- 學位論文
溶菌酶復性程序中之氧化還原對之研究
The Investigation of Redox Pairs in the Lysozyme refolding Process
摘要
為了探討復性環境中，氧化還原對與溶菌酶活性回復率之關聯性，本研究以溶菌酶(Lysozyme)為操作蛋白質，在原態溶菌酶中添加8M尿素及還原態DTT(DTTred)使之變性模擬結構展開之內聚體，並利用直接稀釋法與透析法兩種方法復性。 復性系統中存在著兩組氧化還原對，即DTTred -GSSG和GSH-GSSG，對蛋白質的雙硫鍵進行氧化還原反應，修飾雙硫鍵使復性率上升，實驗結果顯示DTTred -GSSG復性能力較佳，反之GSSG-GSH則效果較差，在不含DTTred的復性環境中，改變GSSG與GSH之比例關係對蛋白質最終之活性回復率並不會有太大影響，然而GSH與雙硫鍵的反應程度會隨著環境中尿素濃度改變而變。在本研究之實驗範圍內，當透析袋內GSSG與DTTred濃度比值大於1或直接稀釋法中GSSG與DTTred濃度比值大於2.5左右時，活性回復率即有顯著的上升，而隨著比值增大，活性回復率趨勢趨於平緩。 此外將變性蛋白質在透析復性前，添加高濃度GSSG入透析袋內，使變性劑中之殘留DTTred與GSSG反應，大幅降低透析袋內DTTred濃度，並將上述復性方法稱為半批次透析復性法。經由半批次透析法復性1 g/l的溶菌酶最終活性回復率可達到80%以上，相對於批次式操作可以提高約20%，此新方法無論在活性回復效果及藥品消耗都比傳統透析法或連續式透析法得到較好的結果，進一步將半批次透析法與控制尿素濃度變化結合可以將復性蛋白質濃度提高，在復性溶菌酶之濃度分別高達4g/l、6 g/l、8 g/l時，經過復性30小時後都得到了接近70%的活性回復率。 然而利用透析法復性時，透析袋內之環境是隨時間變化的，為了掌握透析袋內各物質之濃度變化，利用質傳之觀念得到尿素、DTTred、GSSG、GSH之質傳係數，並發現透析袋內之氧化還原對比例會因各物質之質傳係數不同而改變。此外在直接稀釋法復性時，透過適當之氧化還原對比例，利用4倍的低倍率稀釋即可得到高活性回復效果，使最終溶菌酶濃度1.25 g/l得到約70%之活性回復率、最終溶菌酶濃度0.75 g/l得到約90%之活性回復率。
並列摘要
Currently, protein refolding process is the crucial issue in the bio-industry. In this research, denatured hen egg-white lysozyme was refolded by the direct dilution and the dialysis methods, while the denaturing condition was in 8M urea and reduced dithiothreitol (DTTred) for simulating the solubilized inclusion body. The goal of this work was to investigate the role of redox pairs in the refolding process. There were two redox pairs existing in the refolding system, DTTred -GSSG and GSH-GSSG, shuffling between thiol group and new-forming disulfide bonds to regain the activity. In this research, DTTred -GSSG has better refolding efficiency than GSH-GSSG on lysozyme renaturation. Moreover, in the refolding condition without DTTred, the ratio of GSH to GSSG had little influence on the activity yield. One of the reasons was that the degree of reaction of GSH upon disulfide bonds would depend on the urea concentration. On the other hand, regarding to the redox pair DTTred -GSSG, as the ratios of GSSG to DTTred are higher than 1 inside the dialysis tube and higher than 2.5 in refolding buffer of direct dilution within our experimental conditions, the activity yield would be enhanced significantly. However, while the ratio was higher than the critical value for both methods, little change was obtained in the activity yield. At the beginning of the dialysis refolding operation, high concentration GSSG was added into dialysis tube, which could react with residual DTTred and reduce the concentration of DTTred. The above protein refolding strategy was called fed-batch dialysis refolding method in this work. Fed-batch dialysis was an effective way to refold protein, for example, 80% of refolding yield could be obtained for 1g/l of lysozyme by fed-batch dialysis refolding method, 20% higher than batch operation.Besides, This fed-batch could have better performance than batch dialysis and continuous dialysis in chemicals consumption. Furthermore, via the fed-batch dialysis conbines gradually step-changing concentration of urea, the activity yield of lysozyme with high concentration which are 4g/l、6g/l and 8g/l could reach 70% after 30 hr-refolding process. In addition, in a direct dilution by only 4 times, 70% and 90 % of activity yield could be obtained with an appropriate ratio of GSSG to DTTred for lysozyme concentration of 1.25g/l and 0.75g/l, respectively.