臺灣博碩士論文加值系統

Leucine aminopeptidase (LAP) 為去除胜肽苦味的酵素。Aspergillus oryzae 之 lap 基因在Escherichia coli 大量表現後，會形成內涵體 (inclusion body)。為了提昇LAP在E. coli 的可溶性，將 lap基因構築入 pET32a 表現載體中，形成thioredoxin-LAP (Trx-LAP)融合蛋白質，發現在E. coli 菌體內所表現的Trx-LAP ，高達95%以上為可溶性蛋白質，惟此融合蛋白質之比活性只有12 mU/mg 與由 A. oryzae 純化的LAP (比活性136U/mg)降低了一萬倍。利用 enterokinase 切除 Trx-LAP融合蛋白之 thioredoxin 時， LAP 的比活性比融合蛋白質上昇 10 倍。將純化的融合蛋白質於23℃放置兩天後，Trx-LAP 活性上昇 30至40倍；在受質選擇性方面，Trx-LAP對Leu-r-nitroanilide具有最佳的水解能力，並且與A. oryzae比較其對Lys-r-nitroanilide的水解能力也提昇，另外，將融合蛋白切除thioredoxin 後的LAP，其受質選擇性比較類似A. sojae，因其對Phe-r- nitroanilide的水解能力提高至51.9%。A. oryzae 和 A. sojae的LAP對 Leu-r- nitroanilide 均有最高的水解能力，而對於其他受質的活性則有非常大的差異。比較A. oryzae 及A. sojae LAP 之胺基酸序列，發現在成熟蛋白質上有四個胺基酸的不同，分別是 Ser-152， Asp-183， Gly-192及 Ser-248。將這四個胺基酸分別進行定點突變，發現4種突變的LAP，除了Gly-192-Ser喪失LAP的活性外，其對受質選擇性並沒有太大的變化，但是LAP活性反而下降。其中Ser-152-Phe活性下降80%，Ser-248-Leu活性下降60%， Asp-183-Asn的活性下降50% 。目前正在進一步將這四個突變基因，轉形入A. oryzae ，由轉形株分離變異LAP進行分析。 lap 基因調控方面，發現在鹼性(pH8.0)環境下，以澱粉為碳源，以黃豆蛋白質為氮源時，LAP產量最高。若以胺鹽為氮源時，則會抑制LAP的活性。當以黃豆蛋白質為氮源時，多醣、雙醣或單醣並不會影響LAP的表現。由此可知，就lap基因的表現而言，氮源的調控比碳源來得強勢。A. oryzae 之轉型系統方面，將質體 pDHG-lap轉形入A. oryzae argB-，發現轉形株皆能在CD培養液 (含有2% 麥芽糖) 中表現出LAP的活性，其中以轉形株 A. oryzae argB--lap-3 有最高的LAP產量(688 mU/ml)，與A. oryzae NCHU1002(7.6 mU/ml)及A. oryzae argB-(22 mU/ml) 比較分別提昇了90倍及30倍。南方墨點雜交分析，也證實了轉形株A. oryzae argB--lap-1、-3及-4中的質體pDHG-lap 是游離狀態，而轉形株A. oryzae argB--lap-2、-5及-6則以游離及嵌入兩種狀態存在。為了提昇質體在菌體內的穩定性，已成功將 pOK18sargBlap 質體轉形入A . oryzae argB-。

Leucine aminopeptidase (LAP) could be used to remove the bitterness of peptides. The majority of Escherichia coli overexpressed Aspergillus oryzae LAP formed inclusion bodies while using pQE30 expression system. To promote the solubility of LAP in E. coli, lap gene was constructed into pET32a and expressed in E. coli BL21(DE3). About 95% of thioredoxin-LAP (Trx-LAP) fusion protein was overexpressed in soluble form. However, the relative activity of Trx-LAP was only 12 mU/mg, which decreased over 10,000 folds comparing with purified native A. oryzae LAP (relative activity 136 U/mg). The most suitable substrate of Trx-LAP was Leu-p-nitroanilide. Incubation at 23℃ for 2 days led to about 40 fold increase in the activity of Trx-LAP. Removal of thioredoxin from Trx-LAP by enterokinase could also increase LAP activity. Meanwhile, the hydrolytic activity toward Lys-p-nitroanilide was improved. After the removal of thioredoxin, the substrate specificities of the enzyme are similar with A. sojae LAP owing to the hydrolytic activity to Phe-p-nitroanilide increased to 51.9% (compared with Leu-p-nitroanilide). Both native A. oryzae and A. sojae LAPs have the highest activities to Leu-p-nitroanilide, but difference in the activities to other substrates. Only four residues are different (at positions 152,183,192, and 248) between the two LAPs. Each of the four residues were changed by site-directed mutagenesis. The Gly-192-Ser mutant lost the LAP activity completely, while the other mutants showed indifference on substrate specificities. The activities of Ser-152-Phe, Ser-248-Leu, and Asp-183-Asn decreased 80, 60, and 50%, respectively. These mutated genes had already been transformed into A. oryzae. The enzyme activities will be determined in the future. The lap gene was highly expressed by starch as carbon source and soybean as nitrogen source at pH 8.0. Whereas ammonium down regulated the LAP expression. The expression of LAP was not influenced by the carbon source while soybean was used as nitrogen source. Obviously, nitrogen source play a more important role than carbon source in the regulation of lap expression. A. oryzae argB- harboring pDHG-lap could express LAP in the CD medium contaning 2% maltose. The LAP activity of A. oryzae argB--lap-3 reached 688 mU/ml which was 90- and 30- fold highler than those of untransformed cell A. oryzae NCHU1002 and A. oryzae argB-, respectively. According to the Southern blot analysis, the plasmid in A. oryzae argB--lap-1, A. oryzae argB--lap-3, and A. oryzae argB--lap-4 were in an episomal form. While in A. oryzae argB--lap-2, A. oryzae argB--lap-5, and A. oryzae argB--lap-6, the plasmid were in both episomal and integrated forms.

中文摘要 1
英文摘要 3
前言 5
研究策略 13
材料與方法
一. 藥品 15
二. 菌株、質體 15
三. A. oryzae lap 基因在E. coli 之表現 15
四. lap 基因的定點突變 19
五. lap 基因的調控機制 22
六. A. oryzae 基因轉型系統之建立 25
結果
一. A. oryzae lap 基因在E. coli 之表現 30
1. LAP 可溶性之提昇 30
2. Trx-LAP 的生化性質 30
二. lap 基因的定點突變 31
三. lap 基因的調控機制 32
1. 氮源對lap 基因的影響 32
2. 碳源對lap 基因的影響 32
四. A. oryzae 基因轉型系統之建立 33
1. 嵌入形質體(integration plasmid) 之應用 33
2. 自行複製質體(autonomously replicating plasmid) 之應用35
3. 轉形株的LAP產量 36
4. pH值對轉形株生產LAP的影響 37
5. 碳源對轉形株生產LAP的影響 37
討論
一. lap 基因在E. coli 之表現 39
二. lap 基因的調控 40
三. A. oryzae 基因轉型系統之建立 41
圖表 44
參考文獻 86

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