共價鍵結固定青花菜葉綠素酶1於磁性粒子用以生產葉綠素衍生物

Covalent Immobilization of Brassica oleracea Chlorophyllase 1 (BoCLH1) onto magnetic particles for production of chlorophyll derivatives

黃文鴻

酵素固定化 ； 青花菜綠葉素酶1 ； 磁性氧化鐵粒子 ； 葉綠素衍生物 ； Enzyme immobilization ； Brassica oleracea chlorophyllase 1 (BoCLH1) ； magnetic particles ； Chlorophyll derivatives

義守大學化學工程學系暨生物技術與化學工程研究所學位論文

2017年

碩士

李立群

繁體中文

本研究成功將青花菜葉綠素酶1 (BoCHL1)之重組蛋白以共價鍵鍵結方式固定於APTES包覆的磁性氧化鐵氧化鐵粒子上， 而固定化效率可達到76.73±1.98%。固定化酵素催化水解葉綠素a的活性為40.4±0.09 U / g gel，而比活性為1.4±0.1 U / mg。固定化重組BoCHL1在無水環境下可催化葉綠素a和香茅醇進行轉酯化反應36小時可以明顯偵測到葉綠酸香茅酯的生合成，濃度為0.273 mM。而純化之游離重組BoCLH1的最適反應時間為48小時，只能生合成0.09 mM的葉綠酸香茅酯。比較游離與固定化酵素之生化特性顯示固定化酵素的最適反應溫度為20 °C，而純化游離之重組BoCLH1的最適反應溫度為30 °C；而固定化酵素在高溫環境下(40 和50 °C)的熱穩定度高於純化游離之酵素，且批次操作重複3個週期後，固定化酵素仍可維持約40%的殘留活性。此外，新合成物-葉綠酸香茅酯的抗氧化能力(37.6%)也高於葉綠素a (23.5%) 和香茅醇(31.7%)。由上述結果表明，利用APTES包覆的磁性氧化鐵以共價鍵鍵結方式固定重組BoCHL1可以有效提高酵素的穩定性、轉酯化合成活性、改善熱穩定性以及便於酵素回收而可達到3次的重覆使用率之優點，此技術可做為工業生產葉綠素衍生物之催化反應器，深具研究潛力。

In this study, the recombinant BoCLH1 was successfully immobilized on APTES-coated magnetic iron oxide particles (MIOPs) by covalent binding, led to markedly improve enzyme performance and decrease biocatalyst costs in industrial application. Immobilized enzyme showed high immobilization yield (76.73 ± 1.98%) and confirmed the immobilized recombinant BoCLH1 retained enzymatic activity with 40.4±0.09 U/g gel and the specific activity with 1.4 ± 0.1 U / mg protein by chlorophyllase assay. Immobilized enzyme could catalyze chlorophyll a and citronellol to a novel chlorophyll derivative (citronellylchlorophyl a ) by trans-esterification that was carried out for 36 hours under anhydrous conditions, and the concentration of citronellylchlorophyl a reached to 0.273 mM. However, the optimal reaction time of free enzyme was 48 hours, and the citronellylchlorophyl a prodution was 0.09 mM. Biochemical analysis of the immobilized and free enzyme, the optimum temperature for trans-esterification of the immobilized and free enzyme was at 20 °C and 30 °C, respectively. In addition, compared with free enzyme, the thermal stability of the immobilized enzyme showed higher activity of trans-esterification in high temperature environment (40 to 50 °C). A residual activity of approximately 40% was maintained after 3 cycles in a repeated-batch operation. In addition, the antioxidant capacity of the novel citronellylchlorophyl a (37.6%) was higher than chlorophyll a (23.5%) and citronellol (31.7%). Therefore, MIOPs-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of chlorophyll derivatives.

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