Expression and characterization of extracellular fungal phytase in transformed sesame hairy root cultures.
A recombinant fungal phytase was produced by cultures of sesame hairy roots transformed with Agrobacterium rhizogenes, purified and its molecular properties were characterized. Its transcription level and the phytase production were rapidly increased after 4 weeks of the cultures, suggesting that its transcription and protein synthesis might concur. Western blot analysis provided evidence that the recombinant fungal phytase was secreted into the liquid culture medium of the hairy roots. The phytase enzyme secreted was purified by three steps of ultrafiltration, DEAE-Sepharose ion exchange chromatography, and Sephadex G-100 size-exclusion chromatography. As a result, one single band signal was observed with SDS-PAGE, indicating that the purification step was reasonable. The positive signs of both the zymogram and the PAS staining on SDS-PAGE suggested that the activity of the final product phytase was active and glycosylated. The optimal reaction temperature of the phytase was between 50 and 60 degrees C and at over 60 degrees C its activity was reduced by 30-90%, depending on the temperatures applied. Pre-incubation at temperatures of 20-50 degrees C showed stable catalytic activity, while at over 50 degrees C the phytase activity was gradually decreased by 90%. The optimal pH was between 4 and 5 pH values for the recombinant fungal phytase, while for native phytase it was at pH 5.0. Addition of iron ion inhibited the phytase activity but treatments of some cations, EDTA, and PMSF showed no effect on the activity or slightly stimulated it positively.
Jin
Un-Ho
UH
Department of Biotechnology, Dong-A University, Pusan 604-714, Republic of Korea.
Chun
Jae-An
JA
Lee
Jin-Woo
JW
Young-Byung-Yi
Lee
Shin-Woo
SW
Chung
Chung-Han
CH
D010832:6-Phytase / Q000737:chemistry* / Q000302:isolation & purification*; D015152:Blotting, Northern; D015153:Blotting, Western; D002384:Catalysis; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007501:Iron / Q000737:chemistry; D010503:Periodic Acid-Schiff Reaction; D018517:Plant Roots; D012313:RNA / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D031685:Sesamum / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors
D005656:Fungal Proteins; D007477:Ions; D011994:Recombinant Proteins; D012313:RNA; D004492:Edetic Acid; D007501:Iron; D010832:6-Phytase