- 學位論文
米麴菌白胺酸胺基肽酶之選殖、表現與胞外復性
Molecular Cloning, Gene Expression and in vitro Renaturation of a Recombinant Leucine Aminopeptidase from Aspergillus oryzae
摘要
酶解法是目前產業界常用來提升蛋白質功能性的方法，然而此處理會伴隨苦味胜肽的生成，造成蛋白水解物於食品工業上的應用限制。Aspergillus oryzae 為 GRAS (Generally recognized as safe) 菌種，廣泛使用於東方飲食，可生產改善蛋白質風味的酵素，亦為商業風味蛋白酶 Flavourzyme™ 的生產菌種。本研究選用商業複合酵素 Flavourzyme™ 中的主要酵素— M28 家族中之白胺酸胺基肽酶 A (leucine aminopeptidase A, lapA) ，並且透過 Escherichia coli 異體表現的方式，生產單一重組蛋白，期望純化出單一重組 lapA，可用於改善苦味胜肽的風味。lapA全長為1,134 bp，具337個胺基酸，經預測分析 lapA 為 pre-pro-mature 酵素結構。為確認 pro-peptide 於 lapA的必要性，分別將 pro-mature-peptide (lapAp) 與 mature-peptide (lapAm) 構築至表現載體 pET-22b(+)，並轉形至 E. coli BL21 (DE3)。E. coli BL21 所表現之rHis-lapA 多為不具活性且不可溶之包涵體 (inclusion bodies) 形式，本研究利用尿素回溶包涵體，以親和性管柱進行純化，並透過氧化還原法，成功將rHis-lapAp 復性為具有白胺酸胺基肽酶活性之重組酵素，同時確認 pro-peptide 為蛋白酶形成活性之必要片段。結果顯示 rHis-lapAp 酵素比活性為 2096.02 mU/mg，其最適溫度及最適酸鹼度分別為 50℃ 與pH 6-7，並且於 30℃ 下具有熱穩定性。本研究選殖出之 lapAp 未來可進一步探討是否具改善胜肽苦味之修飾功能，而純化與復性方法可作為 LAPs M28 家族中，以 E. coli 表現胺基肽酶之案例。
並列摘要
Enzymatic hydrolysis has been widely used to improve the functions of protein. However, the enzymatic treatments usually come with the formation of bitter peptides, which seriously limit the application of protein hydrolysates in food industry. Aspergillus oryzae regarded as GRAS (Generally Recognized As Safe) has a long history in the use of fermentation industry in tthe oriental diet. A. oryzae can produce enzymes that reduce the bitterness of hydrolysate. Moreover, commercial complex protease Flavourzyme™ is also derived from A. oryzae. In this study, we chose one of the major enzymes from Flavourzyme™, M28 lapA (leucine aminopeptidase A), as the target. Then lapA gene from A. oryzae ATCC 42149 was cloned and expressed in Escherichia coli system for purifying single recombinant lapA, trying to modify the flavor of protein. Full sequence of lapA was 1,134 bp, and the sequence encoded 378 amino acids residues, which predicted as a pre-pro-mature enzyme. Two truncated lapA, including lapAp (pro-mature peptide) and lapAm (mature peptide), were inserted to the expression vector pET-22b(+) and transformed into E. coli BL21 (DE3) in order to confirm the necessity of pro-peptide. Most of the recombinant rHis-lapA formed as biological inactive inclusion bodies. Hence, urea was used to solubilize inclusion bodies and rHis-lapA was purified by affinity chromatography under denaturing condition. After that, rHis-lapA was refolded and activated successfully by redox method. Meanwhile, we confirmed that pro-peptide was an essential domain for production of active lapA. Renatured rHis-lapAp had a specific activity of 2096.02 mU/mg. It exhibited great stability below 30℃ while optimum temperature and pH was observed at 50℃ and pH 6-7, respectively. This study provided supportive information for future investigation regarding the application to remove bitterness from hydrolysate. Furthermore, the purification and folding treatment of lapAp could be helpful for a better understanding of expressing M28 family LAPs in E. coli.