Purification and characterization of an extracellular esterase with organic solvent tolerance from a halotolerant isolate, Salimicrobium sp. LY19

Abstract

Background: Halotolerant bacteria are excellent sources for selecting novel enzymes. Being intrinsically stable and active under high salinities, enzymes from these prokaryotes have evolved to function optimally under extreme conditions, making them robust biocatalysts with potential applications in harsh industrial processes.

Results: A halotolerant strain LY19 showing lipolytic activity was isolated from saline soil of Yuncheng Salt Lake, China. It was identified as belonging to the genus of Salimicrobium by 16S rRNA gene sequence analysis. The extracellular enzyme was purified to homogeneity with molecular mass of 57 kDa by SDS-PAGE. Substrate specificity test revealed that the enzyme preferred short-chain p-nitrophenyl esters and exhibited maximum activity towards p-nitrophenyl butyrate (p-NPB), indicating an esterase activity. The esterase was highly active and stable over broad temperature (20°C-70°C), pH (7.0-10.0) and NaCl concentration (2.5%-25%) ranges, with an optimum at 50°C, pH 7.0 and 5% NaCl. Significant inhibition of the esterase was shown by ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF) and phenylarsine oxide (PAO), which indicated that it was a metalloenzyme with serine and cysteine residues essential for enzyme activity. Moreover, the esterase displayed high activity and stability in the presence of hydrophobic organic solvents with log P(ow) ≥ 0.88 than in the absence of an organic solvent or in the presence of hydrophilic solvents.

Conclusions: Results from the present study indicated the novel extracellular esterase from Salimicrobium sp. LY19 exhibited thermostable, alkali-stable, halotolerant and organic solvent-tolerant properties. These features led us to conclude that the esterase may have considerable potential for industrial applications in organic synthesis reactions.

Figure 1

Phylogenetic tree based on 16S rRNA gene sequence of the isolate LY19 to other members of the genus Salimicrobium. Accession numbers of the sequences used in this study are shown in parentheses after the strain designation. Numbers at nodes are percentage bootstrap values based on 1,000 replications; only values greater than 50% are shown. Bar 0.001 substitutions per nucleotide position.

Figure 2

Bacterial growth and esterase production of stain LY19 in CM broth containing 4% (w/v) NaCl at 37°C. Results represent the means of three separate experiments.

Figure 3

SDS-PAGE analysis of the purified esterase. Lane 1: molecular mass markers; lane 2: purified esterase.

Figure 4

Substrate specificity of the esterase towards the p-NP esters. Assays were done with 5% NaCl at 50°C and pH 7.0. Results represent the means of three separate experiments.

Figure 5

Effect of temperature (a), pH (b) and NaCl concentration (c) on activity (solid lines) and stability (dotted lines) of the purified esterase. Relative activity was defined as the percentage of activity detected with respect to the maximum enzyme activity. For determining the stability, the enzyme activity without any treatment was taken as 100%. Data are the average of three independent experiments. See “Methods” for further details.