本論文運用細胞表面工程技術，建構以冰核蛋白 (Ice Nucleation Protein, INP) 為主體之細胞表面表現系統，並以此系統進行全細胞酵素之開發及應用於生物轉化之糖化修飾反應。首先，從四株野生菌篩出Xanthomonas campestris BCRC 12608具有較高的轉糖酵素活性，作為細胞酵素與轉糖酵素基因 (aglA) 之來源。其次，利用基因工程技術選殖自X. campestris BCRC 12846之蛋白INP，將INP的N端與C端連接成INPNC融合蛋白作為本論文表面表現系統之載體蛋白，得到表現質體為pETInaXNC1。進一步將選殖自X. campestris BCRC 12608之轉糖酵素基因aglA送入pETInaXNC1，獲得表面表現轉糖酵素之質體，p8XNC-aglA1與p8XNC-aglA2。我們使用E. coli JM109及E. coli BL21作為INPNC-aglA融合蛋白表現之宿主細胞，經誘導表現後可得到蛋白大小約為90 kDa之INPNC-aglA融合蛋白。
重組E. coli細胞經由細胞膜分級分離及免疫螢光法確認融合蛋白INPNC-aglA確實表現在細胞外表面，並以protease accessibility進行重組E. coli細胞酵素殘留活性試驗。在對苯二酚糖化反應中，經proteinase K處理後的細胞其酵素活性降低，為未經過處理細胞酵素活性的31 %，若細胞同時以proteinase K與EDTA處理後，其酵素活性僅存14 %。綜合實驗結果顯示，INPNC-transgluocosidase融合蛋白確實表現於E. coli外表面，表現後的轉糖酵素仍保有原酵素進行糖化修飾的能力。
以對苯二酚 (HQ) 糖化修飾生成熊果素 (arbutin) 為例，比較X. campestris細胞酵素與surface display transglucosidase之大腸桿菌細胞的活性。取0.3 g細胞酵素，以100 mM HQ及1.2 M maltose為基質於40℃及160 rpm，在總體積5 mL phosphate buffer (100 mM, pH 7.2) 條件進行酵素反應。反應1小時的結果顯示，以surface display transglucosidase為酵素時可生成熊果素23 g/L，莫耳轉化率為83 %，相較之下，以X. campestris細胞酵素進行反應僅生成熊果素4 g/L (莫耳轉化率為16 %)。此外，由酵素特徵的試驗結果顯示，無論使用X. campestris細胞酵素或是surface display transglucosidase之大腸桿菌細胞作為對苯二酚糖化修飾生成熊果素之酵素來源皆存有基質抑制與產物抑制的現象發生。
在全細胞表面表現轉糖酵素生產研究，我們採行饋料批式培養策略進行重組E. coli BL21 (p8XNC-aglA1) 之高密度培養，以乳糖作為誘導劑進行融合蛋白表現。在進行饋料批式培養重組E. coli時，採用30℃的起始細胞培養溫度，繼以25℃誘導融合蛋白的表現。以此培養策略，沒有過多的葡萄糖與醋酸累積於發酵液，因而沒有細胞生長與融合蛋白表現抑制的情況發生，並獲得高轉糖酵素活性的重組E. coli細胞。在此培養條件下，可得18 g/L重組E. coli細胞，而所獲得的全細胞酵素之比活性為501 nkat/g cell。因此，利用饋料批式培養表面表現轉糖酵素之重組E. coli細胞及以乳糖誘導融合蛋白表現的方式可以作為大規模融合蛋白表現之全細胞酵素生產的基礎。

A surface anchoring motif using the ice nucleation protein (INP) of Xanthomonas campestris pv. campestris BCRC 12846 for displaying transglucosidase was developed. The transglucosidase gene from Xanthomonas campestris pv. campestris BCRC 12608 was fused to the truncated ina gene. This truncated INP consisting of N- and C-terminal domains (INPNC) was able to direct the expression of transglucosidase fusion protein to the cell surface of E. coli.
Localization of the truncated INPNC-transglucosidase fusion protein was examined by Western blot analysis and immunofluorescence labeling, and by whole-cell enzyme activity in the glucosylation of hydroquinone. The glucosylation reaction was carried out at 40℃ for 1 h, which gave 23 g/L of alpha-arbutin, and the molar conversion based on the amount of hydroquinone reached 83 %. The use of whole-cells of the wild type strain resulted in an alpha-arbutin concentration of 4 g/L and a molar conversion of 16 % only under the same conditions. The results suggested that E. coli displaying transglucosidase using truncated INPNC as an anchoring motif can be employed as a whole-cell biocatalyst in glucosylation.
Recombinant E. coli displaying transglucosidase on the surface was used as whole-cell biocatalyst in hydroquinone glucosylation, and its enzymatic characteristics were also studied. The enzymatic activity of recombinant E. coli was seven to twelve-fold higher than that of X. campestris when using the same amount of cells. In the enzymatic characterization experiments, the optimal temperature was found to be 40℃. The optimum pH for the glucosylation of hydroquinone by E. coli displaying transglucosidase was 7.2. In the study of the effect of hydroquinone on the conversion of alpha-arbutin, it was found that the inhibitory effect of hydroquinone was profound at high concentrations of hydroquinone. In addition, conversion of hydroquinone to arbutin was inhibited slightly by high initial concentration of arbutin in the reaction mixture. Taken together, the results demonstrated that the enzyme was inhibited by both substrate and product.
A fed-batch culture strategy for the high-cell density cultivation of recombinant E. coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for alpha-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as a feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of alpha-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76 % molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.
We have successfully demonstrated the application of fed-batch culture strategy for the production of recombinant E. coli cells anchoring surface-displayed transglucosidase, for use as a biocatalyst in alpha-arbutin synthesis. Although the transglucosylating activity of recombinant cells using lactose as an inducer was slightly lower than that of the biocatalyst produced by IPTG induction, the use of fed-batch culture by lactose induction resulted in a higher productivity of whole cell biocatalysts. Therefore, fed-batch cultivation coupled with lactose induction offers an attractive strategy for the mass production of recombinant E. coli cells for use as whole-cell biocatalysts in biotransformations such as alpha-arbutin synthesis.

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黃思瑀，2004，利用生物觸媒進行對苯二酚醣化反應之研究。清華大學化學工程學系碩士論文。
行政院衛生署，2000，衛署藥字第89028104號。行政院衛生署公報29: (20) 11-12.
財團法人生物技術開發中心，2000，生物技術產業年鑑2000。8-9p.
財團法人生物技術開發中心，2003，生物技術產業年鑑2003。7p.
經濟部工業局，2002，2002生技產業白皮書。5, 7p.