請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/21640
標題: Pseudomonas amyloaderamosa 澱粉枝切酵素基因在 Saccharomyces 現
Expression of Pseudomonas amyloderamosa Isoamylase Gene inerevisiae
作者: 陳佩亨
Chen, Pay-Heng
關鍵字: isoamylase
澱粉枝切酵素
expression
secretion
Sassharomyces cerevisiae
表現
分泌
麵包酵母
出版社: 植物學系
摘要: Isoamylase 為 Pseudomonas amyloderamosa SB15 的胞外酵素,能水解 支鏈澱粉中的 alpha-1,6-糖甘鍵。利用聚合酵素鏈鎖反應,從 P. amyloderamosa 染色體 DNA 中複製 isoamylase 基因 (簡稱iso) 的讀碼 區 (open reading frame,稱 ORF),選殖入 Escherichia coli表現載 體 pBluescriptⅡKS+,獲得重組質體 pKS16 及 pKS18。E.coli HB101 (pKS16) 與 E.coli HB101(pKS18) 的胞外 isoamylase 活性各為 85 U/ ml與 20 U/ml。再將 iso 基因的 ORF 選殖入 Saccharomyces cerevisiae表現載體 pAAH5 (帶有alcohol dehydrogenaseⅠ基因起動子 與終端子) 及 pG-3 (帶有 glyceraldehyde 3-phosphate dehydrogenase 基因起動子及 phosphoglycerate kinase 基因終端子), 獲得重組質體 pHB10 及 pGK8。S.cerevisiae AH22(pHB10) 與 S. cerevisiae YNN27(pGK8) 的胞外 isoamylase 活性各為 51.6 U/ml 與 87.8 U/ml。為改善 isoamylase 在 S.cerevisiae 的分泌能力,將質體 pHB10 iso 基因的訊號序列置換成 Schwanniomyces occidentalis alpha-amylase(簡稱amy) 的訊號序列,獲得重組質體 pHC5。S. cerevisiae AH22(pHC5) 的胞外 isoamylase 活性為 86.4 U/ml。在構築 質體 pHC5 的過程中,發現 amy 基因的訊號序列在 E.coli 菌體內仍具 有分泌功能。經蛋白質電泳分析,S.cerevisiae 確實能生產isoamylase ,並將其分泌到胞外。 Isoamylase of Pseudomonas amyloderamosa SB15 can hydrolyze alpha-1,6-glucosidic bonds of starch. The open reading frame (ORF) of isoamylase gene (iso) was prepared by polymerase chain reaction from P.amyloderamosa SB15 chromosomal DNA. The ORF of iso gene was cloned into Escherichia coli expression vector, pBluescriptII KS+ and two recombinant plasmids pKS16 and pKS18 were obtained. The isoamylase activity of those clones were about 85 U/ml and 20 U/ml, respectively. The ORF of iso gene was also cloned into Saccharomyces cerevisiae expression vectors under the control of alcohol dehydrogenase I gene promoter and glycer- aldehyde-3-phosphate dehydrogenase gene promoter, and the extra- cellular isoamylase activity of transformed S.cerevisiae were 51.6 U/ml and 87.8 U/ml, respectively. In order to improve the secretion efficiency of isoamylase in S.cerevisiae, a recombinant plasmid pHC5 was constructed by replacing the signal sequence of iso gene with that of Schwanniomyces occidentalis alpha-amylase gene. The extracellular isoamylase activity of S.cerevisiae AH22 (pHC5) was incrased. Native-PAGE revealed that isoamylase could be secreted into culture broth by S.cerevisiae.
URI: http://hdl.handle.net/11455/21640
顯示於類別:生命科學系所

文件中的檔案:
沒有與此文件相關的檔案。


在 DSpace 系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。