Diversity Of Cultivable Protease-producing Bacteria And Lipase-producing Bacteria In The Sediments Of Bohai Sea And The North Yellow Sea

Huang Bohai domain is terrestrial origin shallow sea of China,and the seabed sediments containing rich microbial resources,which plays important role in nitrogen cycle of marine ecological system.This study combined traditional plate culture isolation and modern molecular biology methods to analyze the diversity of extracellular protease-producing bacteria and lipase-producing bacteria in the sediments of Bohai Sea and the North Yellow Sea.This study not only improves our knowledge of the diversity of extracellular proteaseproducing bacteria and lipase-producing bacteria,but also provides ocean protease,lipase producing microbial resources.A total of 66 protease-producing bacteria were isolated from 5 sediment samples of the North Yellow Sea with screening medium.The phylogenetic analyses based on 16 S rRNA gene sequence indicated that the 66 strains were classified into the 7 genera of four phyla including Bacteroidetes,Proteobacteria,Actinobacteria and Firmicutes,with Pseudoalteromonas(69.9%),Sulfitobacter(12.1%)and Salegentibacter(10.6%)as the dominant.The richness of the cultivable protease-producing bacteria reached 104 CFU/g in all sediment samples.The protease diversity were evaluated through the inhibition tests for 39 strains with higher enzymatic activities by using four type inhibitors: phenylmethylsulfonyl fluoride(PMSF,serine protease inhibitor),1,10-phenanthroline(O-P,metalloproteinase inhibitor),E-64(cysteine protease inhibitor)and pepstatin A(aspartic protease inhibitor).Furthermore the degradation abilities for different protein substrates such as casein,gelatin and elastin were evaluated through observing hydrolytic zones.The experimental results suggested that 39 strains of bacteria produced enough enzymes to do protease inhibit study.The inhibition tests indicated that all the tested 39 strains produced serine protease and/or metallo protease,only a few strains produced cysteine protease or aspartic protease.Three type substrate hydrolysis experiments showed that all strains can degrade gelatin and/or casein.Pseudoalteromonas and Salegentibacter strains have the ability to hydrolyze elastin,Salegentibacter strains has a stronger hydrolytic ability.With the same method,121 protease producing bacteria were isolated from 7 sediment samples of LaizhouBay,they were belonged to 17 genera from 4 phyla including Bacteroidetes,Firmicutes,Actinobacteria and Proteobacteria.Bacillus,Pseudoalteromonas and Photobacteriumwerethe predominant groups.Protease inhibitors experiments showed that there are 62 strains produced enough amount of enzymes that can be used in protease inhibition study.PMSF inhibition rate was 19.36%~100%,and 27strains' protease enzyme activity was inhibited by OP,the inhibition rate was 21.12%~66.37%,E-64 inhibited 7 strains' enzyme activity,the inhibition rate was 12.14%~65.11%.Pepstatin A inhibited the enzyme activity of 7 strains,the inhibition rate was 12.91%~34.5%.Thus all the tested strains mainly produce serine protease and/or metallo proteases.Through the study of the three types of substrates of all strains of hydrolysis experiments showed that the strains of the genus Bacillus have higher casein,gelatin,and elastin hydrolysis abilities.51 lipase producing bacteria strains were isolated using Tween 80 medium from eight in the Bohai sea sediment samples.Based on the 51 isolates of 16 S rRNA gene phylogenetic analysis showed that these strains belonged to 8 genera from 3 phyla including Bacteroidetes,Proteobacteria and Firmicutes.Pseudoalteromonas(35.2%),Marinobacter(23.5%)and Sulfitobacter(17.6%)were predominat.The enzyme activity determination based on the nitrophenol method showed that all measured strains secreting lipase,strain 70623 with the highest lipase enzyme activity of 42.4 U/mL.In conclusion,protease and lipase producing bacteria in the sediment of the Bohai Sea and the North Yellow Sea were uncovered in this study.Serine protease and/or metallo proteases were the main proteases produced by tested protease producing bacteria,and an efficient lipase producing strain Marinobacter sp.70623 was isolated,which has promising potential applications.