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研究生:靳其傑
論文名稱:基因重組Candidarugosa脂肪酉每表現,純化和特性研究
論文名稱(外文):Purufucatuin and Characterization of Recombinant Candida rugosa Lipase in Yeast
指導教授:唐世杰
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產生物技術研究所
學門:生命科學學門
學類: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:英文
論文頁數:59
中文關鍵詞:脂肪酵素酵素動力學
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  脂肪酵素(Lipases)廣泛存在於各生物體中,且在分子大小,受質專一性和催化速率上有所不同,相似於serine蛋白,脂肪酵素屬□/□水解蛋白構形,且含催化三元體和陰電性氧區,由於具有水解反應和酯類合成反應的特性,脂肪酵素便實際地應用在醫學,工業和其他用途上。Candida rugosa是一種非產生孢子的酵母菌,且可分泌體外的脂肪酵素( CRLs )。目前CRLs至少有七種同質異形體,但僅有五種被定序出來,其中氨基酸為百分之六十六相同,百分之八十四相似,一種不尋常的遺傳密碼CTG在Candida rugosa基因中被轉譯成serine而非leucine。所以要把CTG密碼改換能轉譯成leucine密碼才能將CRLs表現於S. cerevisiae以利轉譯後的修飾。張騰元學長和林經凱學長已將位於CRLipase4上的19個CTG密碼藉由PCR方式來置換成leucine密碼,而林經凱學長將此重組的CRLipase 4 (19) 構築於一載體(pYES2)上,並加上一訊息蛋白片段來取代原CRLipase 4 (19) 上所有的,為使純化方便,將此重組的載體加上一標簽而形成一重組蛋白His-LIP 4 (19)。由於分泌性體外重組His-LIP 4 (19)有限,所以採用長期和大量培養方式,除了離子交換和疏水性交互作用純化外,鎳離子親和層析法並無達到預期效果。將純化後的His-LIP 4 (19)粗略估計來進行單位時間內的酸鹼滴定,以定出其酵素動力學。


  Lipases (triacylglycerol acylhydrolases, EC 3. 1. I. 3) broadly found in many organisms display the variety of sizes, substrate specificities and catalyeic rates. Lipases belong to □/□ hydroase fold protein and contain the catalytic triad and the oxyanion hole similar to other serine proteases. Due to the abilities of lipases in catalyzing hydrolysis and synthesis of ester, they are used virtually for medical, industria and others applications. Candida rugosa, a non-spore forming yeast, can secrete extracellular lipases (CRLs), which exist with multiple isoforms and encoded from different genes. There are seven genes at least of CRLs found, but only five ones whose amino acids sequences are 66% identical and 84% similar had been isolated to date. The unusual codon usage of Candida rugosa is CTG codon codes for serine instead of leucine. To express the CRLs in other expression model systems, like E. coli or S. cerevisiae, the CTG codons on CRLs gene have to be converted to encode for leucine. Ten-Yan Chang and Chin-Kai Lin had made the nineteen CTG codons mutated by PCR technology to obtain LIP 4(19), then C,K Lin had manipulated the □-factor, a signal peptide which came from S. cerevisiae, to take place the original leader sequence of lipase 4, and constructed the recombinant lipase 4 (19) gene with a vector p YES2, to obtain the pYES2/α f-lip 4 (19) for expressing the LIP 4 (19) with post-translations and modifications in S. cerevisiae. For the purpose of facilitated purification, the p YES2/□ f-lip 4(19) had been reconstructed by adding a tag to obtain the p YES2/ f-His-lip 4(19). The longterm incubation with a large-scale culture medium was operated because the amount of the secrete recombinant His-LIP 4(19) was still limited. Beside the ion exchange column and hydrophobic interaction column, nickel-affinity column could not accomplish as previous expectation. The ratio of the recombinant His-LIP 4(19) had been estimated approximately in the final elution solution, and the recombinant His-LIP 4(19) had been analyzed by pH-stat titration for the determination of enzyme kinetics.

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