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

极地地区新型冷适应微生物的探索及其有用酶的研究

基本信息

批准号：
17404021
负责人：
KURIHARA Tatsuo
金额：
$ 8.26万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17404021/
关键词：
psychrotroph psychrophile cold-active enzyme useful enzyme useful gene applied microbiology unusual environment

项目摘要

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.

1.寻找适合低温的微生物作为生产低温活性酶的宿主。我们从西伯利亚冻土带土壤、阿拉斯加冻土带土壤、芬兰北极圈土壤等分离到了在接近0℃的低温下快速生长并大量分泌蛋白质的微生物。在奥地利因斯布鲁克的一个冰川地区采集了土壤样本，用于筛选适应寒冷的微生物。从南极海水中分离纯化出可用于寒冷地区等废水处理的冷活性过氧化氢酶，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形芽孢杆菌B9在培养上清液中产生大量的S-层蛋白同系物。这种细菌被认为是在低温下细胞外生产外来蛋白质的有用宿主。建立了该菌的转化方法。我们利用不动杆菌构建了一个蛋白表达系统。6号菌株为寄主。我们分离到了假单胞菌。Ak26，在阿拉斯加冻土带土壤的培养上清液中产生大量的溶血素共调节蛋白同系物。为了利用该菌株在低温下作为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。该菌株与含有大宿主范围载体pJRD215的大肠杆菌S17-1接合转化。构建了在假单胞菌中高效表达的OprD基因启动子序列和SD序列的表达载体。Ak26.7。我们分析了从南极海水中分离到的雪万氏杆菌Ac10的冷适应机制，并以该菌株为宿主构建了蛋白质表达系统。