Exploration of Novel Cold-adapted Microorganisms That Inhabit the Polar Regions and Investigation of Their Useful Enzymes

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1. We searched for cold-adapted microorganisms useful as the host for the production of cold-active enzymes. We isolated microorganisms that grow rapidly at low temperatures close to 0℃ and secrete proteins abundantly from Siberian tundra soil, Alaskan tundra soil, soil from the Arctic Circle in Finland, etc.2. Soil samples for screening of cold-adapted microorganisms were collected in a glacier area in Innsbruck, Austria.3. Cold-active catalase useful for waste-water treatment in cold districts etc. was purified from Shewanella livingstonensis Ac10 from Antarctic seawater and characterized.4. We found that Bacillus globisporus B9 from soil from the Arctic Circle in Finland produces a large amount of S-layer protein homolog in the culture supernatant. The bacterium is supposed to be useful as the host for the extracellular production of foreign proteins at low temperatures. We established the method for transformation of this bacterium.5. We constructed a protein expression system by using Acinetobacter sp. strain no. 6 as the host.6. We isolated Pseudomonas sp. Ak26, which produces a large amount of a hemolysin-coregulated protein homolog in the culture supernatant, from Alaskan tundra soil. In order to use this strain as the host for extracellular production of heterologous proteins at low temperatures, we developed genetic engineering methods for this bacterium. The strain was transformed by conjugation with Escherichia coli S17-1 harboring a broad-host-range vector, pJRD215. An expression vector was constructed by introducing the promoter sequence and the SD sequence of the oprD gene, which is overexpressed in Pseudomonas sp. Ak26.7. We analyzed cold-adaptation mechanisms of Shewanella livingstonensis Ac10 isolated from Antarctic sea water and constructed a protein expression system by using this strain as the host.