Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/99570
標題: 應用Bacillus subtilis WB800N生產人造纖維素體酵素與其固定化酵素之特性與動力學研究
Deciphering enzymatic characteristics and kinectics of the artificial cellulosome from Bacillus subtilis WB800N and its immobilization
作者: 林家騏
Chia-Chi Lin
關鍵字: 人造纖維素體酵素
內切葡聚醣酶
外切葡聚醣酶
木聚醣酶
微藻
再生纖維膜
酵素固定化和酵素動力學
artificial cellulosome
endoglucanase
exoglucanase
xylanase
microalgae
Regenerated cellulose membrane
immobilized enzyme
and enzymatic kinetics
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摘要: 纖維素體,一種多重酵素複合物,可以協同作水解纖維素成小分子化合物。重組Bacillus subtilis WB800N攜帶一個質體pGETS118 纖維素體酵素基因可產生人造纖維素體酵素。而纖維素體酵素結構中包含有6個纖維素水解酵素蛋白。此一蛋白質重組結構目前各個研究單位都在探索其可以運用的範疇,期望能成為生質能源開發的重要工具。 因此,本研究將分為三個部分。第一部分為探討酵素特性分析Bacillus subtilis WB800N纖維素體酵素的特徵,由結果顯示發現添加1mM IPTG的LB培養基為對纖維素體酵素生產之最佳條件。進一步分析,在收取菌株的發酵液後發現,發酵液經離心後可以分別在上清液與細胞壁上偵測到纖維素體酵素的活性。在酵素活性分析的結果中發現,上清液的纖維素體酵素及細胞壁上的纖維素體酵素的活性分別為3.544±0.17 U / mL和1.931±0.072 U / mL。其中,內切葡聚醣酶和木聚醣酶的有最佳活性。藉由HPLC分析酵素反應的最終產物中,僅有寡糖和纖維二糖在酵素反應中產生的。在動力學研究中,上清液纖維素體酵素的內切葡聚醣酵素的Vmax值(8.98 gL-1 h-1)比錨定於細胞壁的纖維素體酵素高(0.603 g L-1h-1)。然而,錨定於細胞壁的纖維素體酵素的內切葡聚醣酶,其Km值較低,為0.359 g L-1,此數值顯示錨定於細胞壁的纖維素體酵素比上清液纖維素體酵素具有較佳的基質親和力(11.87 g L-1)。 第二部分,則著重於探討纖維素體酵素對微藻油脂萃取殘留物水解程度分析,由於微藻經油脂萃取後所殘留廢棄細胞壁經FTIR成分分析,仍含有纖維素的成分。為了進一步增加廢棄微藻的利用率,因此以此成分為酵素水解反應基質。在酵素活性結果分析中,在上清液纖維素體酵素對微藻油脂萃取殘留物可得到為2.4 U / mL活性。在動力學研究中,在吸附於細胞壁上和發酵液中的纖維素體酵素水解微藻油脂萃取殘留物的Vmax值分別為0.182和3.522 g L-1 h-1。吸附於細胞壁上和發酵液中的纖維素體酵素對水解微藻油脂萃取殘留物所得到的Km值分別為34.66和14.83 g / L。此一章節的結果顯示出吸附,細胞壁上與分泌到發酵液中的纖維素體酵素對水解微藻油脂萃取殘留物相比,後者可以獲得較大的Vmax 以及較佳的基質親和力。 第三部分為了增加纖維素體酵素使用的便利性,因此將發酵液中的纖維素體酵素固定於再生纖維膜上,並且將微藻油脂萃取殘留物先行使用酸水解將其降解成質地較為鬆散與含有多醣的狀態下,增加與酵素碰撞反應的機率。經固定化的纖維素體酵素在反應溫度為50℃,反應pH 值為5的條件下,經酸處理後的微藻油脂萃取殘留物反應後獲取較佳之水解效率。並且,酵素水解反應時間可以維持24hr之內皆可以獲得水解產物。進一步經由HPLC分析,其水解產物仍是以寡糖為主。
Cellulosome, a multienzyme complex, can synergistically hydrolyze polysaccharide cellulose into small compounds. The recombinant Bacillus subtilis WB800N harboring pGETS 118 cellulosome genes can produce artificial cellulosome, including six cellulose hydrolysis enzyme subunits. At present, the researchers are exploring the scope of application of this recombinant protein structure, hoping to become an important tool for bio-energy development. Therefore, this study will be divided into three parts. The first part is to discuss the deciphering characteristics of the designer cellulosome from Bacillus subtilis WB800N via enzymatic analysis. The results were shown the conditions for the production of recombinant B. subtilis was tested and the LB medium with 1mM IPTG was found to be the best condition for cellulosome production. It was noted that cellulosome activity can be found in the supernatant (SC) of the harvest broth and anchored cellulosome (AC). The results showed that the best activities of endoglucanase and xylanase obtained in SC as 3.544±0.17 U/mL and 1.931±0.072 U/mL, respectively. In the kinetic studies, the Vmax value (8.98 g L-1 h-1) for endoglucanase in SC was higher than that in AC (0.603 g L-1 h-1). However, the endoglucanase of AC gave a lower Km value of 0.359 g L-1, indicating a higher substrate affinity than that of SC (11.87 g L-1). In the second part, we focused on the analysis of the hydrolysis degree of lipid-deprived residuals of microalgae by the cellulosome. The profile of the lipid-deprived residuals of microalgae was still contained cellulose by FTIR analysis. In order to further increase the utilization of microalgae, this component such as substrate was applied to degrade by cellulosome hydrolysis. The results were shown SC to obtain cellulosome activity of 2.4 U/mL. In the kinetic studies, the anchoring cellulosome (AC) and secreted cellulosome (SC) of Vmax were obtained 0.182 and 3.522 g L-1 h-1, respectively. The Kms were measured as 34.66 and 14.83 g / L in AC and SC, respectively. The activation energy of the cellulosome to hydrolyze microalgae LDRs was calculated as a 32.804 kJ/mol. The results were shown that the SC hydrolyzed microalgae LDRs can obtain a larger Vmax and a better substrate affinity than AC. In the third part, in order to increase the convenience of the use of SC, the SC was immobilized on the regeneration cellulose membrane. The microalgae LDRs were pretreared with acid before immobilized enzyme hydrolsis. For this reason, the degraded microalgae LDRs were became looser and increase the probability of collision with the enzyme reaction. The acid treatment LDRs can be hydrolyzed with the immobilized cellulosome at the reaction temperature as 50 ℃ and the reaction pH value as 5. The hydrolysis products could be obtained under enzymatic hydrolysis reaction for 24 hours.
URI: http://hdl.handle.net/11455/99570
文章公開時間: 2020-07-05
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