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.

scholarly journals Purification and characterization of a high-Mr proteinase inhibitor of pro-phenol oxidase activation from crayfish plasma

1987 ◽  
Vol 248 (1) ◽  
pp. 223-228 ◽  
Author(s):  
H G Hergenhahn ◽  
A Aspan ◽  
K Söderhäll
Keyword(s):  
Proteinase Inhibitor ◽  
Serine Proteinase ◽  
Hydrophobic Interaction Chromatography ◽  
Phenol Oxidase ◽  
Purification And Characterization ◽  
Recognition Mechanism ◽  
Heat Stable ◽  
Apparent Homogeneity ◽  
Activation Cascade

Crayfish plasma was found to contain a proteinase inhibitor, which was purified to apparent homogeneity by acid precipitation, affinity chromatography on concanavalin A-Sepharose and hydrophobic-interaction chromatography. The inhibitor is a monomeric protein with an Mr of about 155,000 and a pI in the range 4.6-4.8. It is heat-stable and tolerant to low pH. It inhibits the serine proteinases trypsin and chymotrypsin, but not thrombin or subtilisin. Furthermore, it is efficient in decreasing the activity of a proteinase from crayfish haemolymph that is involved in the activation cascade of pro-phenol oxidase and can also block pro-phenol oxidase activation by this serine proteinase. This cascade is believed to play a central role in the recognition mechanism of non-self material in crustaceans and insects. The data presented give some evidence that the new proteinase inhibitor is involved in the regulation of this system.

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+.

Purification and Characterization of a β-Glucosidase Specific for 2,4-Dihydroxy- 7-methoxy-1,4-benzoxazin-3-one (DIMBOA) Glucoside in Maize

1999 ◽  
Vol 54 (3-4) ◽  
pp. 181-185 ◽  
Author(s):  
Akira Oikawa ◽  
Kenkichi Ebisui ◽  
Masayuki Sue ◽  
Atsushi Ishihara ◽  
Hajime Iwamura
Keyword(s):  
Zea Mays ◽  
Ion Exchange ◽  
Cation Exchange ◽  
Hydroxamic Acid ◽  
Zea Mays L ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Purification And Characterization ◽  
Apparent Homogeneity

Occurrence and properties of hydroxamic acid glucoside glucosidase were investigated in 10-day-old, autotrophic maize (Zea mays L.) in which 2,4-dihydroxy-7-methoxy-1,4- benzoxazin-3-one glucoside (DIMBOA-G) is a major benzoxazinone component. Crude extracts of both leaves and roots showed glucosidase activity for both DIMBOA-G and 2,4- dihydroxy-1,4-benzoxazin-3-one glucoside (DIBOA-G). A cation-exchange chromatography after cryoprecipitation of the extract from leaves gave a peak with both activities, and further purification by ion-exchange and hydroxyapatite chromatography gave a fraction with an apparent homogeneity, the purification being 560 fold. The Km values (mᴍ) of the purified glucosidase were 0.16 for DIMBOA-G, 0.68 for DIBOA-G and 2.96 for p-nitrophenyl-β-ᴅ-glucopyranoside. The activity on salicin and esculin was too low to be detected. The data indicate that a glucosidase specific for DIMBOA-G comes into contact with constitutive benzoxazinone glucosides producing defensive aglycone when plants are damaged by microbial or insect attacks.

scholarly journals Enzymatic Synthesis and Characterization of Different Families of Chitooligosaccharides and Their Bioactive Properties

2021 ◽  
Vol 11 (7) ◽  
pp. 3212
Author(s):  
Noa Miguez ◽  
Peter Kidibule ◽  
Paloma Santos-Moriano ◽  
Antonio O. Ballesteros ◽  
Maria Fernandez-Lobato ◽  
...  
Keyword(s):  
High Performance ◽  
Chemical Characterization ◽  
Amperometric Detection ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enzymatic Production ◽  
Bioactive Properties ◽  
Substrate Properties ◽  
Chitin Hydrolysis

