title: Purification and characterization of uridine phosphorylase from the ice-nucleating bacterium, Pantoea agglomerans NBRC12686.
abstract: The ice-nucleating bacterium, Pantoea agglomerans NBRC12686 responds to a decrease in temperature with the induction of proteins, which are classified as cold-induced proteins. When the temperature of the strain NBRC12686 culture was lowered from 30 degree C to 12 degree C, the viability after freezing treatment significantly improved. By the use of SDS-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC), we analyzed the cold acclimation response in strain NBRC12686. After a shift from 30 degree C to 12 degree C, several proteins and saccharides were synthesized. After 48 h of cold acclimation, the induction level of proteins increased. In addition, ribose-1-phosphate was fractionated by HPLC using a TSK gel Sugar AXG column. Cell-free extracts were prepared from a cold acclimation culture (30 degree C to 12 degree C) and a non-cold acclimation culture (30 degree C), and then subjected to SDS-PAGE. A protein of approximately 29.7-kDa was present in the cold acclimation culture but was not present in the non-cold acclimation culture. The 29.7-kDa protein was purified by various chromatographies. We found that apparent molecular mass of the protein was approximately 119-kD constructed of 4 subunits of 29.7-kDa each. Based on the analysis of the N-terminal amino acid sequences of proteins, the 29.7-kDa protein had 83 percent identity with that of uridine phosphorylase (UPase) obtained from Escherichia coli K-12. We confirmed that the 29.7-kDa protein was novel, judged by molecular mass different from the already-known UPase or cryoprotectants. The cryoprotective activity of UPase of 29.7-kDa protein for LDH was approximately 30 percent at 5.0 microgram per ml of the protein. Furthermore, UPase had a high level of cryoprotective activity even after treating at 70 degree C for 30 min, but had no activity after treating at 100 degree C. We could elucidate that UPase from strain NBRC12686 had a cryoprotective activity as well as an enzyme activity, and it seems that UPase works in two different mechanisms for freezing tolerance.
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka 564-8680, Japan. obata@ipcku.kansai-u.ac.jp
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Obata
authors.forename: Naomi
authors.initials: N
authors.lastname: Muryoi
authors.forename: Hidehisa
authors.initials: H
authors.lastname: Kawahara
authors.forename: Ayako
authors.initials: A
authors.lastname: Nishiyama
mesh_terms: D000222:Adaptation, Physiological / Q000502:physiology; D003451:Cryoprotective Agents / Q000302:isolation & purification; D005615:Freezing; D020636:Pantoea / Q000201:enzymology*; D014543:Uridine Phosphorylase / Q000302:isolation & purification*
chemical_list: D003451:Cryoprotective Agents; D014543:Uridine Phosphorylase