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Cloning of two pectate lyase genes from the marine Antarctic bacterium Pseudoalteromonas haloplanktis strain ANT/505 and characterization of the enzymes

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Le, V. T. , Ho, T. , Helmke, E. , Le, T. B. and Schweder, T. (2001): Cloning of two pectate lyase genes from the marine Antarctic bacterium Pseudoalteromonas haloplanktis strain ANT/505 and characterization of the enzymes , Extremophiles, 5 (1), pp. 35-44 . doi: 10.1007/s007920000170
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Abstract:

A marine Antarctic psychrotolerant bacterium(strain ANT/505), isolated from sea ice-covered surfacewater from the Southern Ocean, showed pectinolytic activ-ity on citrus pectin agar. The sequencing of the 16S rRNAof isolate ANT/505 indicates a taxonomic affiliation toPseudoalteromonas haloplanktis. The supernatant of thisstrain showed three different pectinolytic activities aftergrowth on citrus pectin. By activity screening of a genomicDNA library of isolate ANT/505 in Escherichia coli, two dif-ferent pectinolytic clones could be isolated. Subcloning andsequencing revealed two open reading frames (ORF) of1,671 and 1,968 nt, corresponding to proteins of 68 and75 kDa, respectively. The deduced amino acid sequence ofthe two ORFs showed homology to pectate lyases fromErwinia chrysanthemi and Aspergillus nidulans. The pectatelyases contain signal peptides of 17 and 26 amino acids thatwere correctly processed after overexpression in E. coliBL21. Both enzymes were purified by anionic exchangechromatography. Maximal enzymatic activities for bothpectate lyases were observed at 30°C and a pH range of 9to 10. The Km values of both lyases for pectate and citruspectin were 1 g l 1 and 5 g l 1 , respectively. Calcium wasrequired for activity on pectic substrates, whereas theaddition of 1 mM ethylenediaminetetraacetic acid (EDTA)resulted in complete inhibition of the enzymes. These twoenzymes represent the first pectate lyases isolated and char-acterized from a cold-adapted marine bacterium.

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