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

1.我们寻找冷适应微生物作为生产冷活性酶的宿主。我们从西伯利亚冻原土壤、阿拉斯加冻原土壤、芬兰北极圈土壤等中分离到了能在接近0℃的低温下快速生长并大量分泌蛋白质的微生物。在因斯布鲁克的冰川地区采集了用于筛选冷适应微生物的土壤样品。从南极希瓦氏菌（Shewanella livingstonensis Ac 10）中分离纯化了低温活性过氧化氢酶，并对其性质进行了研究.我们发现来自芬兰北极圈土壤的球孢芽孢杆菌B 9在培养上清液中产生大量的S层蛋白同系物。该细菌被认为是有用的宿主在低温下的外源蛋白质的胞外生产。建立了该菌的转化方法.以不动杆菌6号菌株为宿主菌，构建了表达系统.我们从阿拉斯加冻原土壤中分离出假单胞菌Ak 26，它在培养上清液中产生大量的溶血素协同调节蛋白同源物。为了利用该菌株作为低温胞外生产异源蛋白的宿主，我们开发了该细菌的基因工程方法。该菌株通过与携带广宿主范围载体pJRD 215的大肠杆菌S17-1接合来转化。通过引入oprD基因的启动子序列和SD序列构建表达载体，所述oprD基因在假单胞菌属Ak26.7中过表达。本文分析了从南极海水中分离到的希瓦氏菌Ac 10的冷适应机制，并以该菌为宿主构建了蛋白表达系统。

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.