Purification and characterization of a constitutive polygalacturonase associated with the infection process of French bean leaves by Botrytis cinerea

1990 ◽  
Vol 68 (9) ◽  
pp. 1921-1930 ◽  
Author(s):  
G. Leone ◽  
E. A. M. Schoffelmeer ◽  
J. Van den Heuvel
Keyword(s):  
Botrytis Cinerea ◽  
Reaction Rate ◽  
French Bean ◽  
Infection Process ◽  
Purification Procedure ◽  
Purification And Characterization ◽  
Titration Curves ◽  
Optimal Ph ◽  
Bean Leaves

The constitutively produced polygalacturonase isoenzyme PG2 was isolated from culture filtrates of Botrytis cinerea, purified to homogeneity, and characterized. The shape of titration curves of PG2 and two other polygalacturonase isoenzymes explained the difficulties found in separating superimposed pectic enzyme activities during the purification procedure. PG2 hydrolyzed sodium polygalacturonate more quickly than pectin. The optimal pH for PG2 activity with polygalacturonate was 4.5 and with pectin, 4.0. PG2 activity was also influenced by the presence of NaCl or CaCl2 in the reaction mixture. Analysis of the breakdown products by paper chromatography and a comparison of the reaction rate by viscosimetry and reducing group assay revealed that PG2 has an endocatalytic mode of action on polygalacturonate. The isoelectric point and the molecular mass of PG2 were estimated to be 9.1 and 23.0 kDa, respectively. Key words: Botrytis cinerea, chromatofocusing, endopolygalacturonase, purification, substrate specificity, titration curve.

scholarly journals Purification and Characterization of a Monooxygenase Involved in the Biosynthetic Pathway of the Antitumor Drug Mithramycin

2003 ◽  
Vol 185 (13) ◽  
pp. 3962-3965 ◽  
Author(s):  
David Rodríguez ◽  
Luis M. Quirós ◽  
Alfredo F. Braña ◽  
José A. Salas
Keyword(s):  
Biological Activity ◽  
Biosynthetic Pathway ◽  
Flavin Adenine Dinucleotide ◽  
Chain Molecule ◽  
Aliphatic Chain ◽  
Purification Procedure ◽  
Reaction Products ◽  
Purification And Characterization ◽  
Optimal Ph

ABSTRACT A monooxygenase encoded by the mtmOIV gene from the mithramycin gene cluster of Streptomyces argillaceus was purified 21-fold by a three-step purification procedure. This monooxygenase catalyzes the oxidative cleavage of the fourth ring of premithramycin B. The enzyme was dependent on NADPH and flavin adenine dinucleotide for activity with optimal pH at 9.5, and the Km values for NADPH and premithramycin B were 269.22 and 23.35 μM, respectively. The reaction catalyzed by MtmOIV yields two possible isomers of the same basic shortened aliphatic chain molecule. One of the reaction products showed important biological activity, thus highlighting the importance of the cleavage of the fourth ring of the aglycon for biological activity.

scholarly journals Purification and characterization of histidine decarboxylase from mouse kidney

1986 ◽  
Vol 234 (2) ◽  
pp. 349-354 ◽  
Author(s):  
S A M Martin ◽  
J O Bishop
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Histidine Decarboxylase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Mouse Kidney ◽  
Purification Procedure ◽  
Purification And Characterization ◽  
A Subunit

Histidine decarboxylase was purified 800-fold from the kidneys of thyroxine-treated mice. The purification procedure included precipitation of protein from a crude supernatant after heating it to 55 degrees C at pH 5.5, fractionation with (NH4)2SO4, phosphocellulose column chromatography, chromatofocusing, DEAE-Sepharose column chromatography, gel filtration on Sephacryl S-300 and preparative polyacrylamide-gel electrophoresis. The native enzyme had an estimated Mr of 113 000. The protein was analysed in SDS/10%-polyacrylamide gels and formed a single band corresponding to a subunit Mr of 55 000, indicating that it is a dimer. Three forms of the enzyme were resolved on isoelectrofocusing gels, with pI 5.3, 5.5 and 5.7.

Characterization of the nutrients required by Botrytis cinerea to infect broad bean leaves

1981 ◽  
Vol 19 (2) ◽  
pp. 153-167 ◽  
Author(s):  
A.M. Harper ◽  
R.N. Strange ◽  
P. Langcake
Keyword(s):  
Botrytis Cinerea ◽  
Broad Bean ◽  
Bean Leaves

scholarly journals Purification and characterization of a new endoglucanase from Clostridium thermocellum

1992 ◽  
Vol 283 (1) ◽  
pp. 69-73 ◽  
Author(s):  
M P M Romaniec ◽  
U Fauth ◽  
T Kobayashi ◽  
N S Huskisson ◽  
P J Barker ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Reducing Sugars ◽  
Rapid Decrease ◽  
Purification And Characterization ◽  
Temperature Optimum ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Hydrolysis Of ◽  
Optimal Ph

An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from the thermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants. No carbohydrate is associated with the endoglucanase. A molecular mass of 76,000 Da was determined by SDS/PAGE. The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05. The enzyme has a temperature optimum of 70 degrees C and retains approx. 50% of its activity after 48 h at 60 degrees C. Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity. The endoglucanase shows little ability to hydrolyse highly ordered cellulose. Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity. The enzyme is completely inactivated by 1 mM-Hg2+ and is inhibited by a thiol-blocking reagent.

Purification and characterization of an extracellular proteinase from Brevibacterium linens

1990 ◽  
Vol 36 (7) ◽  
pp. 510-512 ◽  
Author(s):  
Ondrej Juhasz ◽  
Bohumil Škárka
Keyword(s):  
Gel Electrophoresis ◽  
Serine Proteinase ◽  
Purification And Characterization ◽  
Extracellular Proteinase ◽  
Brevibacterium Linens ◽  
Caseinolytic Activity ◽  
Inhibition Studies ◽  
Substrate Proteins ◽  
Optimal Ph

The alkaline proteinase of Brevibacterium linens was partially purified through ultrafiltration and chromatography on Sephacryl S-200. The molecular weight of this enzyme was determined by gel electrophoresis in polyacrylamide to be 52 000 – 55 000. The proteinase hydrolyses natural polypeptides such as casein, haemoglobin, bovine serum albumin, and ovalbumin. An apparent Km for casein was determined to be 1.3 mg∙mL−1. The optimal pH for caseinolytic activity was between 7.0 and 8.5 at the optimal temperature of 45 °C. The isolated enzyme is thermolabile: incubation at 50 °C destroyed proteolytic activity. Substrate proteins have a stabilizing effect. Our inhibition studies revealed that the B. linens proteinase belongs to the serine proteinase class. Key words: Brevibacterium linens, proteinase, purification.

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