Characterization of the pectinolytic enzymes of the marine psychrophilic bacterium Pseudoalteromonas haloplanktis strain ANT/505

• Main Author: Van Truong, Le
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: 2006
• Subjects: Pectat-Lyase; Pektinesterase; ddc:570; Antarctic; Arctic; Southern Ocean; Antarc*; Sea ice
• Online Access: https://epub.ub.uni-greifswald.de/frontdoor/index/index/docId/254; https://nbn-resolving.org/urn:nbn:de:gbv:9-000312-0; https://epub.ub.uni-greifswald.de/files/254/Truong_thesis_20.9.06.pdf

The biodiversity of marine microorganisms opens a promising potential for the discovery of new technical enzymes. During this study a characterization of marine microorganisms, isolated from Arctic or Antarctic ice, sea water or sediment from the ocean was performed based on a comprehensive strain collection at the Alfred-Wegener-Institut für Polar- und Meeresforschung. These marine psychrophilic bacteria indicated a wide spectrum of extracellular cold-active enzymes. 16S rRNA sequencing revealed that many of these psychrophilic bacteria represent new species. Characterization of selected isolates by means of transmission electron or raster electron microscopy showed remarkably pleomorphic cellular structures throughout their growth. The major part of this thesis focuses on a marine Antarctic, psychrophilic bacterium (strain ANT/505) isolated from sea ice covered surface water from the Southern Ocean, which was identified to express a very uncommon enzymatic activity for the marine environment, namely a pectinolytic activity. The sequencing of the 16S rRNA of isolate ANT/505 and biochemical tests indicated a taxonomical affiliation to the specie Pseudoalteromonas haloplanktis. The supernatant of this bacterial isolate showed after growth on citrus pectin three different pectinolytic activities. 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 reading frames of 1671 and 1968 nt corresponding to proteins of 68 and 75 kDa. The deduced amino acid sequence of the two orfs showed homology to pectate lyases from Erwinia chrysanthemi and Aspergillus nidulans. The pectate lyases contain signal peptides of 17 and 26 amino acids length that were correctly processed after overexpression in E. coli BL21. Both enzymes were purified by anionic exchange chromatography. Maximal enzymatic activities for both pectate lyases were observed at a temperature of 30°C and a pH range of 9-10. The Km ...