Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2

Abstract

The structural gene encoding the extracellular lipase of Aeromonas hydrophila MCC-2 was cloned and found to be expressed in Escherichia coli using its own promoter. When the cloned gene (lip) was expressed in E. coli minicells, an 80 kDa protein was identified. Subcellular fractionation of E. coli carrying the lip gene indicated that the Lip protein was mainly associated with the membrane fraction. Nucleotide sequence analysis revealed that the gene is 2253 bp long, coding for a 79.9 kDa protein with an estimated pl of 1036. The deduced protein contains two putative signal peptide cleavage sites; one is a typical signal peptidase cleavage site and the other bears a strong resemblance to known lipoprotein leader sequences. Radioactivity from [3H]palmitate was incorporated into the Lip protein when expressed in E. coli.The deduced protein contains a sequence of VHFLGHSLGA which is very well conserved among lipases. It shows 67% and 65% overall identity to the amino acid sequences of lipase from A. hydrophila strains H3 and JMP636, respectively, but shows little homology to those of other lipases. The Lip protein was purified to homogeneity from both A. hydrophila and recombinant E. coli. In hydrolysis of p-nitrophenyl esters and triacylglycerols, using purified enzyme, the optimum chain lengths for the acyl moiety on the substrate were C,, to C,, for ester hydrolysis and C, to C,,, for triacylglycerol hydrolysis.

Detection of antibiotic resistance, virulence gene determinants and biofilm formation in Aeromonas species isolated from cattle

“…Lipases are involved in changes of the host plasma membrane and consequently increase the severity of infection (Pemberton et al 1997;Chaung et al 1997;Nawaz et al 2010). Lipases also participate in an integrated and coherent role in the establishment of pathogenicity in aeromonads (Pemberton et al 1997;Nawaz et al 2010).…”

Structural Basis for Action of the External Chaperone for a Propeptide-deficient Serine Protease from Aeromonas sobria

“…We observed that the ORF2 N-terminal extension containing the two antiparallel ␤-sheets (Ser 12 23 ) is the region in which the potential formation of a ␤-strand was implied by SSP analysis (data not shown). Interestingly, the ORF2 ⌬N43 and ⌬N63 deletion mutants obviously lost the ability to mediate folding, although their binding constants were reduced by an order of magnitude or less.…”

“…The main syndrome caused by infection with Aeromonas is gastroenteritis, although in severe cases sepsis may occur (9 -11). Aeromonas species secrete a number of putative virulence factors, including hemolysins, enterotoxins, and proteases (12)(13)(14)(15). We purified a 65-kDa serine protease from the culture supernatant of A. sobria (ASP) 3 and found that the enzyme induced vascular leakage, reduced blood pressure through activation of the kallikrein/kinin system (16), promoted human plasma coagulation through activation of prothrombin (17), and caused the formation of pus and edema through the action of anaphylatoxin C5a (18).…”

Sequence Analysis of Amplified DNA Fragments Containing the Region Encoding the Putative Lipase Substrate-Binding Domain and Genotyping of Aeromonas hydrophila

“…A. hydrophila produces a variety of extracellular enzymes, including protease (22), elastase (6), amylase (15), chitinase (24), lipases (2,9), phospholipases (18,23), hemolysins (3,17), and enterotoxins (7,8,26,27).…”

“…The lip and lipH3 genes code for extracellular lipases, which are similar in that they have esterase but not phospholipase activities (2,9). The apl-1 gene codes for a lipase and exhibits nonhemolytic phospholipase C activity (18).…”