Cold-active serine alkaline protease from the psychrotrophic bacterium Shewanella strain ac10: gene cloning and enzyme purification and characterization

Abstract

The gene encoding serine alkaline protease (SapSh) of the psychrotrophic bacterium Shewanella strain Ac10 was cloned in Escherichia coli. The amino acid sequence deduced from the 2,442-bp nucleotide sequence revealed that the protein was 814 amino acids long and had an estimated molecular weight of 85,113. SapSh exhibited sequence similarities with members of the subtilisin family of proteases, and there was a high level of conservation in the regions around a putative catalytic triad consisting of Asp-30, His-65, and Ser-369. The amino acid sequence contained the following regions which were assigned on the basis of homology to previously described sequences: a signal peptide (26 residues), a propeptide (117 residues), and an extension up to the C terminus (about 250 residues). Another feature of SapSh is the fact that the space between His-65 and Ser-369 is approximately 150 residues longer than the corresponding spaces in other proteases belonging to the subtilisin family. SapSh was purified to homogeneity from the culture supernatant of E. coli recombinant cells by affinity chromatography with a bacitracin-Sepharose column. The recombinant SapSh (rSapSh) was found to have a molecular weight of about 44,000 and to be highly active in the alkaline region (optimum pH, around 9.0) when azocasein and synthetic peptides were used as substrates. rSapSh was characterized by its high levels of activity at low temperatures; it was five times more active than subtilisin Carlsberg at temperatures ranging from 5 to 15 degreesC. The activation energy for hydrolysis of azocasein by rSapSh was much lower than the activation energy for hydrolysis of azocasein by the subtilisin. However, rSapSh was far less stable than the subtilisin.

FIG. 1

Phylogenetic relationship between the 16S rDNA sequence of Shewanella strain Ac10 and the 16S rDNA sequences of other Shewanella strains and selected Vibrio strains. The balanced cladogram was constructed by using a matrix of pairwise genetic distances generated by the Clustal method with the MEGALIGN program. The scale indicates percentages of sequence divergence. The numbers are the GenBank accession numbers of the 16S rDNA sequences of various bacteria.

FIG. 2

PAGE of rSapSh. Lane 1, precipitate obtained from cell extract of E. coli BL21(DE3)/pSapSh3; lane 2, soluble active preparation of rSapSh after bacitracin-Sepharose column chromatography; lane 3, molecular weight markers. The position of the inclusion body formed from the preproprotein of rSapSh is indicated by an arrow.

FIG. 3

(A) Nucleotide and deduced amino acid sequences of SapSh. The potential ribosome binding site is indicated by a different typeface. The stop codon is indicated by an asterisk. The predicted signal peptide cleavage site is indicated by an arrow. The N terminus of the mature protein is indicated by +1. The N-terminal amino acid sequences of the preproenzyme and the mature enzyme are underlined with two lines and one line, respectively. The catalytic triad (D, H, and S) is indicated by boldface type. (B) Schematic diagram of the deduced amino acid sequence of SapSh.

FIG. 4

Effect of pH on the activity of rSapSh toward azocasein (⧫) and the synthetic peptide AAPF (•).

FIG. 5

(A) Thermal stabilities of rSapSh (•) and subtilisin Carlsberg (■). The enzymes were incubated at 60°C in 50 mM Tris-HCl buffer (pH 9.0) supplemented with 2 mM CaCl₂ for different periods of time, and the residual activities were determined at 60°C with AAPF as the substrate. (B) Denaturation of rSapSh (•) and subtilisin Carlsberg (■) by urea. The enzymes were incubated with various concentrations of urea for 30 min, and then the reactions were started by adding AAPF.

FIG. 6

Effect of temperature on the activities of rSapSh (•) and subtilisin Carlsberg (■) toward azocasein.