Abstract
BackgroundHalophiles are extremophiles that thrive in environments with very high concentrations of salt. Although the salt reliance and physiology of these extremophiles have been widely investigated, the molecular working mechanisms of their enzymes under salty conditions have been little explored.Methodology/Principal FindingsA halophilic esterolytic enzyme LipC derived from archeaon Haloarcula marismortui was overexpressed from Escherichia coli BL21. The purified enzyme showed a range of hydrolytic activity towards the substrates of p-nitrophenyl esters with different alkyl chains (n = 2−16), with the highest activity being observed for p-nitrophenyl acetate, consistent with the basic character of an esterase. The optimal esterase activities were found to be at pH 9.5 and [NaCl] = 3.4 M or [KCl] = 3.0 M and at around 45°C. Interestingly, the hydrolysis activity showed a clear reversibility against changes in salt concentration. At the ambient temperature of 22°C, enzyme systems working under the optimal salt concentrations were very stable against time. Increase in temperature increased the activity but reduced its stability. Circular dichroism (CD), dynamic light scattering (DLS) and small angle neutron scattering (SANS) were deployed to determine the physical states of LipC in solution. As the salt concentration increased, DLS revealed substantial increase in aggregate sizes, but CD measurements revealed the maximal retention of the α-helical structure at the salt concentration matching the optimal activity. These observations were supported by SANS analysis that revealed the highest proportion of unimers and dimers around the optimal salt concentration, although the coexistent larger aggregates showed a trend of increasing size with salt concentration, consistent with the DLS data.Conclusions/SignificanceThe solution α-helical structure and activity relation also matched the highest proportion of enzyme unimers and dimers. Given that all the solutions studied were structurally inhomogeneous, it is important for future work to understand how the LipC's solution aggregation affected its activity.
Highlights
Lipolytic enzymes primarily catalyze both the hydrolysis and synthesis of ester compounds and the catalytic processes are widespread in various organisms including animals, plants, and microorganisms
We present the cloning, heterologous expression of the lipolytic gene lipC from halophilic Archaeon Haloarcula marismortui (ATCC 43049) using mesophilic host Escherichia coli BL21 (DE3)
We undertook the blast analysis of our enzyme LipC in the hydrolysis database ESTHER and the results showed that the LipC sequence belonged to the H block of the lipase/esterase family corresponding to the family IV of bacterial lipolytic enzymes according to Arpigny and Jaeger classification (1999) [31] and that the catalytic triad of LipC was Ser128, Asp224 and His254
Summary
Lipolytic enzymes (lipases and esterases) primarily catalyze both the hydrolysis and synthesis of ester compounds and the catalytic processes are widespread in various organisms including animals, plants, and microorganisms. 6. Stability of LipC The above studies have assessed the changes of activity with respect to temperature, pH and ionic strength and a typical combination of the optimal conditions for achieving the highest enzyme activity is at pH 8.5, 45 C and [NaCl] = 3.4 M.
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