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標題: Production of N-carbamoyl-D-HPG with Recombinant Escherichia coli
作者: 尹邦定
Yin, Bang-Ding
關鍵字: 大腸桿菌;Escherichia coli;固定化;穿透性;D-內醯尿脢;藻朊酸鈣;immobilization;permeabilization;D-hydantoinase;calcium alginate
出版社: 化學工程學系
Recombinant Escherichia coli cells expressing D-hydantoinase were used as the biocatalysts for the production of N-carbamoyl-D-hydroxyphenylglycine from DL-p-hydroxyphenylhydantoin. The optimal D-hydantoinase activity was observed at 40℃and pH 8.5. Thermostability and reusability of D-hydantoinase were significantly increased upon immobilization. However, the rate of N-carbamoyl-D-HPG production is relatively low, due to low substrate solubility and high substrate mass transfer resistance. In this study we have confirmed that the use of cosolvents such as DMSO can significantly enhance the rate of D-amino acid precursor production with whole cell D-hydantoinase. It was also demonstrated that the use of permeabilized cells as biocatalysts can enhance production rate by reducing mass transfer resistance and can improve product purity by eliminating the secretion of metabolites. The permeabilization of cells also lead to reduced enzyme stability toward organic and thermal denaturation and shift in optimal pH toward lower pH.

D-內醯尿脢 (D-Hydantoinase,簡稱 DHTase) 為製藥工業上用來生產β-lactam抗生素前驅物之重要酵素。目前酵素法合成D-p-HPG的主要困難為低反應基質水溶性,且以全細胞 (whole cell) 做為生物觸媒催化反應時,反應基質進入胞內與酵素反應之質傳阻力相當高。本研究以藻朊酸鈣將細胞固定化以增加酵素之熱安定性與重複使用性。並探討添加二甲亞風 (dimethyl sulfoxide,DMSO) 以增加反應基質在反應液中的濃度及以N-cetyl-N,N,N-trimethylammounium bromide (CTAB) 與戊二醛 (glutaraldehyde) 處理細胞,以改變反應基質對細胞之穿透性 (permeability) 對反應速率的影響。具D-hydantoinase活性之重組大腸桿菌細胞經固定化後熱安定性及重複使用性有顯著的增加。當DMSO濃度低於2 % 時,對細胞之酵素活性沒有負面的影響,但卻可大幅提高基質的溶解度而提高比反應速率,發現在含有1.5 % 之DMSO的反應系統中比反應速率比對照組高83 %。經CTAB與glutaraldehyde處理之高穿透性細胞催化生物轉換之比反應速率在35℃,pH 7.0下約是未經處理之細胞在相同條件下的1.6倍,細胞經穿透性處理後,最適反應pH由7.5下降至6.5,最適反應溫度也由40℃下降至35℃。
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