南极微生物产低温蛋白酶菌株的筛选、分子鉴定及部分酶活特性

Characterization, screening and molecular classification of low-temperature-protease strains from Antarctic microorganism

从南极获得的260株低温细菌中筛选到107株具有蛋白酶活性菌株,其中5株菌所产蛋白酶的活性高于45U/mL。对其进行16SrRNA基因序列的同源性和系统发育分析,结果表明,菌株NJ276、NJ59、NJ1670、NJ345属于假交替单胞菌属(Pseudoalteromonas),NJ341属于科尔韦尔氏属(Colwellia)。对其中NJ276、NJ341、NJ1670、NJ345这4株产蛋白酶南极嗜冷菌的生长及分泌蛋白酶的部分酶活特性进行研究,结果表明:(1)4株菌最适生长、产酶温度均为10℃左右;培养2～5d,嗜冷菌生长、产酶量一直处于较高的状态。(2)4株南极嗜冷菌分泌的蛋白酶的酶活反应最适pH值为9。(3)菌株NJ276、NJ59分泌的蛋白酶最适酶活温度为50℃;菌株NJ341、NJ345分泌的蛋白酶最适酶活温度为40℃;在0℃时蛋白酶活性是最高活性的30%左右,蛋白酶热稳定性较差,因此菌株NJ341、NJ345分泌的蛋白酶属于低温蛋白酶。

The continental Antarctica is considered the unique, mostly pristine and extreme environment, thus it would be the new and potential sources of low-temperature enzymes from Antarctic microorganism. In this study, bacteria samples were isolated from the sea ice and sea water in Antarctica during 2001-2002. These strains were purified and conserved in the Key Laboratory of Marine Bioactive Substances, State Ocean Administration (SOA)of China.One hundred and seven strains producing protease were screened from 260 strains of Antarctic psychrotrophic bacteria, among which proteolytic activity of five strains was more than 45?U/ mL. The 16S rRNA gene sequences homology and phylogenetic analysis of the five Antarctic psychrotrophic bacteria showed that NJ276,NJ5-9,NJ16-70 and NJ345 belonged to the described genus Pseudoalteromonas and NJ341 belonged to genus Colwellia. The growth and the protease characteristics of four Antarctic psychrotrophic bacteria were studied, and the results showed that the optimal temperature for growth and proteaseproduction of the four strains was about 10?℃. Their growth and proteaseproducing were still high during 2-5 days of incubation. The maximum proteolytic activity occurred at pH 9 for the four Antarctic psychrotrophic bacteria. The optimal temperature of protease action for Strains NJ276 and NJ5-9 was about 50?℃,however,the optimal temperature of protease action of Strains NJ341 and NJ345 was about 40?℃, and their proteolytic activity under 0?℃ exhibited nearly 30% of the maximum activity, but their thermal stabilities were weaker. These results indicated that proteases from NJ341 and NJ345 were low-temperature protease.