以固定化細胞Pichia fermentans L-5 生產乙酸苯乙醇及乙酸苯乙酯之研究

Production of 2-Phenylethyl alcohol and 2-Phenylethyl acetate by immobilization cultures of Pichia fermentans L-5

李建育(Chien-Yu Lee)

指導教授 ： 張志雄

大同大學/工程學院/化學工程學系所/碩士(2007年)

摘要

將含有Pichia fermentans L-5 的藻酸鈉滴入氯化鈣中形成菌體顆粒。連續五天的實驗得知，藻酸鈉以及氯化鈣濃度、顆粒大小會直接影響到菌體顆粒的流失與固定後菌體的殘餘量。藉由均勻設計（Uniform Design）方法，找出最佳的固定化條件(顆粒大小1.5mm、藻酸鈉4.4 (w/v)、氯化鈣2.0 (w/v)、菌體濃度42 (A600))。
使用最佳的固定化條件，置入100mL 的轉化液中，在200rpm、30℃下轉化培養24 小時，反應後產物為乙酸苯乙醇及乙酸苯乙酯。與未固定菌體的產物相比，固定化的乙酸苯乙醇達到88％ 而乙酸苯乙酯則有63％ 的產量。雖然未固定菌體反應的產量較高，但是反應後的產物對菌體生長有害，導致無法進行2 次轉化反應。另一方面，固定化的菌體進行連續5 天的轉化反應，乙酸苯乙醇的產量為242~67mg/L 而乙酸苯乙酯的產量為226~167mg/L。由於連續的反應使菌體活性降低，所以把固定後的菌體分成兩組反應。當第1 組進行轉化時，第2 組在培養液(5% 葡萄糖、0.25% yeast extract)內進行活化培養。每組轉化5次，共反應10 天，乙酸苯乙醇產量從267到206 mg/L 而乙酸苯乙酯產量從219到190 mg/L。

關鍵字

乙酸苯乙酯 ； 均勻設計 ； 藻酸鈉 ； 乙酸苯乙醇

並列摘要

Pichia fermentans L-5 was entrapped by drop-wise addition of an aqueous mixture of sodium alginate to a hardening solution of a Ca2+ salt. Effects of immobilization conditions such as alginate concentration, CaCl2 concentration, ratio by weight of cell to alginate (C/A) and bead size on the average loss of the immobilized cell and the residual cell immobilized in the bead over five cultivation days were investigated. Six experiments located by the uniform design (UD) method were carried out to find the optimized immobilization condition (alginate concentration 4.4 (w/v), CaCl2 concentration 2.0 (w/v), cell concentration 42 (A600), bead size 1.5mm).
Adopting the same culture conditions (100mL culturing medium in a 250mL Erlenmeyer flask, shaking at 200rpm at 30 for 24h per run) and the obtained optimized immobilization condition, production of 2-phenylethanol and 2-phenylethyl acetate by the immobilized and free Pichia fermentans L-5 were carried out. It was found that the immobilized cell can give 88% (246/280(mg/L)) and 63% (220/350(mg/L)) production efficiency of 2-phenylethanol and 2-phenylethyl acetate respectively with respect to the free cells cultivation. Although the immobilized cell cultivation cannot maintain the same production efficiency as the free cells cultivation, the used free cells cannot generate any products in the second run (another 24 h) because the free cells may be poisoned by their products. On the other hand, the same immobilized cells can be cultivated for 5 runs continuously to produce 2-phenylethanol (242~67mg/L) and 2-phenylethyl acetate (226~167mg/L). The cultivation process using the immobilized cells can be improved by two sets of the immobilized cell beads. When the first set of the immobilized cell beads were cultivated, the second set of the immobilized cell beads were maintained in the preculturing medium containing 5% glucose and 0.25% yeast extract. By this operating mode (5 runs for each set in 10 days), the productions of 2-phenylethanol and 2-phenylethyl acetate were ranged from 267 to 206mg/L and 219 to 190mg/L respectively.

並列關鍵字

sodium alginate ； 2-phenylethanol ； 2-phenylethyl acetate ； Uniform Design

參考文獻

林子楊，”在發酵槽中以高密度細胞Pichia fermentans L-5生產乙酸苯乙醇及乙酸苯乙酯之研究,” 碩士論文，大同大學生物工程研究所，民國93年。

Berger, R.G. Why novel biotechnology of aroma? In Aroma biotechnology. Springer Verlag Berlin Heidelberg, NewYork. 1995, 35-41.

Chang, J. S.; Lin, J. P. Product and Process Development via Pseudo-Sequential Uniform Design. Ind. Eng. Chem. Res. 2004, 43, 4278-4292.

Chou, C. C.; Huang, C. J.; Lee, S. L. Prouction of 2-phenylethanol, a flavor ingredient, by Pichia fermentans L-5 under various culture conditions. Food Research International. 2001, 34, 277-282.

Demuth, M.; and Rotthaus, O. Efficient cyclization of squalene epoxide to lanosterol with immobilized cells of baker’s yeast. Tetrahedron. 2002, 58, 7291-7293.