Expression and characterization of recombinant Candida rugosa

• Main Authors: Lo, Yu-Chi, 羅玉枝
• Other Authors: Tang Shye-Jye
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/69196433633646261894

碩士 === 國立海洋大學 === 生物技術研究所 === 85 === CRL( candida rugosa lipase4),是由一種不產生孢子的酵母菌 Candida rugosa, 所分泌的多樣性胞外脂肪酵素之一.一般的生 物 遺傳密碼CTG被轉譯成Leu, C.rugosa卻產生Ser, CRL4有19個 CTG 密碼, 包括活性中心Ser209, 故要以E.coli獲得重組脂肪酵 素的 大量表現,必須克服CTG密碼的問題.本實驗將學長提供19個 CTG密 碼完全被置換的CRL4基因(lip4),接入pTrxFus, pET23a表 現質體, 分別轉形進入原核細胞之GI698,GI724,BL21(DE3)pLysS ,AD494( DE3)pLysS等E. coli菌株進行蛋白質的表現.藉由SDS- PAGE分析, 在57kDa及69kDa位置,有重組脂肪水解酵素及重組融 合蛋白質被 大量表現.經活性染色分析此被大量表現的蛋白質, 除了在BL21( DE3)pLysS所表現的脂肪酵素外,其它宿主所表現的 蛋白質均具有 活性,大量表現的蛋白質並以Western blotting的 方法進一步確認 是Lip4. AD494(DE3)pLysS所表現蛋白質具有活 性,似乎是因為 AD494(DE3)pLysS允許雙硫鍵在細胞質形成,使蛋 白質folding更正 確.我們藉由定位基因突變的方法更直接確認, 防止C60-C97雙硫鍵 的生成,會導致所表現的脂肪酵素不具活性. 另外於BL21(DE3) pLysS所表現之融合蛋白質,以活性染色分析發 現具有活性,說明 thioredoxin可以在不允許雙硫鍵生成的環境 下,使脂肪水解酵素 具有活性.因此這些表現系統相當具有潛力 生產量多質純,單一基 因表現和價格便宜的脂肪酵素,以利工業 上的利用. CRL4(Candida rugosa lipase4) is one of the multiple extracellular lipases produced by Candida rugosa. Three expression vectors, pTrxFus-Lip4, pET23a-thioLip4 and pET23a-Lip4, containing CRL4 gene which CTG codon had been replaced with TCG codon, were constructed. The recombinant CRL4 were expressed as a 69kDa fusion protein with thioredoxin or a 57kDa re- combinant Lipase4 in E. coli strains (GI698, GI724, BL21(DE3)pLysS and AD494( DE3)pLysS), as being demonstrated by SDS-PAGE, in soluble or inso- luble fractions. By activity staining assay, the fusion proteins and re- combinant Lipase4 were revealed to possess significant lipase activity, except recombinant Lipase4 expressed by BL21(DE3)pLysS. Using Western blotting, the fusion products in soluble fraction were confirmed to contain CRL4, indicating that the fusion of thioredoxin to CRL4 may improve the solubility of originally insoluble CRL4. Because recombinant Lipase4 in ADA494(DE3)pLysS shows significant lipase activity, it seems AD494, which allow disulfide-bond formation in cytoplasm, may help the proteins to fold at the right conformation. This observation is further proved by that disruption of the disulfide-bond formation between C60-C97 using site- directed mutagenesis will make the recombinant Lipase4 in AD494( DE3)pLysS inactive. In addition, the fusion products expressed by BL21(DE3)pLysS still possess lipase activity shows that thioredoxin will help Lipase4 to gain the lipase activity, by an unidentified mechanism, under the circum- stance disallowing disulfide-bond formation. In conclusion, these expre- ssion systems can be potentially applied to produce a large amount of lipase for a broad range of industrial applications.