醱酵培養對於基因重組E. col i生產endo-glucanase之研究

The Study of endo-glucanase Production by Recombinant Escherichia coli in Fed-batch Cultivation

鄭宇哲(Yu-Che Cheng)

指導教授 ： 陳志成

大同大學/工程學院/生物工程學系所/碩士(2011年)

摘要

本篇研究採用基因重組大腸桿菌利用醱酵槽來進行有關纖維水解酵素生產之研究。於實驗中，探討培養基種類、誘導劑種類及饋料的方式與組成對菌體生長及纖維水解酵素表現之影響，並使用固定流速饋料來達成自動化生產之目標。從研究結果顯示，使用Luria Bertani (LB)培養基於醱酵12小時後菌體乾重達到3.56 g/L，使用誘導劑為IPTG，於醱酵12小時後取出醱酵液於搖瓶中誘導48小時的纖維水解酵素活性為570 U/L，蛋白量為1888 mg/L，而於醱酵槽內整體培養基誘導48小時的酵素活性為567 U/L，蛋白量為1967 mg/L，可看到兩種誘導方式的差異不大。在培養基探討上，改用Terrific Broth (TB) 培養基於醱酵12小時後的菌體乾重達到8.84 g/L，酵素活性於醱酵第4小時之菌體誘導48小時後，其活性達到944 U/L，蛋白量為1099 mg/L，但是第12小時之醱酵溶液之活性誘導則下降到624 U/L。在誘導劑選擇上，使用廉價之Disaccharide作為誘導劑添加於TB培養基中，於醱酵第4小時之菌體誘導48小時後，其活性達到1075 U/L，蛋白量為1814 mg/L，但是於第12小時之醱酵溶液之活性誘導則下降到694 U/L，可看到以雙醣作為誘導劑的酵素活性與IPTG誘導差異不大。在改變饋料探討上，使用醇類作為饋料，設定醇類濃度維持於3 g/L範圍，於醱酵第4小時之菌體經雙醣誘導48小時後，其活性達到888 U/L，蛋白量為1447 mg/L，在醱酵12小時後酵素活性維持在1073 U/L，蛋白量為2003 mg/L，與使用葡萄糖為饋料比較，酵素活性可維持而不下降。在醇類設定濃度探討上，發現提升醇類設定濃度為 4 g/L broth，於醱酵第6小時之菌體誘導48小時後，其活性達到1380 U/L，蛋白量為1992 mg/L，醱酵12小時後酵素活性略降為1276 U/L，蛋白量為2571 mg/L，整體來看表示醇類濃度提升也可提升酵素活性。在固定流速饋料(醇類)探討上，饋料瓶醇類濃度為50 g/L，幫浦流速為1.33 g/min，可得雙醣誘導之最大酵素活性為1296 U/L，蛋白量為2362 mg/L。若提升饋料瓶中的醇類濃度為 75 g/L，幫浦流速為1.37 g/min，於醱酵第10小時之菌體經雙醣誘導48小時後可得之最大酵素活性為1588 U/L，蛋白量為2140 mg/L。所以固定流速之醇類饋料有助於纖維水解酵素自動化量產程序之開發。

關鍵字

纖維水解酵素 ； 大腸桿菌

並列摘要

This study used recombinant E. coli BL 21cultured in jar fermentor to produce endo-glucanase. In this experiment, medium composition，inducer and feeding components were tested their effect on the production of endo-glucanase. By using Luria Bertani (LB) broth after 12 hours of fermentation the dried cell weight reached 3.56 g/L; in using IPTG as inducer in the 12 hrs fermentation broth, the induced enzyme activity was 570 U / L and protein concentration is 1888 mg/L after 48 hrs induction in the flask culture. Meanwhile when the induction was performed in the whole broth within fermentor for 48 hrs，the induced enzyme activity was 567 U/L and protein concentration was 1967 mg/L. Therefore, there are similar result of induced enzymatic activity during two types of induction. By using Terrific Broth (TB) broth after 12 hours of fermentation, the dried cell weight could reach 8.84 g/L. In using IPTG as inducer in the 4 hrs fermentation broth with induction for 48 hrs, the induced enzyme activity was 944 U/L and protein concentration was 1099 mg/L，but in 12 hr fermentation broth with induction for 48 hrs, the induced enzyme activity decreased to 624 U/L。When using Disaccharide as inducer in the 4 hrs fermentation broth with induction for 48 hrs, the induced enzyme activity reached 1075 U/L and protein was 1814 mg/L，then in the 12 hr fermentation broth with induction for 48 hrs, the induced enzyme activity decreased to 694 U/L. Therefore, there was similar result of induced enzymatic activity by using IPTG or Disaccharide as the inducer. By using alcohols as the feeding carbon source，by setting controlled alcohols concentration in the rage of 3 g/L and using Disaccharide as inducer, in the 6 hrs fermentation broth with induction for 48 hrs, the induced enzyme activity was 888 U/L and protein concentration was 1447 mg/L, but in the 12 hrs fermentation broth with induction for 48 hrs, the induced enzyme activity reached 1073 U/L and protein concentration is 2003 mg/L. By controlling concentration of alcohols in the range of 4 g/L，in the 6 hrs fermentation broth and induction for 48 hrs, the induced enzyme activity was 1380 U/L and protein concentration is 1992 mg/L，but in the 12 hrs fermentation broth with induction for 48 hrs, the induced enzyme activity decreased slightly to 1276 U/L and protein concentration was 2571 mg/L. By using a fixed flow rate of feed (50 g/L alcohols) and pumping flow rate of 1.33 g/min，in the 12 hrs fermentation broth with induction for 48 hrs, the induced enzyme activity was 1296 U/L and protein concentration was 2362 mg/L. Enhancing the concentration of stocked alcohols concentration to 75 g/L and pumping flow rate of 1.37 g/min，in the 10 hrs fermentation broth with induction for 48 hrs, the induced enzyme activity was 1588 U/L, and protein concentration was 2140 mg/L. Therefore the fixed flow rate of alcohols in the feeding stream can help for the development of automatic production process.

並列關鍵字

escherichia coli ； cellulase

參考文獻

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