterization of an α2-macroglobulin-like proteinase inhibitor from plasma of the crayfish Pacifastacus leniusculus

1988 ◽  
Vol 255 (3) ◽  
pp. 801-806 ◽  
Author(s):  
H G Hergenhahn ◽  
M Hall ◽  
K Söderhäll
Keyword(s):  
Proteinase Inhibitor ◽  
Hydrophobic Interaction Chromatography ◽  
Acid Precipitation ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Pacifastacus Leniusculus ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Α2 Macroglobulin

An alpha 2-macroglobulin (alpha 2M)-like proteinase inhibitor from plasma of the crayfish Pacifastacus leniusculus was purified to apparent homogeneity by acid precipitation, hydrophobic interaction chromatography, affinity chromatography on concanavalin A-Sepharose and anion-exchange chromatography. The subunit Mr is about 190,000. Pore-size-limit electrophoresis proved the native protein to be a dimer. The purified protein resembled vertebrate alpha 2 Ms in that it protected trypsin from inhibition by soyabean trypsin inhibitor, and in its sensitivity to methylamine treatment. Methylamine also prevented the protein from being autolytically cleaved into Mr 60,000 and 140,000 fragments when subjected to heat treatment. The amino acid composition showed similarities with both human alpha 2 M and an alpha 2 M-like protein from the arthropod Limulus polyphemus. These data indicate that this Pacifastacus alpha 2M-like protein (P alpha 2M) may be a distantly related homologue of vertebrate alpha 2Ms.

Partial Purification and Some Properties of Brassica napus Lipase

1994 ◽  
Vol 49 (5-6) ◽  
pp. 293-301 ◽  
Author(s):  
Cornelia Fuchs ◽  
Gerd Hansen
Keyword(s):  
Brassica Napus ◽  
Size Exclusion Chromatography ◽  
Lipase Activity ◽  
Sodium Deoxycholate ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Partial Purification ◽  
Exclusion Chromatography ◽  
Molecular Masses

Abstract Lipase (triacylglycerol acylhydrolase EC 3.1.1.3) from rape (Brassica napus cv. Ceres) was isolated from cotyledons of dark-grown seedlings. The enzyme was partially purified by poly­ ethylene glycol precipitation. Delipidation of the lipase with n-hexane was required prior to further purification by anion exchange chromatography and size exclusion chromatography. A purification factor of 337 was ultimately achieved and the purification process was moni­tored by SDS-PAGE. Here, at least two protein bands with molecular masses of 62 and 64 kD a respectively were found in the active fraction obtained by size exclusion chromatography. Sodium deoxycholate was found to stimulate the lipase activity, but appeared to cause aggregation of the enzyme. It was not possible to estimate the isoelectric point of the dialyzed rape lipase due to the high molecular mass of the aggregates. Two simple methods to detect lipase activity directly on polyacrylamide gel were applied. No esterase activity was found by using p-nitrophenyl acetate as substrate.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

scholarly journals Pemurnian Parsial dan Karakterisasi Enzim Xilanase dari Bakteri Laut Bacillus safencis strain LBF P20 Asal Pulau Pari Jakarta

2017 ◽  
Vol 37 (1) ◽  
pp. 31
Author(s):  
Fitria Fitria ◽  
Nanik Rahmani ◽  
Sri Pujiyanto ◽  
Budi Raharjo ◽  
Yopi Yopi
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Centrifugal Filter ◽  
Sds Page ◽  
Enzyme Specific Activity ◽  
Hydrolysis Of

