臺灣博碩士論文加值系統

在本研究裡已將E. coli ATCC11105染色體中的pac基因片段植株到質體
pBR322的 適當位置，形成重組質體pCLL32系列與pCLL29系列，而取
pCLL3201及pCLL2902為 研究測試之代表菌株。對於重組質體pCLL2902在
轉形至HB101後，已被證實擁有非常良好的pac基因表現，但在改換成其他
種宿主細胞後，其結果則不盡理想；而 pCLL3201轉形至宿主細胞中，大
致均有不錯之表現。利用酵素活性定量分析，得 知HB101(pCLL3201)的
pac基因表現受到苯乙酸誘導，而不被葡萄糖所抑制，並且 要於低溫下才
能生產PAC酵素；而HB101(pCLL2902)則均不受苯乙酸的誘導及葡萄 糖的
抑制，但同樣須要在低溫下才能生產PAC酵素。對於HB101(pCLL2902)，在
適 當環境下，其PAC酵素產量可超出HB101(pCLL3201)或ATCC11105許多，
然而其胞生長情況卻不盡理想，可能因素為PAC酵素的基因表現對於細胞
的毒性影響了重組細胞的生長，這可能亦是日後欲高度表現該酵素基因時
將遭遇到的瓶頸問題。最後 為了在pac基因轉錄層次上作進一步的改良，
以改善PAC酵素的生產，我們擷取 pCLL2902及pCLL3201上含有pac基因
之DNA片段，副植株入pTrc99A的適當位置， 形成了重組質體
pTrcPAC3201與pTrcPAC2902，使得pac之基因表現受tac啟動子之 控拆，
實驗結果發現該二株質體之pac基因表現不佳，此部份則有待更進一步的
研究。

Recombinant DNA technology was applied for the production of an
industrial enzyme of penicillin acylase (PAC). The PAC-encoding
gene (pac) from an E. coli strain of ATCC11105 was cloned
using the PCR technology in this study. We made two
clones, pCLL3201 and pCLL2902, containing the pac gene which
was suitably expressed in many of the E. coli host strains.
The PAC expression level could be greatly enhanced, at
more than 3 folds compared to ATCC11105, using an
appropriate gene expression system. We also subcloned the pac
gene into an expression vector of pTrc99A by making two
recombinant plasmids of pTrcPAC3201 and pTrcPAC2902 in which
the pac gene expression was under the regulation of the tac
promoter system. The PAC enzyme activity was however minimally
detected for the two clones, therefore further
investigation will be required.