A serine protease gene from the Exiguobacterium sp. strain LP15: cloning, sequencing, and characterization

• Main Authors: Mei-Lun Lee, 李美倫
• Other Authors: Fu-Shyan Wen
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/80946154601866286116

碩士 === 國立中興大學 === 植物學系 === 90 === Proteases are enzymes which catalyze the hydrolysis of peptide bonds.It constitute one of the most important groups of industrial enzymes, accounting for at least a quarter of the total global enzyme production which are commercially used in the food industry, chemical industry, and medical utilization. But protease is a kind of protein and its’ activity will be affected by temperature, pressure, ion strength, and storage problem as they are easily prone to inactivation by self-digestion (autolysis). Therefore, if we can find some more stable protease, it will be unlimited potential. LP15 is a psychrotrophic bacterium isolated in our psychro -philic bacteria-screening project. As determined by 16S rRNA sequencing and Biolog system, LP15 is more closely related to the genus Exiguobacterium (96％-98％ similarity)and was designated as Exiguobacterium sp. LP15. It is a gram-positive, rod-shaped, non-sporing bacterium and forms orange colonies on LB agar plate. It has proteolytic activity on skim milk agar plate whose optimum temperature for growth is about 30℃. In order to isolate a protease gene from the genomic DNA of LP15 by PCR analysis, the following degenerated primers were synthesized on the basis of the consensus amino acid sequences of subtilase family, which can amplify 483 bp DNA fragment. After constructing and screening the genomic DNA library, we can get a 5403 bp DNA fragment. And then, we can find an open reading frame of 2412 bp DNA sequence, which can encode a 804 amino acid. This amino acid exhibits sequence 39.7％ and 38.7％ similarities with cell wall-associated protease of Bacillus haloduraus and Bacillus subtilis. There is a high level of conservation in the region around a putative active site after searching the protease sequence in the conserved domain database. Therefore, we consider the gene should be a kind of serine protease related to subtilase family in preliminary analysis.