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研究生:陳光興
論文名稱:應用固定化脂酉每化反應之探討
論文名稱(外文):Studies on the Esterification by Using Immobilizd Lipase
指導教授:劉益善蔡珊珊蔡珊珊引用關係
指導教授(外文):Liu, I-ShanTsay, San-San
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:59
中文關鍵詞:固定化脂酉每矽藻土疏水性矽膠己酸以酯正己烷酒精溶液超臨界二氧化碳
外文關鍵詞:immobilized lipasecelitehydrophobic silanen-hexanealcoholic solutionsupercritical fluidethyl caproate
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本研究係探討四種不同微生物來源(Pseudomonas spp, Candida rugosa, Mucor javanicus, Rhizopus arrhizus)之商用脂酉每(PS、CR、MJ及RA),經由矽藻土吸附固定或利用疏水性矽膠來包蔽固定後,對其在有機溶劑正己烷中、不同濃度之乙醇水溶液中或在超臨界二氧化碳系統中來進行己酸乙酯合成時之影響。
四種游離脂酉每(PS-P、CR-P、MJ-P及RA-P)在正己烷中進行己酸乙酯之合成反應時,以PS-P的活性最高,其次依序為MJ-P、CR-P及RA-P。在反應至8h時,PS-P之己酸乙酯轉換率可達80%,每毫克酵素每分鐘可合成0.0462μM的己酸乙酯。
各種脂酉每經以每克擔體固定200mg酵素後,其酯化反應速率均可加快,其中以疏水性矽膠來包蔽固定PS脂酉每(PS-E)的活性為最強,在反應至1.5h時,其己酸乙酯之轉換率可達90%,每克固定化脂酉每每分鐘可合成298.3μM的己酸乙酯(298.3μM/min.g),約高出等量游離PS脂酉每(PS-P)活性的43倍,較以矽藻土吸附固定之PS脂酉每(PS-A)的活性高出約11倍。在有機溶劑中添加微量水,可使吸附固定PS脂酉每的活性上升,但水分含量達0.062%以上時,其脂酉每活性不再隨水分增加而上升。
以矽藻土吸附固定PS脂酉每時,每克擔體之酵素最佳固定用量為400mg。而使用矽膠包蔽固定PS脂酉每時,每克擔體可固定脂酉每之用量達800mg以上,且固定後之酵素活性隨酵素用量增加而增加。
四種游離脂酉每中,只有PS脂酉每在40%乙醇溶液中可進行酯化反應,其己酸乙酯之轉化率最高為15.4%。經矽藻土吸附固定後之轉化率可提高為23%,而用矽膠包蔽固定後之轉化率可更提高達40%。在不同濃度之乙醇溶液中,使用矽膠包蔽固定之PS脂酉每,其合成己酸乙酯之量較穩定,合成速率也隨著乙醇濃度增加而上升,在60%乙醇溶液中,其己酸乙酯之轉換率在第5h時可達43%。
以疏水性矽膠包蔽固定之PS脂酉每來進行己酸乙酯合成之重複批次;試驗時,其酵素活性之半衰期在正己烷中為40批次,而在40%乙醇溶液中為16批次。至於以矽藻土吸附固定之脂酉每,無論在何種溶劑中,其活性在第二批次反應時即已明顯下降到原活性的一半以下。將兩種不同固定化PS脂酉每應用於超臨界二化碳系統中來進行己酸乙酯之合成時,以疏水性矽膠包蔽固定之PS脂酉每為佳,在反應至8h時之轉化率可達80%,在持續反應至50h時仍可維持50%的轉化率。
Four different commercial lipases of Pseudomonas spp (PS-P), Candida rugosa (CR-P), Mucor javanicus (MJ-P) and Rhizopus arrhizus (,RA-P) have been immobilized by adsorption with celite (PS-A, CR-A, MJ-A, RA-A) and by entrappment with hydrophobic silane (PS-E, CR-E, MJ-E, RA-A). The conversion rate of capronic acid and ethanol to ethyl caproate of thase free and immobilized lipase in organic solvent n-hexane, in aqueous alcohol solution, and in the supercritical fluid of carbon dioxide system have been measured and compared in this study.
 The free PS lipase (PS-P) is found to have the highest esterification activity in n-hexane. The conversion rate can reach 80% after reaction for 8h. Each mg o f PS-P can synthesize 0.0462 μM of ethyl caproate per minute.
 The esterification activity of lipase is increased greatly after immobilization, especially for the entrapped PS lipase (PS-E). The conversion rate of PS-E can reach 90% at 1.5h, and each gram of PS-E can synthesize 298.3 μM of ethyl caproate per min. The optimum enzyme dose for adsorbed lipase is 400mg/g of celite, but for entrappment in hydrophobic silane is more than 800mg. Adding trace amount of water in n-hexane can increase the esterification activity of celite adsorbed lipase up to water content to 0.0625%.
 Among these four lipase, only PS lipase show some esterification activity in 40% alcohol solution. The conversion rate of caproic acid to ethyl caproate for PS-P, PS-A and PS-E are 15.4%, 23% and 40% respectively. The ethyl caproate synthesized by PS-E is more stable than PS-P and PS-A, and it''s rate of ester formation is increased with the increasing in alcohol content. In 60% alcohol solution, the conversion rate for PS-E at 5h is 43%.
 The immobilized lipases have been used repeatly batchwise for ethyl caproate synthesis in n-hexane or in 40% alcohol solution. For celite adsorbed lipase (PS-A), more than half enzyme activity is lost at the second batch no matter how is the polarity of the reaction medium. But for the hydrophobic silane entrapped PS lipase (PS-E), the half life of enzyme activity in n-hexane is 40 batches and in 40% alcohol solution is 16 batches.
 The immobilized lipases have also been applied in the supercritical fluid of carbon dioxide system for ethyl caproate synthesis. Better results can be obtained for the entrapped PS lipase (PS-E, the conversion rate can be got to 80% after starting for 8h and maintained at 50% when reaction proceed for 50h.
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