Chitooligosaccharides (COS) are homo- or hetero-oligomers of D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) that can be obtained by chitosan or chitin hydrolysis. Their enzymatic production is preferred over other methodologies (physical, chemical, etc.) due to the mild conditions required, the fewer amounts of waste and its efficiency to control product composition. By properly selecting the enzyme (chitinase, chitosanase or nonspecific enzymes) and the substrate properties (degree of deacetylation, molecular weight, etc.), it is possible to direct the synthesis towards any of the three COS types: fully acetylated (faCOS), partially acetylated (paCOS) and fully deacetylated (fdCOS). In this article, we review the main strategies to steer the COS production towards a specific group. The chemical characterization of COS by advanced techniques, e.g., high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry, is critical for structure–function studies. The scaling of processes to synthesize specific COS mixtures is difficult due to the low solubility of chitin/chitosan, the heterogeneity of the reaction mixtures, and high amounts of salts. Enzyme immobilization can help to minimize such hurdles. The main bioactive properties of COS are herein reviewed. Finally, the anti-inflammatory activity of three COS mixtures was assayed in murine macrophages after stimulation with lipopolysaccharides.

scholarly journals Occurrence of fatty acid epoxide hydrolases in soybean (Glycine max). Purification and characterization of the soluble form

1992 ◽  
Vol 282 (3) ◽  
pp. 711-714 ◽  
Author(s):  
E Blée ◽  
F Schuber
Keyword(s):  
Glycine Max ◽  
Gel Filtration ◽  
Soluble Form ◽  
Soluble Enzyme ◽  
Purification And Characterization ◽  
Epoxide Hydrolases ◽  
Major Activity ◽  
Apparent Homogeneity ◽  
A Minor

Epoxide hydrolases catalysing the hydration of cis-9,10-epoxystearate into threo-9,10-dihydroxystearate have been detected in soybean (Glycine max) seedlings. The major activity was found in the cytosol, a minor fraction being strongly associated with microsomes. The soluble enzyme, which was purified to apparent homogeneity by (NH4)2SO4 fractionation, hydrophobic, DEAE- and gel-filtration chromatographies, has a molecular mass of 64 kDa and a pI of 5.4.

scholarly journals Purification and Characterization of Asparaginase fromPhaseolus vulgarisSeeds

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Saleh A. Mohamed ◽  
Mohamed F. Elshal ◽  
Taha A. Kumosani ◽  
Alia M. Aldahlawi
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Potential Candidate ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Wide Range ◽  
Optimum Ph ◽  
Glutaminase Activity ◽  
Low Activity

L-asparaginase from bacteria has been used in treatment of acute lymphoblastic leukemia. The aim of this study was to purify and characterize L-asparaginase fromPhaseolus vulgarisseeds instead of microbial sources. L-asparaginase was purified to apparent homogeneity. The enzyme has molecular mass of 79 kDa. The purified asparaginase had very low activity toward a number of asparagine and glutamine analogues. L-asparaginase was free from glutaminase activity. Kinetic parameters, Km andVmax of purified enzyme, were found to be 6.72 mM and 0.16 μM, respectively. The enzyme had optimum pH at 8.0. The enzyme showed high stability at alkaline pH (pH 7.5–9.0) when incubated for up to 24 h. L-asparaginase had the same temperature optimum and thermal stability at 37°C. K+was able to greatly enhance the activity of asparaginase by 150% compared with other metals tested. In conclusion, L-asparaginase showed no glutaminase activity and good stability over a wide range of physiological conditions, and thus it could be used as a potential candidate for treatment of acute lymphoblastic leukemia.

scholarly journals Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

2013 ◽  
Vol 10 (3) ◽  
pp. 844-853
Author(s):  
Baghdad Science Journal
Keyword(s):  
Aspergillus Flavus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Stability ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Original Activity ◽  
A Value ◽  
Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

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