Enzyme xylanase (EC 3.2.1.8) is widely used in various industrial  fields for the hydrolysis of xylan (hemicellulose) into xylooligosaccharide and xylose. The aims of this study were to  conduct partial purification and characterization of xylanase from marine Bacillus safencis strain LBF P20 and to obtain the  xylooligosaccharide types from xylan hydrolysis by this enzyme.  Based on this research, the optimum time for enzyme production  occurred at 96 hours with the enzyme activity of 6.275 U/mL and  enzyme specific activity of 5.093 U/mg. The specific activities were  obtained from precipitation by amicon® ultra-15 centrifugal filter devices, gel filtration chromatography and anion exchange chromatography that were increased by 15.07, 34.7, and 96.0  U/mg. The results showed that the highest activity at pH 7, temperature of 60 °C, and stable at 4 °C. Type of  xylooligosaccharide produced by this study were xylohexoses, xylotriose, and xylobiose. SDS-PAGE analysis and zimogram  showed that the molecular weight of xylanase protein were about  25 kDa. ABSTRAKEnzim xilanase (EC 3.2.1.8) digunakan dalam hidrolisis xilan  (hemiselulosa) menjadi xilooligosakarida dan xilosa. Penelitian  ini bertujuan untuk melakukan purifikasi parsial dan karakterisasi xilanase dari bakteri laut Bacillus safencis strain LBF P20 serta uji  hidrolisis untuk mengetahui jenis xilooligosakarida yang  dihasilkan oleh enzim tersebut. Berdasarkan hasil penelitian, waktu optimum untuk produksi enzim terjadi pada jam ke 96  dengan aktivitas enzim sebesar 6,275 U/mL dan aktivitas spesifik enzim sebesar 5,093 (U/mg). Aktivitas spesifik enzim hasil  pemekatan dengan amicon® ultra-15 centrifugal filter devices,  kromatografi filtrasi gel dan kromatografi penukar anion  mengalami peningkatan berturut-turut sebesar 15,1; 34,7 dan96,0 U/mg. Hasil karakterisasi menunjukkan aktivitas  tertinggi pada pH 7, suhu 60 °C dan stabil pada suhu 4 °C. Analisis SDS-PAGE dan zimogram menunjukkan berat molekul protein xilanase berkisar 25 kDa. Jenis gula reduksi yang  dihasilkan yaitu xiloheksosa, xilotriosa, dan xilobiosa.

scholarly journals Purification and Characterization of Transglutaminase from Streptomyces sp. TTA 02 SDS 14

2016 ◽  
Vol 11 (3) ◽  
Author(s):  
Lia Siti Nur'amaliyah ◽  
Dewi Seswita Zilda ◽  
Nisa Rachmania Mubarik
Keyword(s):  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Phenacyl Bromide ◽  
Optimum Temperature ◽  
Purification And Characterization ◽  
Streptomyces Sp ◽  
Exclusion Chromatography ◽  
Sulfonyl Fluoride

Streptomyces sp. TTA 02 SDS 14 is a transglutaminase producing bacteria which previously had been  screened along with more than one hundred isolates. This research aimed to purify and characterize transglutaminase from this strain. Transglutaminase was purified from crude enzyme by ultrafiltration, Q-Sepharose ion exchange chromatography and Sepacryl S200 size exclusion chromatography sequentially, obtaining yield and purification fold of  1.36%  and 27 folds, respectively. The molecular weight of the purified transglutaminase was 72 kDa detected by zymogram gel electrophoresis. The optimum temperature and pH were 50°C and 6. The transglutaminase was stable at 45°C and could be activated in the presence of 5 mM and 10 mM of Na+, K+, Li+,Ca2+, Mg2+, BPB (4-bromo-phenacyl bromide), and IAA (iodo acetamide acid), but the activity was inhibited by  the presence of Cu+, Zn2+, and PMSF (phenyl methyl sulfonyl fluoride).

N-Acetyl-ß-ᴅ-hexosaminidases of the Brine Shrimp Artemia: Partial Purification and Characterization

1991 ◽  
Vol 46 (9-10) ◽  
pp. 781-788 ◽  
Author(s):  
Klaus-Dieter Spindler ◽  
Brigitte Funke-Höpfner
Keyword(s):  
Brine Shrimp ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Strong Inhibitor ◽  
Molecular Masses ◽  
Dose Dependent ◽  
Enzyme I

Abstract N-Acetyl-β-ᴅ-hexosaminidases (EC 3.2.1.52) from Artemia nauplii were isolated and char­acterized. Three different enzymes I, II1 and II2 were separated according to their behaviour on anion exchange chromatography and gel filtration columns. Their apparent molecular masses were 83,000 ± 7000, 110,000 ± 10,000 and 56,000 ± 5000 Da with corresponding S-values of 8.6, 11.9 and 7.9. All three enzymes also differ in their apparent pH-optima (5.1, 4.5 and 6.1) and they all bind to concanavalin A. The three enzymes have about the same affinities (app. Km between 0.16 and 0.72 mmol/1) for the three substrates (p-nitrophenyl-N-acetyl-β-ᴅ-glucosamine or p-nitrophenyl-N-acetyl-β-ᴅ-galactosamine and N ,N′-diacetyl-chitobiose) and are therefore N-acetyl-β-ᴅ-hexosaminidases. In contrast, the three enzymes behave quite differently, both in terms of their inhibitor constants and the type of inhibition. The substrates inhibit both enzymes II1 and II2 but not enzyme I. On the other hand, N-acetyl-β-ᴅ-galactosamine inhibits enzyme I in a non-competi­tive way but not enzymes II1 and II2. All three enzymes are inhibited by the end product N-acetyl-β-ᴅ-glucosamine, enzyme I in a competitive manner, both enzymes II1 and II2 in a non-competitive way. 2-Acetamido-2-deoxy-ᴅ-galactonolactone is a strong inhibitor for en­zyme I (Ki = 13 μtmol/l) with much lower affinities towards enzymes II1 and II2 (Ki = 0.63 and 1.03 mmol/l). All three enzymes are inhibited in a dose-dependent way and completely reversi­ble by α-methyl-mannoside.

