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Characterization of a recombinantly expressed proteinase K‐like enzyme from a psychrotrophic Serratia sp.
Author(s) -
Larsen Atle Noralf,
Moe Elin,
Helland Ronny,
Gjellesvik Dag Rune,
Willassen Nils Peder
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.05044.x
Subject(s) - enzyme , biochemistry , molecular mass , biology , escherichia coli , pmsf , peptide sequence , proteinase k , chemistry , microbiology and biotechnology , gene
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 ∼ 56 kDa peptidase and includes both C‐terminal domains. Purified enzyme is converted to the ∼ 34 kDa form by autolytic cleavage when incubated at 50 °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 °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 k cat at the temperature range 12–37 °C, but a fivefold higher K m results in a slightly lower catalytic efficiency ( k cat / K m ) of SPRK compared to PRK.