- 期刊
Overexpression and Secretion of Escherichia coli Phytase Gene Using Glyceraldehyde-3-phosphate Dehydrogenase Promoter in Protease-deficient Yeast Pichia pastoris SMD1168H
以甘油醛3-磷酸啟動子在蛋白酶缺陷赤壁酵母中表達與分泌大腸桿菌植酸酶基因
摘要
Phytase is any type o f phosphatase that catalyzes the hydrolysis o f phytic acid. It is used as animal feed supplement to poultry feeds which can efficiently utilize phytate-phosphorus thus minimize environmental pollution. In this study, an Escherichia coli phytase gene was cloned to pGAPZ-aA vector, using a glyceraldehyde-3-phosphate dehydrogenase promoter constitutively and transformed to protease-deficient Pichia pastoris SMD1168H. After electroporation, six recombinant transformants possessed with higher phytase activity were isolated. The highest phytase activity among those six transformants was observed at 6th day by incubating in 50 ml BMGY medium. When one isolate of Pichia constitutive transformant SD9B was incubated in the BMY medium supplied with methanol as carbon source, the highest phytase activity was obtained about 2005 U/ml cultural broth using methanol as carbon source in a 5-1 fermentor at 274 hours. From SDS-PAGE analysis, the phytase presented three major bands by different glycosylated forms. Recombinant phytase after treated with Endo H f was shown only one band about Mw 43 KDa. The deglycosylated recombinant phytase activity was decreased about 20% after Endo H f treatment for 24 h. In addition, the thermostability of recombinant enzyme expressed from Pichia pastoris transformant was superior to that o f the deglycosylated recombinant phytase.
並列摘要
植酸酶是一種可催化植酸水解並釋放出磷的酵素，可用於添加於動物飼料中以減少 環境中磷的污染。本研究，將大腸桿菌的植酸酶基因選殖至pGAPZ αA載體上，利用其上游的甘油醛3 -磷酸啟動子活化基因並在蛋白酶缺陷赤壁酵母中進行表達，結果顯示，在電穿孔後，獲得6個可產生髙活性植酸酶活性轉型株。當轉型株培養於50 ml BMGY培養液後6天可獲得最高活性。其中一株轉型株SD9B利用甲醇為碳源時在5公升發酵槽中，可產生2005 U /ml的活性。由SDS-PAGE的分析中可發現，植酸酶呈現出三種不同醣基化狀態。重組酵素經過Endo Hf去除醣基後，酵素大小約為43KDa，然而，去醣基24小時後的植酸酶活性明顯降低20%，而剛表現出的植酸酶活性也明顯優於去除醣基。