Extremophiles as Cell Factories for Novel Thermostable Enzymes
Garo ANTRANIKIAN*, Frank NIEHAUS, Costanzo BERTOLDO, Seffen PROWE, and Fiona DUFFNER
Technical University Hamburg-Harburg and Center for Biotechnology of Extremophiles, Denickestrasse 15, D-21071 Hamburg, Germany
The recent efforts in the last few years have clearly shown that cell components of extreme thermophilic and hyperthermophilic archaea and bacteria are unique and deliver a valuable source for new biocatalysts and compounds. In our research group the physiology and enzymology of a variety of extreme thermophilic, hyperthermophilic and thermoalkaliphilic microorganisms was studied in detail. Most of the enzymes derived from these organisms are active and stable at the boiling point of water and were found to be resistant against chemical denaturants such as detergents. chaotropic agents. organic solvents and extremes of pH.
The thermostable polymer-degrading enzymes amylases, pullulanases, proteases and xylanases have been detected in various hyperthermophilic archaea belonging to the genera Thermococcus, Pyrococcus and Pyrodictium. The genes encoding the thermostable amylase and pullulanase from Pyrococcus woesei have been cloned, sequenced and expressed in a mesophilic host. Both purified recombinant enzymes are optimally active at 100℃ and show considerable activity at 120℃. From the deduced amino acid sequence it is evident that the enzymes are secreted after the cleavage of a signal peptide. Further investigations have shown that the archaeon Pyrodictium abyssi is also able to grow on various polymeric substrates and secrete unique heat-stable amylase, pullulanase and xylanase; the latter enzyme is optimally active at 110℃.
Thermoactive extracellular enzymes (e.g. protease, xylanase, pullulanase) were also detected in extreme thermophilic anaerobic bacteria belonging to the genera Thermotoga and Fervidobacterium. The gene encoding the pullulanase from Fervidobacterium pennavorans Ven5 was cloned and expressed in E. coli. Unlike the pullulanase from Pyrococcus woesei (specific to bothα-1,6- andα-1,4-glycosidic linkages; pullulanase type II) the enzyme from the bacterium F. pennavorans attacks exclusively theα-l, 6-glycosidic linkages in polysaccharides. This bacterial enzyme (pullulanase type I) is the only thermostable enzyme known so far that attacks exclusively branching points in polysaccharides resulting in the formation of long chain linear polysaccharides. In further experiments we were able to isolate a thermoalkaliphilic bacterium (Thermoalkalibacter bogoriae) from lake Bogoria in