scholarly journals Partial purification and characterization of ribonucleases from roots, stem and leaves of cowpea

2001 ◽  
Vol 13 (3) ◽  
pp. 357-364 ◽  
Author(s):  
OCTÁVIO LUIZ FRANCO ◽  
LORRANCE ABREU GONDIM ◽  
KÁTIA REGINA BEZERRA ◽  
MARIA ELANE DE CARVALHO GUERRA ◽  
CARMEM ROGÉLIA FARIAS MACHADO LIMA ◽  
...  
Keyword(s):  
Exchange Chromatography ◽  
Partial Purification ◽  
Ionic Exchange ◽  
Purification And Characterization ◽  
Chemical Denaturation ◽  
Molecular Masses ◽  
Deoxyribonuclease Activity ◽  
Different Tissues ◽  
Optimal Ph

Partial purification and characterization of ribonucleases (RNase; EC 3.1.27.1) present in roots, stem and leaves of 5 day-old Pitiúba cowpea [Vigna unguiculata (L.) Walp.] seedlings are described. Crude extracts from the different tissues were precipitated with ammonium sulfate followed by ionic exchange chromatography (CM-Cellulose) resulting in purification factors of 48-fold for roots, 21 for stem and 42 for leaves. No deoxyribonuclease activity was practically observed. The molecular masses of the RNases did not significantly differ, averaging 16.3 kDa. Leaf RNase was stable up to 50ºC while the others were inactivated at this temperature. The maximal inactivation for both stem and roots RNases was reached at 70ºC while for leaf it occurred at 80ºC. The addition of KCl to the assay medium caused a shift of optimal pH from 6.0 toward the range of 5.2 - 5.6 for the enzymes extracted from the different tissues. RNase activities were strongly inhibited by Hg2+, Zn2+ and Cu2+, partially inhibited by Co2+ and Fe2+ and were not affected by EDTA, Ca2+ or Mg2+. In contrast to the leaf RNase, roots and stem enzymes were inactivated by urea and 2-mercaptoethanol (2-ME). Although there is a great similarity among the enzymes studied, leaf RNase appears to be more stable to heat and to chemical denaturation than root and stem RNases. The results also suggest that the enzymes extracted from different tissues of Pitiúba cowpea seedlings are ribonucleases and not nucleases.

A Fibrinolytic Agent from a Saturnid Caterpillar Partial Purification and Characterization

1973 ◽  
Vol 29 (01) ◽  
pp. 135-142 ◽  
Author(s):  
C. L Arocha-Pinango ◽  
N. A Marsh ◽  
D Robinson
Keyword(s):  
Clinical Syndrome ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Heat Stable ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Human Plasminogen ◽  
The Relationship ◽  
Stable Material

SummaryPurification and characterization studies were performed on a proteolytic agent obtained from a Saturnid moth caterpillar. Starting material possessed caseinolytic, fibrinolytic and plasminogen-activator activities. The fibrinolytic activity was stable over a wide range of pH and temperature. Purification was performed by molecular exclusion chromatography and ion exchange chromatography. A pH- and heat-stable material was obtained having a molecular weight in the range of 16,000-18,000. This material did not possess the ability to activate human plasminogen but retained direct caseinolytic and fibrinolytic activities. It had no effect on thrombin- and arvin- clotting times of human plasma, on partial thromboplastin time or on the whole blood thrombin generation test. Electrophoresis on cellulose acetate agar gel and polyacrylamide showed the material to be very basic and moving away from any detectable protein. The isoelectric point was found to be greater than pH 10. The relationship between the characterized material and the clinical syndrome caused by contact with the caterpillar remains to be determined.

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