Purification and characterization of a glucoamylase secreted by the plant pathogenSclerotinia sclerotiorum

2002 ◽  
Vol 48 (3) ◽  
pp. 212-218 ◽  
Author(s):  
M B Martel ◽  
C Hervé du Penhoat ◽  
R Létoublon ◽  
M Fèvre
Keyword(s):  
Amino Acids ◽  
Sclerotinia Sclerotiorum ◽  
Reaction Products ◽  
Lytic Enzymes ◽  
Purification And Characterization ◽  
Degrading Enzyme

Among the lytic enzymes secreted by the phytopathogen fungus Sclerotinia sclerotiorum, a starch-degrading enzyme has been isolated and characterized. This glycoprotein of 72 kDa is composed of several isoforms ranging from pI 4.8 to 5.4. The enzymatic parameters have been determined. Specificity studies together with the analysis of the reaction products show that it is an α-1,4-glucanohydrolase. This result is also corroborated by the analysis of the N-terminal and two inner amino acids sequences that are very similar to fungal glucoamylase genes or enzymes so far sequenced.Key words: hydrolase, glucoamylase, Sclerotinia sclerotiorum, starch-degrading enzyme.

scholarly journals Purification and Characterization of Two Enantioselective α-Ketoglutarate-Dependent Dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH

2006 ◽  
Vol 72 (7) ◽  
pp. 4853-4861 ◽  
Author(s):  
Tina A. Müller ◽  
Thomas Fleischmann ◽  
Jan Roelof van der Meer ◽  
Hans-Peter E. Kohler
Keyword(s):  
Amino Acids ◽  
Divalent Cations ◽  
Propanoic Acid ◽  
Dichlorophenoxyacetic Acid ◽  
Purification And Characterization ◽  
Subgroup Ii ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Acid Herbicides ◽  
Growth Experiments

ABSTRACT α-Ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and α-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His6-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the α-ketoglutarate-dependent dioxygenases and share the specific motif HXDX24TX131HX10R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent Km values were 99 μM for (R)-mecoprop, 164 μM for (R)-dichlorprop, and 3 μM for α-ketoglutarate for RdpA and 132 μM for (S)-mecoprop, 495 μM for (S)-dichlorprop, and 20 μM for α-ketoglutarate for SdpA. Both enzymes had high apparent Km values for oxygen; these values were 159 μM for SdpA and >230 μM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace α-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdAα-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.

Sign in / Sign up

Export Citation Format

Share Document