Gene cloning, overproduction, and characterization of thermolabile alkaline phosphatase from a psychrotrophic bacterium |
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Authors: | Suzuki Yutaka Mizutani Yoichi Tsuji Tadao Ohtani Naoto Takano Kazufumi Haruki Mitsuru Morikawa Masaaki Kanaya Shigenori |
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Institution: | Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Japan. |
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Abstract: | The gene encoding alkaline phosphatase from the psychrotrophic bacterium Shewanella sp. SIB1 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein was purified and its enzymatic properties were compared with those of E. coli alkaline phosphatase (APase), which shows an amino acid sequence identity of 37%. The optimum temperature of SIB1 APase was 50 degrees C, lower than that of E. coli APase by 30 degrees C. The specific activity of SIB1 APase at 50 degrees C was 3.1 fold higher than that of E. coli APase at 80 degrees C. SIB1 APase lost activity with a half-life of 3.9 min at 70 degrees C, whereas E. coli APase lost activity with a half-life of >6 h even at 80 degrees C. Thus SIB1 APase is well adapted to low temperatures. Comparison of the amino acid sequences of SIB1 and E. coli APases suggests that decreases in electrostatic interactions and number of disulfide bonds are responsible for the cold-adaptation of SIB1 APase. |
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