title: Identification of proteolytic bacteria from the Arctic Chukchi Sea expedition cruise and characterization of cold-active proteases.
abstract: Following collection of seawater samples during an Arctic Chukchi Sea expedition cruise of the Korean icebreaker Araon in 2012, a total of 15,696 bacteria were randomly isolated from Marine Broth 2216 agar plates. Of these, 2,526 (16%) showed proteolytic activity and were identified as mainly Alteromonas (31%), Staphylococcus (27%), and Pseudoalteromonas (14%). Among the proteolytic strains, seven were selected based on their significant ability to grow and produce a halo on skim milk plates at low temperatures (<5°C) owing to cold-active proteases. These strains were affiliated with the genus Pseudoalteromonas and were divided into three groups based on phylogenetic analysis of the 16S rRNA genes. Profiling cell membrane fatty acids confirmed the 16S rRNA-based differentiation and revealed the accordance between the two analyses. Seven genes for serine protease precursors were amplified from the corresponding strains, and based on sequence similarities, these genes were divided into three groups that were identical to those identified by the 16S rRNA phylogenetic analysis. Three protease genes from the representative strains of each group were composed of 2,127-2,130 bp, encoding 708-709 amino acids, and these genes yielded products with calculated molecular weights of approximately 72.3-72.8 kDa. Amino acid sequence analysis suggested that the precursors are members of the subtilase serine endo- and exo-peptidase clan and contain four domains (signal peptide, N-terminal prosequence, catalytic domain, and two pre-peptidase C-terminal domains). Upon expression in E. coli, each recombinant protease exhibited proteolytic activity on zymogram gels.
authors.affiliation: Division of Life Sciences, Korea Polar Research Institute, Incheon, 406-840, Republic of Korea.
authors.forename: Ha Ju
authors.initials: HJ
authors.lastname: Park
authors.forename: Yung Mi
authors.initials: YM
authors.lastname: Lee
authors.forename: Sunghui
authors.initials: S
authors.lastname: Kim
authors.forename: Ah Ram
authors.initials: AR
authors.lastname: Wi
authors.forename: Se Jong
authors.initials: SJ
authors.lastname: Han
authors.forename: Han-Woo
authors.initials: HW
authors.lastname: Kim
authors.forename: Il-Chan
authors.initials: IC
authors.lastname: Kim
authors.forename: Joung Han
authors.initials: JH
authors.lastname: Yim
authors.forename: Dockyu
authors.initials: D
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001110:Arctic Regions; D002462:Cell Membrane / Q000737:chemistry; D016000:Cluster Analysis; D003080:Cold Temperature; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005101:Expeditions; D005227:Fatty Acids / Q000032:analysis; D015870:Gene Expression; D007723:Korea; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D031161:Pseudoalteromonas / Q000145:classification* / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012623:Seawater / Q000382:microbiology*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D005227:Fatty Acids; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D010447:Peptide Hydrolases