Identification Of A Proteolytic Bacterium Hw08 And Characterization Of Its Extracellular Cold-active Alkaline Metalloprotease Ps5

A psychrophilic protease-producing bacterium HW08 was isolated from the sediment of Yellow Sea in eastern China. Based on the16S rDNA sequence analysis and physiological properties, the isolate was identified as Pseudomonas lundensis. The Pseudomonas lundensis HW08 is gram-negative, motile by means of one polarly inserted flagellum. Rod shaped,1.0 by 3 to 5μm, and asporogenous. A greenish tinge can be observed due to the formation of fluorescent pigments. The temperature range for growth is 4 to 33℃, The optimal growth temperature is 25℃. Metabolism is respiratory and never fermentative.HW08 secretes an extracellular alkaline cold-adapted protease, named Ps5. Purification of Ps5 was achieved by a three-step protocol consisting of (NH4)2SO4 precipitation, hyperfiltration and gel filtration. Ps5 is a monomer with an apparent molecular mass of 46 kDa on SDS-PAGE. As a metalloprotease (inhibited by EDTA), the enzyme showed maximum activity at 30℃and pH10.4.The enzyme had no activity loss when exposed at 4℃for 60 days or experiencing repeated (at least 4 times) freezing and thawing. Broad pH (7.0-10.4) and temperature (0-40℃) stability was observed in the presence of 5 mM Ca2+. Furthermore, the enzyme was resistant to the detergent additives such as non-ionic surfactants and bleaches. It shows considerable potential in alkaline-protease needed industry.As the experiment of metal ions influences shown to us, Zn2+ and Ca2+ reduced Ps5 activity rather than enhanced, which is different from other researches, may indicate a new catalytic style. Moreover, concerning the remarkable stability especially supplied with Ca2+, we propose that Ps5 is valuable for further study of enzyme's cold-adapted mechanism.Shot-gun method was used for searching Ps5's gene, but the result was negative. However, the PCR method was success in cloning the gene of Ps5, which has full ORF structure. But the gene couldn't express active protease in the host of E. coli. We deduced that the upstream and downstream genes are needed for the correct expression of Ps5, which was so-called ABC strategy. Further study is undergoing.