Isolation and characterization of proteins involved in cryoprotection from cold resistant mutants of <i style="">Pseudomonas fluorescens </i>ATCC13525

Abstract

The proteins isolated from cold resistant mutants of <i style="">Pseudomonas fluorescens </i>ATCC13525<i style=""> </i>were biologically characterized. These proteins, designated as CRP-1 (41.6 kDa, from CRPF₉) and CRP-2 (14.1 kDa, from CRPF₈), were able to protect freeze-labile alkaline phosphatase (ALP) against freeze denaturation up to approximately 84% activity. Addition of BSA could not yield similar cryoprotection of ALP. However, 60% loss of enzymatic activity was recorded in absence of the proteins. Furthermore, the two proteins in combination protected the enzyme activity up to 86%. The freeze-thaw challenge given to the mutants and wild type cells revealed the mutant to be more resistant to freezing (-20°C). The survival percentage of the mutant cells after repeated freeze thaw was significantly higher (55.6%) than wild type (9.8%). Nevertheless, incubation at 10°C, prior to freezing (-20°C) resulted in a further increase in survival percentage of mutant cells (79.6%). The proteins isolated from these mutants possessed the cryoprotective ability, which could be correlated with increased freezing resistance of the mutants at low temperature.