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  • 學位論文

澱粉水解酵素固定於金屬有機骨架之微反應器的開發與應用

Development and application of α-amylase immobilized on metal-organic-framework microreactors

指導教授 : 黃悉雅
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摘要


金屬有機骨架(Metal-Organic Frameworks, MOFs)材料應用於酵素固定化時,因蛋白質酵素體積過大(~3-10 nm),而無法進入MOFs的微孔(<2 nm)內,因此,較不易以簡單的物理吸附方式固定酵素於MOFs。 為改善此問題,本研究利用改變酵素結構的方式,使團聚的酵素變成較線性的結構以改變酵素尺寸,故能將常見的α-澱粉酶(α-amylase)固定化於MOFs上,形成澱粉酶固定化微反應器。研究中,以質譜儀及紫外─可見光譜儀(UV-Visible spectrometry)對澱粉酶固定化微反應器催化水解澱粉所得之醣產物進行定性及定量,並進行澱粉酶固定化效果及穩定性的比較。文獻中顯示,α-澱粉酶水解澱粉後所得主產物為麥芽醣(Maltose),為確認變性後的α-澱粉酶催化水解出的產物是否改變,故以質譜儀進行確認,再利用Bernfeld法檢測醣化合物的產量,評估本研究所製備的微反應器催化水解澱粉之效果。因α-澱粉酶結構中含有的色胺酸(tryptophan),此胺基酸具螢光特性,故可用螢光儀測得此固定化微反應器所吸附的酵素量為0.0997 mg α-amylase / mg UiO-66。 由實驗結果顯示,以UiO-66材料所製備之澱粉酶固定化微反應器具有最佳的催化效果,其原因推測是此MOFs孔洞尺寸(0.6 nm)吻合變性後澱粉酶的胜肽鏈片段,進而有利於酵素的吸附。此反應器與溶液態澱粉酶的催化效果相互比較後,澱粉酶固定化微反應器在第一次催化上就有80%以上的相對產率(Relative hydrolysis efficiency %),並在重複15次催化反應或重複催化25天後,其產率依然可達70%以上。因此,本實驗所開發出的澱粉酶固定化微反應器,具有可重複催化、高穩定性、易與產物分離等優點。

並列摘要


In the immobilized micro-reactor research, physical adsorption method is not commonly used due to the size of enzyme that is difficult to enter into the synthetic support. In this study, metal-organic frameworks (MOFs) were used as immobilized substrate. The α-amylase was immobilized on MOFs by changing its structure from cluster to linear type by denaturation process. Hererin, mass spectrometry (MS) and UV-visible spectrophotometry (UV-Vis) were used to identity the performance and stability of α-amylase@MOFs bioreators. For qualitative analysis, the native and denatured α- amylase were compared based on their efficiency to hydrolyze the starch. The product was identified as maltose for both α- and denatured- amylase using MS. For the quantitative analysis, Bernfeld method coupled with UV-Vis spectrophotometry, the α-amylase@MOFs efficiency was used to test (i.e : the amount of carbohydrates). Furthermore, the tryptophan, (a amino acid), in α-amylase structure has fluorescent ability, thus, the enzyme loading capacity was determined via fluorescence spectrophotometry. The loading capacity of the denature α-amylase@UiO-66 was 0.0997 mg. After the optimization procedure, the bioreactor has the best catalysis efficiency when UiO-66 was used as substrate which still remained 80% hydrolysis efficiency compared with α-amylase (in-solution). It shows that the window pore size (0.6 nm) was suitable to accommodate the peptide chain size. Finally, the denature α-amylase@UiO-66 has a relative hydrolysis efficiency over 70% even after 15 cycles or 25 days. The developed α-amylase@UiO-66 have many advantages, including reusability, stability and ease sparation from products.

並列關鍵字

metal-organic-framework immobilized amylase

參考文獻


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