Thermostable and low pH tolerant phytase:

Cloning01Complete gene encoding phytase gene (phyA), from A. ficuum NRRL 3135 and A. niger BTCF 5 and acid phosphatase gene (appA) from E. coli XL1 blue was sequenced. The sequencing data was analyzed to predict phyA signal cleavage site and intron. Phytase gene phyA was expressed from E. coli BL21DE3 pLysS cells as inclusion bodies. Acid phosphatase gene with native signal was expressed from pET 20b in Bl21DE3pLysS cells and expressed recombinant enzyme showed an activity of 119.1IU/mL in cytoplasmic fraction and 614.9 IU/mL in periplasmic fraction. Acid phosphatase gene was also expressed in periplasm with maltose binding protein fusion. The linearization of phytase gene cloned in pKLAC1 vector by digestion with restriction enzyme sacII resulted in the formation of an expression cassette which integrated in to the genomic DNA of Kluyveromyces lactis GG799 (Figures 1, 2). K. lactis cells were transformed with linearized plasmid according to the NEB K.lactis transformation protocol. The transformants have to be further screened for the presence of single and multy copy integrants. Work has been initiated to purify and characterize the recombinant acid phosphatase from E. coli.Cloning02

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