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Immobilization of Candida rugosa lipase onto amine functionalized polyvinylidene fluoride membrane for 2-phenylethyl acetate synthesis
|關鍵字:||PVDF membrane;聚偏二氟乙烯;immobilized enzyme;level of hydrophobic;2-phenylethyl acetate;enzyme kinetics;ternary complex mechanism;固定化酵素;疏水程度;二醋酸苯乙酯;酵素動力學;三級複合體機制||出版社:||化學工程學系所||引用:||1. SHAN, H., LU, Y., LI, Z., LI, M., CAI, Y., SUN, X., and ZHANG, Y., “A Novel Temperature-Controlled Ionic Liquid as the Medium for Phenylethyl Acetate Synthesis Catalyzed by Lipase,” Chinese Journal of Catalysis, Vol. 31, No. 3, pp. 289-294 (2010) 2. Mateo, C., Palomo, J.M., Fernandez-Lorente, G., Guisan, J.M., and Fernandez-Lafuente, R., “Improvement of enzyme activity, stability and selectivity via immobilization techniques,” Enzyme and Microbial Technology, Vol. 40, No. 6, pp. 1451-1463 (2007) 3. Yang, J., Ma, X., Zhang, Z., Chen, B., Li, S., and Wang, G., “Lipase immobilized by modification-coupled and adsorption-cross-linking methods: A comparative study,” Biotechnology Advances, Vol. 28, No. 5, pp. 644-650 (2010) 4. 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在本研究中，利用三種不同親疏水性質的薄膜做為酵素固定化之載體，包括再生纖維 (regenerated cellulose, RC) 膜、玻璃纖維 (glass fiber, GF) 膜及聚偏二氟乙烯 (polyvinylidene fluoride, PVDF) 膜。在酵素固定化流程上，三種薄膜均先導入親水性之1,4-diaminobutane (1,4-DA)，之後再導入glutaraldehyde (GA)，使薄膜表具有醛基，最後以脂肪分解酶上之氨官能基與醛基產生共價鍵結，以完成酵素固定化步驟。研究結果顯示，將脂解酵素固定於疏水性之PVDF-DA-GA薄膜展現最高之固定化酵素活性，其活性比起親水性之RC-DA-GA薄膜高出11倍。因此，本研究選擇PVDF-DA-GA薄膜進行後續研究。
酵素固定化之最適化研究係利用實驗設計法，探討固定化時間、溫度、酸鹼值及酵素液濃度對於固定化酵素活性之影響。根據脊型分析可以得到最適化之酵素固定化條件為固定化時間86分鐘、溫度33oC、固定化酸鹼值6.1及固定化酵素液濃度7.1 mg/ml，其活性為59.99 U/g matrix。
最後，本研究以二苯乙醇(2-phenyl ethanol)及乙酸乙烯酯(vinyl acetate)為基質，利用游離或固定化酵素在有機溶劑中催化轉酯化反應，產生高單價之玫瑰香精，二醋酸苯乙酯 (2-phenylethyl acetate)。由酵素動力學研究可以發現，此反應遵循ternary complex機制。從動力學參數可以知道，二苯乙醇對於游離酵素之親合力明顯高出乙酸乙烯酯許多(K2PE = 4.55×10-2 M, KVA = 1.85 M)，近而導致二苯乙醇對於游離酵素之抑制現象；而其對於固定化酵素之親合力較乙酸乙烯酯僅高出一些(K2PE = 1.74 M, KVA = 4.96 M)。酵素經由固定化方式，雖然降低了其與基質之親合力，卻也明顯改善了基質抑制的現象。最後，由ternary complex包含動力學參數之積分式可以預測時間與轉化率之關係，而由實驗結果與預測值比較，亦有相當地高契合度。
In this study, three membranes, regenerated cellulose (RC), glass fiber (GF) and polyvinylidene fluoride (PVDF), were grafted with 1,4-Diaminobutane (DA) and then activated with glutaraldehyde (GA) for lipase covalent immobilization. The efficiencies of lipases immobilized on these membranes with different hydrophobic/hydrophilic properties were compared. The lipase immobilized on hydrophobic PVDF-DA-GA membrane exhibited an over 11-fold increase in activity as compared to its immobilization on a hydrophilic RC-DA-GA membrane. Thus the PVDF-DA-GA membrane was selected for the following study.
The characterizations of native and amine functionalized PVDF membranes were qualitatively determined by using SEM and contact angle measurement. Response surface methodology (RSM) with a 5-level–4-factor central composite rotatable design were employed to evaluate the effects of immobilization parameters, such as immobilization time, temperature, pH, and lipase concentration, on the activity (U/g matrix) of immobilized lipase and based on the analysis of ridge max, the optimum immobilization conditions were as follows: immobilized time 85.9 min, temperature 33.3 ℃, pH 6.1 and lipase concentration 7.1 mg/ml; the highest activity obtained was 59.99 U/g matrix.
Moreover, the catalytic ability of lipase-immobilized membrane for production of 2-phenylethyl acetate (rose flavor) from 2-phenylethaol (2PE) and vinyl acetate (VA) was investigated. The reaction kinetics of 2-phenylethyl acetate via a free or immobilized lipase membrane catalyzed in hexane was evaluated using response surface methodology with a 5-level-2-factor central composite rotatable design. The data obtained indicated that the reaction follows a ternary complex mechanism. The kinetics results showed that the immobilized lipase had the same magnitude affinity for both substrates (K2PE = 1.74 M, KVA = 4.96 M) while the free lipase had a higher affinity for 2PE (K2PE = 4.55×10-2 M, KVA = 1.85 M). The yields predicted from integrated ternary complex mechanism rate equation with the kinetic constants were well fitted with experimental results.
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