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From China's Yellow Sea sediment in the separation of a production temperature alkaline protease Psychrophilic Bacterium No. HW08. By its morphological characteristics, physiological and biochemical identification and 16S rDNA sequence than on the research, we identified the members of its species Pseudomonns lundensis. The study showed that the strains of gram-negative bacilli, width 1μm, long 3-5μm, can take advantage of a polar flagella sports, non-spore colonies were pale green under UV irradiation. The growth temperature is 4-33 ℃, and the optimum growth temperature of 25 ° C. Aerobic but not anaerobic fermentation. Alkaline Low HW08 bacteria secrete an extracellular protease, after centrifugation of the fermentation broth, ammonium sulfate precipitation, gel filtration, concentrated by ultrafiltration to obtain the electrophoresis grade pure extracellular protease, named Ps5. Enzyme optimum temperature is 30 ℃, the optimum pH of 10.4. Ps5 metalloproteinase (its activity by EDTA was significantly inhibited), polyacrylamide gel electrophoresis, its molecular weight of 46 kDa. The optimum temperature for the Ps5 30 ℃ 0 ℃ also maintain 20% of the activity seen, it belongs to a low-temperature enzymes. As a low-temperature enzymes, Ps5 kept for 60 days at 4 ℃ or experienced repeated freezing and thawing (at least 4 times), the activity was not significantly reduced. 5 mM Ca2 exist under the conditions, the enzyme shows a wider range of temperature stability (25-40 ° C) and pH A Stability (7.0-11.0). Further, the protease of the washing additives, such as non-ionic surface active agents and bleaching agents have a certain tolerance. These characteristics make the Ps5 show some potential in industrial applications. In addition, in the study of the influence of metal ions on the enzyme, we found that mostly metalloproteinase: Zn2, Ca2 inhibition of the activity of Ps5, rather than being their activation. The combination more significant Ps5 stability (especially in the presence of Ca2 when), we speculate that this feature should be the mechanism of enzyme catalytic mechanism of its special role worthy of further study. Use the shotgun cloning Ps5 gene, but there is no screening positive clones. Laboratory by PCR method Ps5 gene the complete ORF reading frame. Unable to express active protein in E. coli, however, we believe that this is because the active expression of Ps5 its joint participation on downstream genes belonging to the so-called ABC strategy secretion mechanism. Ps5 on downstream genes being cloned.
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