Study Of The Serine Protease Secreted By Pseudoalteromonas Sp. CF4-3 From The South China Sea

There are various and abundant microorganisms in the sea. They are valuable resources for new enzymes and new medicines, and therefore, may have good biotechnological potential. Pseudoalteromonas sp. CF4-3 was isolated from the sediment of the South China Sea. In this study, a serine protease secreted by P. sp. CF4-3 was purifed through DEAE-Sephadex exchange chromatography, which was named CF43. The N-terminal amino acid sequence of CF43 showed very high identity to the serine protease from Pseudoalteromonas sp. A28 which has algicidal activity. CF43 gene was cloned from the genomic DNA of P. sp. CF4-3, which contains 2145 bp. Sequence analysis showed that this protease is a serine protease of family 8, having 86% amino acid identity with the serine protease from P. sp. A28. The precursor of CF43 has 714 residues with a molecular weight of about 78 kDa, consisting of four domains:a signal peptide sequence, an N-teminal pro-sequence, a catalytic domain, and a C-teminal extension (two repeat PPC domains).CF43 was ligated to vector pET-22b(+) and expressed in Escherichia coli BL21(DE3). Through DEAE-Sephadex exchange chromatography, the mature recombinant CF43 was purified. Analysis of the N-terminal sequence and molecular weight of CF43 showed that the mature protease contains 327 amino acid residues. The optimum temperatue of CF43 was 50℃and its optimum pH was 8.5. The thermostability (t1/2) of CF43 was 60 min at 50℃. CF43 could hydrolyze synthetic subtrates such as FAAF, AAPF, AAPL, AAPK, AAPR efficiently, but it could not hydrolyze AAK, AAV, GGF. Mn2+and Sn2+increased the activity of CF43 while Zn2+,Ni+,Co2+,Sr2+ and Fe2+ had evident inhibitory effect on CF43 activity. Kinetic study showed that, at 50℃, the Km of CF43 towards FAAF was 3.17 mM. CF43 showed algicidal activity towards Spirulina platensis and Anabaena flosaguas. When they were treated with CF43, Spirulina platensis was ruptured into smaller fragments or single cells, and Anabaena flosaguas was degraded into smaller fragments with the vegetable cells being splitted. These results suggest that CF43 may have potential in algal bloom pollution treatment.