請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/21611
標題: Regulation , characterization and expression of Aspergillus oryzae and Aspergillus sojae leucine aminopeptidase gene
Aspergillus oryzae與Aspergillus sojae leucine aminopeptidase 基因的調控,特性與表現
作者: 陳俊彰
Chen, Chun-Chang
關鍵字: Aspergillus
麴菌
leucine aminopeptidase
gene regulation
白胺酸
基因調控
出版社: 分子生物學研究所
摘要: 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.
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。
URI: http://hdl.handle.net/11455/21611
顯示於類別:分子生物學研究所

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