臺灣博碩士論文加值系統

Leucine aminopeptidase (LAP) 是用於去除胜肽苦味的酵素。本研究由Aspergillus sojae選殖 lap 基因，並比較A. sojae LAP的酵素性質與Aspergillus oryzae LAP之差異性。由酵素性質分析與序列比對的結果， LAP應該是屬於鋅離子依賴酵素家族的一員。將A. oryzae的 lap 基因選殖到表現載體pQE 30上，並且利用噬菌體的T5啟動子表現lap基因。發現LAP在Escherichia coli NovaBlue菌體內幾乎全部都會形成內涵體。利用北方墨點雜交法及酵素活性分析法，發現lap基因的表現會受到pH值、碳源及氮源的調控。lap基因在鹼性環境下表現量最多，而碳源(glucose與glycerol)與無機氮源(urea與ammonium sulfate)卻會抑制其表現。本研究也成功的利用Novozyme 234與Yatalase建立A. oryzae原生質體的製備方法。利用UV突變，篩選到A. oryzae抗pyrithiamine的突變株，由突變株中選殖ptrA基因，並將其構築在E. coli - A. oryzae穿梭載體上，作為基因轉形之篩選標誌。

Leucine aminopeptidase (LAP) is used in the debitterring of peptides. Aspergillus sojae lap gene was cloned and the properties of gene product was compared with the purified A. oryzae LAP. Based on enzyme properties and sequence comparison, LAP was proved to be a zinc-dependent enzyme. A. oryzae lap gene was cloned into pQE30 and its expression was under the control of phage T5 promotor. The LAP formed inclusion bodies in transformed Escherichia coli NovaBlue. Northern blotting and activity assay indicated that the expression of lap gene was regulated by pH, carbon source, and nitrogen source. The lap gene was highly expressed in alkaline condition and repressed by carbon source (glucose and glycerol) and inorganic nitrogen source (urea and ammonium sulfate). A. oryzae protoplast could be successfully prepared by the enzyme mixture of Novozyme 234 and Yatalase. Pyrithiamine resistant mutants of A. oryzae were generated by UV mutation and their ptrB genes were cloned for the construction of the E. coli — A. oryzae shuttlt vector.

