Purification and characterization of a novel extracellular carboxylesterase from the moderately halophilic bacterium Thalassobacillus sp. strain DF-E4

Abstract

An extracellular carboxylesterase from the moderately halophile Thalassobacillus sp. strain DF-E4 was purified to 8-fold with 11% recovery and specific activity of 2,046 U mg −1 . The molecular mass of the native enzyme was approximately 49 kDa as determined by analytical ultracentrifugation, while SDS-PAGE analysis showed a single protein band corresponding to a molecular mass of 45 kDa, suggesting that the enzyme was a monomer. Among the pNP (p-nitrophenyl) esters tested, p-nitrophenyl butyrate (C 4 ) was hydrolyzed most effectively, with K m and V max values of 0.69 mM and 0.84 μmol min −1 mg −1 , respectively, and the optimum activity occurred at pH 8.5, 40°C and 0.5 M NaCl (w/v). The enzyme activity appeared to be stable over pH 6.0-9.5 and up to 45°C for 1 h. Tests of substrate specificity and inhibitor susceptibility revealed it was a serine-type carboxylesterase (EC 3.1.1.1), rather than a lipase. None of the divalent cations tested enhanced the enzyme activity, while most of them had no effect or slightly inhibited the activity. The enzyme activity was strongly inhibited by PMSF, PAO and DEPC, implying that serine, cysteine and histidine residues at the active site were essential for catalysis.

Purification and characterization of a thermo-halophilic, alkali-stable and extremely benzene tolerant esterase from a thermo-halo tolerant Bacillus cereus strain AGP-03, isolated from ‘Bakreshwar’ hot spring, India

“…As considering the B. cereus this was a novel characteristic and probably the first report on halotolerant esterase from B. cereus. This unique finding was comparable to the halotolerant esterase from halophiles [31,32] rather than other Bacillus sp. This verdict supported the opinions of Elend et al [33], stated that targeting habitat related characteristics, in many cases, leads to an underestimate of the enzyme's potential.…”

“…The purified enzyme activity was almost inhibited by PMSF, which indicated that the purified esterase was serine enzyme. Besides, it was also strongly inhibited in the presence of PAO, a cysteine enzyme inhibitor [32], suggesting that serine and cysteine residues were essential for its catalytic activity. Moreover, the enzyme lost its full activity in the presence of reducing agent like DTT, 2-mercapto ethanol and urea, again in the presence of SDS, enzyme activity almost inhibited.…”

Extracellular production of beta-amylase by a halophilic isolate, Halobacillus sp. LY9

“…Complete inhibition of the b-amylase by DEPC, a histidine modifier [32], and PAO, a cysteine modifier [12], were observed, indicating that histidine and cysteine residues at the active site were essential for the enzyme catalysis. The activity was stimulated by Ca 2?…”

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

“…After incubation at 80°C for 2 h, about 40% activity still retained. In contrast, other halophilic esterases described previously were inactive under temperatures higher than 70°C [12,15]. Excellent thermostability may favor its application in processes that lead to inactivation of enzymes with increasing temperature.…”