Expression of Bacillus subtilis phytase gene in Lactobacillus casei with a food grade recombinant plasmid

• Main Authors: Shih-Hsein Ku, 辜世賢
• Other Authors: Chii-Ling Jeang
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/12444370403642407867

碩士 === 國立中興大學 === 食品科學系 === 89 === Lactobacillus is a well vehicles for the delivery of biologyically active compounds, such as antibodies, vaccines and enzymes. In this study, we used pHG as the vector for heterogeneous genes expessed in Lactobacillus casei ATCC 27092. We attempt at improving the electroporation of Lb. casei by weakening the cell wall with glycine and lysozyme. The addition of 3% glycine in culture had the best effect in improving electroporation efficiency. And transfomant-- Lb. casei (pHG) was able to directly secret CGTase into medium. Then cgt gene was served as reporter gene for truncation of selective marker gene to develop a food grade expression system. And the recombinant plasmid was named pHGa-t-. After database search and alignment, we designed proper primers to clone phytase gene candidate (1.2 kb) from Bacillus subtilis DB2 into pHG with Polymerase chain reaction, and the clone was named pHP. The deduced amino acid sequence of phytase gene candidate had 70% similarity with phytases from B. subtilis and Bacillus sp.. It revealed that it may be phytase. Then the Lb. casei (pHP) was growed on PSM plate and stained with coloring reagent. It showed that it was able to secret phytase like enzyme on plate. The medium of Lb. casei (pHP) culture was assay by phytase specific method. The phytase activity was 1306.5U/L. So the phytase gene candidate, in this study, must be a phytase. And Lb. casei (pHP) has the ability to produce phytase.