請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/50764
標題: Cytophaga sp.生澱粉水解基因於Bacillus subtilis中之表現
Expression of raw starch-digesting amylase gene feom Cytophaga sp. in Bacillus subtilis
作者: Hung, Hui-Chen
洪慧貞
關鍵字: 雙向載體
shuttle vector
枯草桿菌
表現
生澱粉水解酵素
Cytophaga sp.
轉錄起始點
胞外分泌
B. subtilis
expression
raw starch- digesting amylase
Cytophaga sp.
transcription start site
extra-cellular
出版社: 食品科學系
摘要: 在先前(陳, 1998)的研究中,已將Cytophaga sp.菌株中rsda選殖於pAcUW21質體,並轉形於Escherichia coli中,重組質體命名為pARMH10,表現RSDA於胞內系統。為簡化純化流程,本實驗進行B. subtilis胞外分泌系統之載體構築。首先以限制 剪切質體pARMH10 (包含3.2 kb rsda基因),與可在B. subtilis進行複製之質體pWP18連接,可得重組質體pAWR。基於工業使用之安全考量,構築另一重組質體表現RSDA。以重組質體-pARMH10為模板,利用聚合 鏈鎖(PCR)反應合成一段約2.7 kb片段的rsda基因,將此基因選殖於一雙向載體- pHY300PLK,可得重組質體pHR轉形入B. subtilis中。兩個雙向重組載體於B. subtilis皆可表現RSDA於胞外。Primer extension實驗結果顯示,rsda基因之轉錄起始點位於起始碼上游第34個鹼基,預測啟動子區域為原來Cytophaga sp.所選殖的基因片段,-35 TTGACT -10 TATAGA。培養重組質體於B. subtilis之2YT培養基中進行培養,生產酵素量以pAWR高於pHR於菌體中的表現。經60小時培養於未添加抗生素之培養基,pAWR於B. subtilis中僅殘留48%的相對穩定性,而pHR則殘留95%之相對穩定性。 B. subtilis (pAWR)培養液經不同純化步驟分離可得單一染色帶之蛋白質,回收率為33%,純化倍數為54倍。將純化之RSDA進行N端胺基酸序列分析得AATNGTMMQYFEWYVPN,與Cytophaga sp.生產RSDA之N端基酸序列相同。將不同來源之酵素進行生化性質之分析,B. subtilis與Cytophaga sp.所生產的RSDA性質較相近,而E. coli表現之RSDA生化性質較不同。上述種種結果,說明枯草桿菌胞外分泌系統可運用於澱粉加工業RSDA之大量生產。
In our previous study, the raw-starch digesting amylase gene from a soil bacterium, Cytophaga sp., was selected and constructed in plasmid, pAcUW21, and then transformed into the host cells, Escherichia coli. The recombinant plasmid was designated as pARMH10. E. coli harboring the pARMH10 expressed the gene product, raw-starch digesting amylase (RSDA) intracellular. For simplifying the followed-up purification procedures, we subclone the rsda gene into Bacillus subtilis which often secret the foreign proteins. There were two shuttle recombinant plasmids constructed in this work. A DNA fragment of 3.2 kb in length was deleted from pARMH10. The shortened product was ligated with pWP18, a plasmid containing replication region of B. subtilis. The reconstructed plasmid was designated as pAWR, and transformed into B. subtilis. Besides, a PCR product of 2.7 kb including the rsda structural gene and 5'-upstream region obtained by using pARMH10 plasmid as template was ligated with pHY300PLK, a shuttle vector. The recombinant plasmid was designated as pHR and transformed into B. subtilis. The transformants, B. subtilis (pAWR) and B. subtilis (pHR), produce RSDA extra-cellular. Primer extension experiments indicated that the transcription start site lies 34 bp upstream of the ATG start codon, and allowed the identification of —35 (TTGACT) and —10 (TATAGT) promoter-like sequences. Higher amount of RSDA was produced by B. subtilis carrying pAWR grown on 2YT medium than B. subtilis carrying pHR. Fourty-eight and ninety-five percentage of recombinant plasmids was retained as B. subtilis (pAWR) and B. subtilis (pHR), respectively, grown on 2YT medium for 60 hr without adding tetracycline. B. subtilis RSDA was purified through three steps, the purification folds was about 54 and activity recovery was 33% . The N-termnial amino acid sequence of B. subtilis RSDA determinated was AATNGTMMQYFE WYVPN, which is consistent with that of Cytophaga sp. The enzyme biochemical properties produced from different hosts shows that RSDA produced by Cytophaga sp. and B. subtilis are the same ,however, E.coli (pAWR) are different . From above results, we proposed the system could be employed in the production of large amount of enzyme for starch industrial processed application.
URI: http://hdl.handle.net/11455/50764
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