Characterization of a recombinantly expressed proteinase K-like enzyme from a psychrotrophic Serratia sp.
The gene encoding a peptidase that belongs to the proteinase K family of serine peptidases has been identified from a psychrotrophic Serratia sp., and cloned and expressed in Escherichia coli. The gene has 1890 base pairs and encodes a precursor protein of 629 amino acids with a theoretical molecular mass of 65.5 kDa. Sequence analysis suggests that the peptidase consists of a prepro region, a catalytic domain and two C-terminal domains. The enzyme is recombinantly expressed as an active approximately 56 kDa peptidase and includes both C-terminal domains. Purified enzyme is converted to the approximately 34 kDa form by autolytic cleavage when incubated at 50 degrees C for 30 min, but retains full activity. In the present work, the Serratia peptidase (SPRK) is compared with the family representative proteinase K (PRK) from Tritirachium album Limber. Both enzymes show a relatively high thermal stability and a broad pH stability profile. SPRK possess superior stability towards SDS at 50 degrees C compared to PRK. On the other hand, SPRK is considerably more labile to removal of calcium ions. The activity profiles against temperature and pH differ for the two enzymes. SPRK shows both a broader pH optimum as well as a higher temperature optimum than PRK. Analysis of the catalytic properties of SPRK and PRK using the synthetic peptide succinyl-Ala-Ala-Pro-Phe-pNA as substrate showed that SPRK possesses a 3.5-4.5-fold higher kcat at the temperature range 12-37 degrees C, but a fivefold higher Km results in a slightly lower catalytic efficiency (kcat/Km) of SPRK compared to PRK.
Larsen
Atle Noralf
AN
Department of Molecular Biotechnology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, Norway.
Moe
Elin
E
Helland
Ronny
R
Gjellesvik
Dag Rune
DR
Willassen
Nils Peder
NP
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000378:metabolism / Q000494:pharmacology; D019286:Endopeptidase K / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D012705:Serratia / Q000737:chemistry / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D004492:Edetic Acid; D012697:Serine Endopeptidases; D019286:Endopeptidase K