Characterization of a cloned subtilisin‐like serine proteinase from a psychrotrophic Vibrio species

Abstract

The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli. The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa. The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence. Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity. The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases. With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same. Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio-proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues. The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index. These factors may contribute to the adaptation of these proteinases to different temperature conditions.

Evaluation of the vaccine potential of a cytotoxic protease and a protective immunogen from a pathogenic Vibrio harveyi strain

“…In support of this hypothesis, in silico analyses indicated that Vhp1 is an extracellular peptidase of the subtilisin family and is highly homologous to a number of known extracellular proteases. A study on a subtilisin-like proteinase from a psychrotrophic Vibrio species showed that the mature enzyme is a peptide corresponding to the protease region of the protein [40,41]. Similarly, in our study, we found that the protease domain of Vhp1 possesses apparent proteolytic activity when purified as a recombinant protein.…”

“…Similarly, in our study, we found that the protease domain of Vhp1 possesses apparent proteolytic activity when purified as a recombinant protein. Like many subtilisin proteases that are known to be calcium-dependent [40,[42][43][44], we found that Vhp1 was most active in the presence of calcium and completely blocked in activity by the presence of EDTA. Since other divalent metal ions failed to activate Vhp1 in a magnitude approaching that caused by calcium, the effect of calcium on Vhp1 is probably specific.…”

Comparative thermal unfolding study of psychrophilic and mesophilic subtilisin-like serine proteases by molecular dynamics simulations

“…The cold-adapted VPR from a psychrotrophic Vibrio sp. PA-44 (Arnórsdóttir et al, 2002;Kristjansson, Magnusson, Gudmundsson, Alfredsson, & Matsuzawa, 1999) and its mesophilic counterpart, the proteinase K (PRK) (Ebeling et al, 1974) from Tritirachium album limber, are closely related, with sequence identity being 43% and C α atom root mean square deviation (RMSD) of .84 Å between the determined X-ray crystal structures (Arnórsdóttir, Kristjánsson, & Ficner, 2005;Betzel et al, 2001). However, these two enzymes are characterized by significantly different catalytic activities and stability: the cold-adapted VPR has been found to be up to two times more catalytically efficient in the temperature range 15-55°C and much more labile towards both heat and denaturants than the mesophilic PRK, reflecting their different temperature adaptions (Kristjansson et al, 1999).…”

Ca 2+ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

“…Because subtilisins are commercially valuable enzymes, extensive attempts to improve their activities and stabilities with proteinengineering technology have also been made (10, 55, 60). The subtilisin family includes subtilisins from (hyper)thermophiles (13,27,28,35) and psychrophiles (3,14,29,37). The crystal structures of some of them have been determined (2,4,50,57).…”