ASChi61酵素水解幾丁質生產N-乙醯幾丁六醣之最佳化

Optimization of N-Acetylchitohexaose Production from chitin Hydrolysis by ASChi61 Enzyme

藍志瑜(Chih-Yu Lan)

指導教授 ： 莊瑞鑫

元智大學/工程學院/化學工程與材料科學學系/碩士(2011年)

摘要

本論文主要是探討ASChi61水解幾丁質所產生之N-乙醯幾丁六醣的最適化條件。進行條件擇定的方法是採用反應曲面法來分析，以探討在不同的膠態幾丁質濃度、反應溫度、反應時間、pH值以及酵素活性之影響，先利用Minitab分析而預測最佳條件後，再次實驗操作驗證。
將膠態幾丁質經ASChi61水解後，透過HPLC以及MALDI-TOF可以有效地並確定在第12至14分鐘可以透過管柱將N-乙醯幾丁六醣從反應液中分離、回收。
透過實驗設計中的部分因子設計法，將第1至8組與9至16組，測其還原醣量所得之OD420代入Minitab，畫出陡峭圖並交叉比對分析後，可以看出膠態幾丁質濃度與pH值具有顯著之影響，且彼此之間有交絡現象。
進一步將膠態幾丁質濃度與pH值利用反應曲面實驗設計法得最佳化條件分別為：3.5mg/ml、pH6.55，並進行實驗驗證，結果顯示透過反應曲面實驗設計所得之最適化條件，能將N-乙醯幾丁六醣產量提升至42.175mg/L，其誤差值為0.273%，而N-乙醯幾丁六醣的含量更是原本的1.59倍，因此顯示出反應曲面法具有高度可靠性。

關鍵字

幾丁質 ； ASChi61 ； N-乙醯幾丁六醣 ； 反應曲面法

並列摘要

Chitin is an insoluble linear homopolymer of N-acetyl-D-glucosamine (GlcNAc). It distributes worldwide and is one of the most abundant polysaccharides. Derivatives of chitin, inclusive of polysaccharides, oligosaccharides and monosaccharides, have been investigated to enhance the functions of immunological system in the host and they displayed tumoricidal agents recently. The physiology effects of chitin-oligosaccharides vary with the number of the oligomerization. Usually, the hexamers is the most significant. Recently, a chitinase, ASCHI61, produces hexamer was identified.
Classically, optimization of expression has been achieved linearly by changing one factor at a time. However, the interaction between different factors is overlooked. The experimental design is developed in past decades. In the work, the conditions for N-acetylchitohexaose from chitin degraeded with ASChi61 were optimized. The parameters, colloidal chitin concentration, reaction temperature, reaction time, pH, and enzyme amount, were evaluated. The optimal conditions were predicted with Minitab analysis by response surface methodology. And then they were verified by the experiments.
N-acetylchitohexaose was obtained from colloidal chitin hydrolyzed with ASCHI61 by HPLC. Its retention time was at the 12th and 14th minutes. The hexameric saccharides were quantified by measure the reducing sugar at OD420. According to the Minitab, the steep map analysis and cross-analysis, colloidal chitin concentration and pH were significant. And the interlacing was observed. By response surface experimental design, the optimal conditions for hexaoligochitin producing were predicted with colloidal chitin concentration of 3.5mg/ml at pH6.55. The conditions were verified by experiments. N-acetylchitohexaose of 42.175mg/L obtained. The error was 0.273% and the amount of hexamers was produced 1.59 times than the condition reported before.

並列關鍵字

chitin ； ASChi61 ； N-acetylchitohexaose ； Response surface methodology

參考文獻

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