- 學位論文
重組Candida rugosa 脂肪同功酶之功能多樣性及蛋白質工程之研究
Functional diversity and protein engineering of recombinant Candida rugosa lipase isoenzymes
摘要
假絲酵母菌 Candida rugosa 所生產之脂肪酶(lipase, LIP)為重要工業用酵素，已被廣泛應用於生物技術領域。目前已知該酵素的組成包含了至少七種同功酶LIP1-LIP7，該七種同功酶之胺基酸序列相似度極高，但受質專一性均不相同。LIP2 已知為工業化應用上極具潛力之酵素。LIP2之結構顯示，其受質結合位置(substrate binding site) 之胺基酸L127、 L132及G450在受質選擇性上扮演重要的角色。 本研究首先致力於對LIP2之受質結合位置進行特定位置飽和誘變(site-specific saturation mutagenesis)以改變其受質專一性，並提高其受質應用性。 透過mutagenic degenerate primer 對L132及G450進行正向演化，接著在Pichia pastoris 表現系統表達具功能性之LIP，並於本實驗中挑選出突變脂肪酶L132A; L132I; G450S及 G450A。 於三酸甘油質水解活性測試中，相較於wild-type LIP2，L132A及L132I 之最佳受質由短碳鏈受質改變成中碳鏈受質；G450S 及G450A之受質選擇性雖未改變，但G450A對於tributyrin (C4) 具有更高活性。另外，於p-nitrophenyl (p-NP) esters 活性測試中，L132A; L132I 及 G450S與wild-type LIP2相比未有明顯不同，但G450A之最佳受質則由長碳鏈受質改變成中碳鏈受質。此外，相較於wild-type LIP2，所有的突變株之催化活性、最適反應溫度及pH穩定性都有所提升。 本實驗室已成功表現具催化能力之重組C.rugosa LIP1-LIP 4，但C. rugosa LIP5之特性分析仍未完成。因此本研究第二部分則致力於重組脂肪酶C. rugosa LIP5之研究。本實驗室已成功利用密碼子最佳化技術成功表現LIP5，並比較市售脂肪酶之生化特性及受質選擇性。相較於市售脂肪酶之最適溫度為37℃，LIP5以p-NP butyrate (C4)作為受質時，最適溫度為50℃。於LIP5之受質選擇性分析中發現，LIP5對p-nitrophenyl butyrate; butyryl-CoA (C4); cholesteryl laurate (C12), 及N-carbobenzoxy-l-tyrosine-p-nitrophenyl ester (L-NBTNPE)具有較佳水解活性。有趣的是，LIP5對於胺基酸衍生物的活性高於LIP1-4，但卻對水解三酸甘油酯之活性極低。由此得知，LIP1-5對於不同受質皆有其特異性及催化特性。
並列摘要
Candida rugosa (formerly Candida cylindracea) lipase (CRL) is an important industrial enzyme that is widely used in biotechnological applications. CRL comprises at least seven isozymes (LIP1-LIP7), which share a similar amino acid sequence but with different specificities for substrates. The catalytic versatility of recombinant C. rugosa LIP2 has been known to have potential applications in industry. Structure showed that residues located at positions 127, 132 and 450 where situated in the hydrophobic pocket are responsible for the differences in substrate specificity. In first study, site-specific saturation mutagenesis on residues L132 and G450 of recombinant LIP2 has been employed to investigate the impact of both residues on substrate specificity of LIP2. Point mutations on L132 and G450 were done separately using mutagenic degenerate primer sets containing 32 codons to generate two libraries of mutants in Pichia pastoris. Replacements of amino acid on these mutants were identified as L132A, L132I, G450S, and G450A. In lipase activity assay, L132A and L132I mutants showed a shift of preference from short- to medium-chain triglyceride, whereas G450S and G450A mutants retained preferences as compared to wild type LIP2. Among mutants, G450A has the highest activity on tributyrin. However, in p-nitrophenyl (p-NP) esters hydrolysis assay, compared to wild-type LIP2, L132A, L132I, and G450S did not show differences of preferences over medium- to long-chain esters. In addition to G450A, this prefers only medium-chain ester as compared to wild-type LIP2. All mutants showed an enhanced catalytic activity and higher optimal temperature and pH stability as compared to wild type LIP2. Recombinant CRLs has been functionally expressed along with CRL1-4 isoforms in our laboratory. However, the characterization and codon optimization of LIP5 have not been done. In second study, LIP5 was codon-optimized, expressed, characterized and compared with commercial lipase. The optimal reaction temperature of hydrolysis of p-NP butyrate by LIP5 was optimal at 50℃as compared with 37℃ of the commercial lipase. Several assays were also performed to determine the substrate specificity of LIP5. p-NP butyrate (C4), butyryl-CoA (C4) and L-NBTNPE were found as preferred substrates of LIP5. Interestingly, LIP5 exhibited the highest activity on hydrolysis of amino acid-derivative substrates among all lipase isoforms, but it had very weak preference on hydrolyzing triacylglycerol substrates. The characterization of LIP5 along with that of LIP1-LIP4 previously identified shows that each lipase isoform has a distinct substrate preference and catalytic activity.