Solvent-Stable <i>Pseudomonas aeruginosa</i> PseA Protease Gene: Identification, Molecular Characterization, Phylogenetic and Bioinformatic Analysis to Study Reasons for Solvent Stability

Abstract

We have previously isolated a solvent-stable protease from a novel solvent-tolerant strain of Pseudomonasaeruginosa (PseA). Here we report cloning and characterization of the gene coding for this solvent-tolerant protease. A homology search of the N-terminal amino acid sequence of the purified PseA protease revealed an exact match to a P. aeruginosa PST-01 protease gene, lasB. The c-DNA sequence of the PST-01 protease was used to design primers for the amplification of a 1,494-bp open reading frame encoding a 53.6-kDa, 498-amino-acid PseA LasB polypeptide.The deduced PseA LasB protein contained a 23-residue signal peptide (2.6 kDa) followed by a propeptide of 174 residues and a 33-kDa mature product of 301 residues. A phylogenetic analysis placed PseA lasB closest to the known zinc metalloproteases from P. aeruginosa. This gene was also found to contain a conserved HEXXH zinc-binding motif, characteristic of all zinc metallopeptidases. The 3D structure analysis of PseA protease revealed the presence of 7 α-helices (36% of the sequence). The molecule was found to have two disulfide bonds (between Cys-227 and Cys-255 and between Cys-467 and Cys-494) and had a number of hydrophobic clusters at the protein surface. These hydrophobic patches (21% of the sequence) and disulfide bonds may possibly be responsible for the solvent-stable nature of the enzyme.

Organic solvent-tolerant elastase efficiently hydrolyzes insoluble, cross-linked, protein fiber of eggshell membranes

“…The N-terminal amino acid residue of mature ME-4 protease (Cheng et al 2009) is located at position 198 of the deduced amino acid sequence, which suggested that ME-4 protease is synthesized as a pre-pro-protein, as previously observed for elastases from P. aeruginosa (Schad et al 1987;Jellouli et al 2008). The sequence of the first 20 N-terminal residues of the 197 amino acid pre-pro-region was similar to the signal peptide sequences of Pseudomonas elastases (BAB79621, AAG07111, and AAZ78231), and was followed by a 177 amino acid pro-peptide (Bever and Iglewski 1998;Gupta et al 2008). The primary structure of the mature region of the ME-4 protease has a high level of identity to the primary structure of elastases from P. aeruginosa: LasB of P. aeruginosa PST-01 (BAB79621, 99% identity, Online Resource 1), LasB of P. aeruginosa PA01 (AAG07111, 99%), and LasB of P. aeruginosa MN7 (AAZ78231, 99%), and similarity (Fig.…”

“…The probe hybridized to 3.2 kb PstI, 5.0-kb EcoRI, and 6.0 kb ClaI fragments (data not shown), which indicated that strain ME-4 carries one gene encoding pre-pro-LasB_ME4. These findings indicate that native ME-4 protease might be identical to one of the Pseudomonas elastases (Gupta et al 2008;Jellouli . 2008).…”

“…S1) to that of the metalloprotease from Aeromonas veronii AeG1 (AB103464, 66%), Aeromonas hydrophila AG2 (AAF07184, 66%), and Vibrio cholerae 3083 (P24153, 63%). Although Pseudomonas elastases have been characterized as organic-solvent-tolerant proteases (Ogino et al 2000;Gupta et al 2008;Jellouli et al 2008), little is known about their mechanical stability and the hydrolysis of insoluble cross-linked eggshell membrane.…”

Optimization of upstream and downstream process parameters for cellulase-poor-thermo-solvent-stable xylanase production and extraction by Aspergillus tubingensis FDHN1

“…They have reported that the disulfide bonds and amino acid residues located on the surface of the molecule play an important role in the organic solvent stability of the enzymes. Gupta et al [35] have proposed that the presence of hydrophobic clusters on the protein surface and disulfide bonds was responsible for A B Figure 3 Optimal temperature (A) and thermal stability (B) of the xylanase from A. tubingensis FDHN1. Xylanase activity was measured at different temperatures to determine its optimal range.…”

Extreme Environments: Goldmine of Industrially Valuable Alkali-stable Proteases

“…They reported that disulfide bonds and amino acid residues located on enzyme surface play important roles in organic solvent stability. The structural analysis of P. aeruginosa protease (after treatment with organic solvent) revealed the presence of two disulfide bonds and hydrophobic patches at the protein surface, and was thought to be a key factor for solvent-stable nature of the enzyme [246,247]. Gaur et al [247] carried out bioinformatic analysis to find the structural basis for the solvent tolerant nature of P. aeruginosa PseA aminopeptidase, and found that its primary structure contained 52% hydrophobic residues (folded in a conformation that favored its stability in organic solvents).…”