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

A marine Antarctic psychrotolerant bacterium (strain ANT/505), isolated fro m sea ice-covered surface water from the Southern Ocean, showed pectinolyti c activity on citrus pectin agar. The sequencing of the 16S rRNA of isolate ANT/505 indicates a taxonomic affiliation to Pseudoalteromonas haloplankti s. The supernatant of this strain showed three different pectinolytic activ ities after growth on citrus pectin. By activity screening of a genomic DNA library of isolate ANT/505 in Escherichia coli, two different pectinolytic clones could be isolated. Subcloning and sequencing revealed two open read ing frames (ORF) of 1,671 and 1,968 nt, corresponding to proteins of 68 and 75 kDa, respectively. The deduced amino acid sequence of the two ORFs show ed homology to pectate lyases from Erwinia chrysanthemi and Aspergillus nid ulans. The pectate lyases contain signal peptides of 17 and 26 amino acids that were correctly processed after overexpression in E. coli BL21. Both en zymes were purified by anionic exchange chromatography. Maximal enzymatic a ctivities for both pectate lyases were observed at 30 degreesC and a pH ran ge of 9 to 10. The K-m values of both lyases for pectate and citrus pectin were 1 g l(-1) and 5 g l(-1), respectively. Calcium was required for activi ty on pectic substrates, whereas the addition of 1 mM ethylenediaminetetraa cetic acid (EDTA) resulted in complete inhibition of the enzymes. These two enzymes represent the first pectate lyases isolated and characterized from a cold-adapted marine bacterium.