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研究生(外文):Mei-Lun Lee
論文名稱(外文):A serine protease gene from the Exiguobacterium sp. strain LP15: cloning, sequencing, and characterization
指導教授(外文):Fu-Shyan Wen
外文關鍵詞:Exiguobacteriumserine protease genecloning
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LP15為本實驗室分離低溫菌計畫中篩得的一株能產生胞外蛋白質分解的陸生耐冷菌,經BIOLOG系統及16S rRNA定序法鑑定後,將此菌命名為Exiguobacterium sp. LP15。LP15為革蘭氏陽性菌,菌體呈短桿狀,菌落為黃橘色,最適生長溫度約為30℃。本實驗依據subtilase family的蛋白分解活性部位的高度保留胺基酸序列設計合成退化性引子(degenerated primer),進行初步的選殖與定序,再以PCR-基因庫篩選法及南方墨點法,從LP15的染色體基因庫篩選得到選殖株,共得到一段長為5403 bp的序列,其中有一段長為2412 bp的序列可以轉譯出包含804 個胺基酸的蛋白質。將此蛋白質的胺基酸序列利用fasta3程式到SWISS-Pro蛋白質資料庫比對,發現和Bacillus haloduraus及B. subtilis的胞壁蛋白分別有約39.7%及38.7%的相似度。另外與NCBI的蛋白質保留區資料庫比對,也顯示和subtilase家族的活性區域有高度的相似性,所以推論LP15的蛋白分解是一種絲胺酸蛋白。

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.

中文摘要(chineae abstract)---------------------------- 1
英文摘要(English abstract)---------------------------- 2
Ⅰ 前言----------------------------------------------- 3
一.環境微生物資源利用------------------------------ 3
二.蛋白分解之應用-------------------------------- 4
三.蛋白分解之限制與改善策略---------------------- 8
四.實驗目的---------------------------------------- 8
Ⅱ 材料----------------------------------------------- 9
Ⅲ 方法----------------------------------------------- 10
一.菌株之篩選-------------------------------------- 10
二.菌株型態觀察------------------------------------ 10
三.菌株保存---------------------------------------- 11
四.菌種鑑定---------------------------------------- 11
五.細菌生長測定------------------------------------ 16
六.增幅部分LP15蛋白分解基因--------------------- 17
七.南方墨點法-------------------------------------- 17
八.北方墨點法-------------------------------------- 18
九.基因庫的構築------------------------------------ 19
十.PCR-基因庫篩選法-------------------------------- 22
Ⅳ 結果----------------------------------------------- 25
一.土壤細菌的篩選---------------------------------- 25
二. LP15的型態特徵--------------------------------- 25
三. LP15的菌種鑑定--------------------------------- 25
四.生長曲線---------------------------------------- 26
五LP15蛋白分解基因之選殖------------------------ 27
六基因庫的構築------------------------------------- 29
七基因庫的篩選------------------------------------- 29
八溶菌斑篩選結果----------------------------------- 29
九LP15蛋白分解基因之分析------------------------ 30
十LP15蛋白分解胺基酸序列之分析------------------ 31
十一LP15蛋白分解嫌水性預測分析------------------- 32
Ⅴ討論------------------------------------------------ 33
Ⅶ表與圖---------------------------------------------- 44
Ⅷ 附錄----------------------------------------------- 67
附錄A 菌種、噬菌體與質體------------------------- 67
附錄B 引子--------------------------------------- 69
附錄C 培養基配方--------------------------------- 70
附錄D 實驗試劑----------------------------------- 72

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