中文摘要-----------------------------------------------------------------------1
英文摘要-----------------------------------------------------------------------2
緒論------------------------------------------------------------------------------3
一. 蛋白質與胜肽類食品--------------------------3
1. 具有生物活性的胜肽--------------------------3
2. 生物活性胜肽在日本市場發展的情形------------------4
3. 生物活性胜肽的生產---------------------------------------4
4. 蛋白質水解液的介紹---------------------------------------5
5. 胜肽的呈味研究---------------------------------------------6
6. 苦味胜肽的研究---------------------------------------------7
7. 苦味胜肽的去除---------------------------------------------9
二. Leucine aminopeptidase (LAP)的簡介-------------10
1. LAP在生理上的重要性-----------------------------------10
2. LAP在食品工業上的應用--------------------------------11
三. A. oryzae------------------------------------------------12
1. A. oryzae的型態與分類------------------------------------12
2. A. oryzae的安全性------------------------------------------12
四. 環境pH值對絲狀真菌基因表現之調控-----------13
五. 氮源對絲狀真菌基因表現之調控---------------------14
六. 碳源對絲狀真菌基因表現之調控---------------------15
七. A. oryzae異源性蛋白表現系統-----------------------16
八. 前人研究--------------------------------------------------------18
九. 研究策略--------------------------------------------------------18
1. LAP的純化與酵素特性分析-----------------------------18
2. 利用E. coli 來表現lap基因-----------------------------18
3. 探討影響LAP生產的因素--------------------------------19
4. 建立A. oryzae異源性蛋白表現系統--------------------19
材料與方法--------------------------------------------------------------------21
一. 藥品---------------------------------------------------------------21
二. 菌株、質體及培養基---------------------------------------21
三. E. coli 質體 DNA 的抽取及轉形作用-------------22
四. DNA片段的回收及純化-------------------------------------22
五. 核酸純度、定量及洋菜凝膠電泳分析---------------23
六. 引子 (Primer) 的設計------------------------------------23
七. 聚合酶鏈鎖反應-----------------------------------------------23
八. DNA的黏接反應------------------------------------------------24
九. Aspergillus sp. 染色體DNA的抽取----------------25
十. A. sojae lap 基因的選殖------------------------------ 26
十一. 蛋白質的電泳分析-------------------------------------------26
十二. A. oryzae培養液的濃縮------------------------------------28
十三. LAP的純化-----------------------------------------------------28
1. A. oryzae LAP的純化-----------------------------------------28
2. A. sojae LAP的純化-------------------------------------------29
十四. LAP 之活性分析-----------------------------------------------29
1.蛋白質濃度的測定---------------------------------------------29
2. ρ-nitroaniline的標準曲線的製作-----------------------29
3.酵素呈色法------------------------------------------------------30
4.LAP 活性染色---------------------------------------------------30
十五. 南方轉漬雜交法------------------------------------------------31
十六. A. oryzae總RNA的萃取------------------------------------31
十七. 北方轉漬雜交法------------------------------------------------33
十八. lap 基因在 E. coli 的表現-----------------------------34
十九. 蛋白含醣量之分析---------------------------------------------35
二十. A.oryzae的抗藥性與氮源利用能力--------------------35
二十一. ptrA基因的選殖-----------------------------------------------35
1. UV的照射劑量與致死率的關係------------------------------35
2.利用UV篩選突變株並選殖ptrA基因-----------------------36
二十二. A. oryzae表現載體的建構-------------------------------36
二十三. A. oryzae 的轉形作用-------------------------------------37
結果-----------------------------------------------------------------------------------39
一. A. sojae lap 基因的選殖----------------------------------39
二. A. sojae lap基因的結構-----------------------------------39
三. A. sojae 與A. oryzae lap基因的比較----------------40
四. A. oryzae LAP的純化------------------------------------------40
五. A. sojae LAP的純化--------------------------------------------41
六. A. oryzae與A. sojae LAP的酵素性質分析-----------41
1. LAP的分子量------------------------------------------------------41
2. LAP的最適反應pH值-------------------------------------------42
3. LAP的最適反應溫度--------------------------------------------42
4. LAP對受質的選擇性--------------------------------------------43
5.金屬離子對LAP活性的影響-----------------------------------43
6. LAP的熱穩定性--------------------------------------------------44
7. LAP的pH穩定性------------------------------------------------44
8. NaCl對LAP活性的影響 ---------------------------45
9. H2O2對LAP活性的影響----------------------------45
七. LAP 3D結構的預測-----------------------------------------------46
八. 利用E. coli表現A. oryzae的lap基因---------------------46
九. 醣化作用對於LAP活性的影響--------------------------------47
十. lap基因在染色體上的套數---------------------------------------47
十一. 孢子的接種量與LAP活性的關聯性-----------------------48
十二. pH值、氮源與碳源對lap表現的影響--------------------48
十三. 原生質體的再生-----------------------------------------------------50
十四. A. oryzae的抗藥性及氮源利用能力-----------------------50
1.Zeocin------------------------------------------------------------------50
2.Pyrithiamine---------------------------------------------------------51
十五. ptrA基因的選殖-----------------------------------------------------51
1. UV的照射劑量與致死率的關係----------------------------------51
2.由抗pyrithiamine之突變株選殖ptrA基因----------------------52
3. Pyrithiamine對突變株的最小抑制濃度------------------------52
4. ptrA基因的序列分析-------------------------------------------------52
十六. A. oryzae - E. coli穿梭載體的建構----------------------------53
1. amyB基因的選殖-----------------------------------------------------53
2. amyB基因中止子的選殖--------------------------------------------53
3. amyB基因啟動子的選殖--------------------------------------------54
4. lap基因的選殖--------------------------------------------------------54
十七. 轉形株的確認----------------------------------------------------------55
討論---------------------------------------------------------------------------------------56
一. A. sojae的 lap基因-----------------------------------------------56
二. LAP的純化------------------------------------------------------------56
三. LAP的醣化作用-----------------------------------------------------57
四. LAP的性質-----------------------------------------------------------58
五. LAP在食品工業上的應用--------------------------------------62
六. LAP的生理角色------------------------------62
七. ptrA 基因--------------------------------------------------------------63
圖表----------------------------------------------------------------------------------------66
參考文獻---------------------------------------------------------------------------------112

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