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A novel esterase gene selected from metagenomic sequences of deep-sea hydrothermal vents was successfully expressed in Escherichia coli. The recombinant protein (est-OKK), which belongs to the lipolytic enzyme family V, exhibited high activity toward pNP-esters with short acyl chains and especially p-nitrophenyl butyrate. Site-mutagenesis results confirmed that est-OKK contains the nonclassical catalytic tetrad predicted by alignment and computational modeling. The est-OKK protein is a moderately thermophilic enzyme that is relatively thermostable, and highly salt-tolerant, which remained stable in 3 mol/L NaCl for 6 hr. The est-OKK protein showed the considerable alkalistability, displayed optimal activity at pH 9.0 and maintained approximately 70% of its residual activity after incubation at pH 10 for 4 hr. Furthermore, the est-OKK activity was strongly resistant to a variety of metal ions such as Co 2+ , Zn 2+ , Fe 2+ , Na + , and K + ; nonionic detergents such as Tween-20, Tween-80; and organic solvents such as acetone and isopropanol. Taken together, the novel esterase with unique characteristics may give us a new insight into the family V of lipolytic enzymes, and could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions or food and pharmaceutical industries., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Bacterial esterases and lipases, especially extremozymes attract increasing attention due to various advantages both in good properties and wide applications. In the present study, a cold-adapted, alkali-stable and highly salt-tolerant esterase (Est700) was cloned from Bacillus licheniformis, expressed and purified with a molecular mass of 25 kDa. The optimal temperature of Est700 was 30 °C, with 35% maximal activity retaining at 0 °C. Its optimal pH was 8.0 and showed high stability at pH 5.0-11.0. Noticeably, Est700 was highly activated by 3.5 M NaCl and the extent of this activation is much stronger than that of currently reported halophilic ones. It was also stable in 5 M NaCl with no activity loss. High salt concentrations changed the secondary structure and folding properties of Est700 with formation of more α-helix and less β-sheet domains. With salt incubation, its melting temperature was estimated to be 57.2 °C, which is 12.8 °C higher than that of native one. Interestingly, Est700 lacks the acidic surface that is considered essential for enzyme stability at high salinity. However, it has a mainly positive surface electrostatic potential, which is probably different from most reported halotolerant esterases. These multiple properties make Est700 a valuable candidate in both basic research and industrial applications., (Copyright © 2018. Published by Elsevier B.V.)

Esterases are widely used in different industries. Here, a novel esterase, Esth, with low identity with previously reported esterases, was identified and characterized. The results showed that Esth was a cold-adapted esterase and retained 50% of its maximum activity at 0°C. Besides, Esth showed great activity and stability in high concentrations of NaCl. When treated with some organic solvents, detergents and metal ions, Esth showed high activity as well. The kcat/Km value of Esth was 29.5s -1 mM -1 , suggesting that it has higher catalytic efficiency than all the previously reported esterases from the same family, lipolytic enzyme family VI. The structural modeling showed that changing Ala129 into Gly would form a new hydrogen bond between ser125 and Gly129 and make theα-helix longer, which might influence on the thermostability of enzymes (Kumar, 2000). To confirm this, the mutant Esth A129G was obtained by site-directed mutagenesis. The result indicated that Esth A129G retained over 70% of the activity versus 12% for Esth after incubation at 55°C for 120min, showed a nearly six fold increase when compared with wild type. Overall, Esth shows a potential application prospect in extreme conditions and the mutation research can provide some structural information about thermostable enzymes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A salt-tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p-nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three-dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat-resistant features., (© 2015 American Institute of Chemical Engineers.)

Background: In recent years, cold-active esterases have received increased attention due to their attractive properties for some industrial applications such as high catalytic activity at low temperatures. Results: An esterase-encoding gene (estS, 909 bp) from Serratia sp. was identified, cloned and expressed in Escherichia coli DE3 (BL21). The estS encoded a protein (EstS) of 302 amino acids with a predicted molecular weight of 32.5 kDa. It showed the highest activity at 10 °C and pH 8.5. EstS was cold active and retained ~92 % of its original activity at 0 °C. Thermal inactivation analysis showed that the T1/2 value of EstS was 50 min at 50 °C (residual activity 41.23 %) after 1 h incubation. EstS is also quite stable in high salt conditions and displayed better catalytic activity in the presence of 4 M NaCl. To improve the thermo-stability of EstS, variants of estS gene were created by error-prone PCR. A mutant 1-D5 (A43V, R116W, D147N) that showed higher thermo-stability than its wild type predecessor was selected. 1-D5 showed enhanced T1/2 of 70 min at 50 °C and retained 63.29 % of activity after incubation at 50 °C for 60 min, which were about 22 % higher than the wild type (WT). CD spectrum showed that the secondary structure of WT and 1-D5 are more or less similar, but an increase in β-sheets was recorded, which enhanced the thermostability of mutant protein. Conclusion: EstS was a novel cold-active and salt-tolerant esterase and half-life of mutant 1-D5 was enhanced by 1.4 times compared with WT. The features of EstS are interesting and can be exploited for commercial applications. The results have also provided useful information about the structure and function of Est protein. [ABSTRACT FROM AUTHOR]

Highlights: [•] A genomic library of a psychrotrophic bacterium Psychrobacter celer 3Pb1 was constructed. [•] Est12, a phylogenetically new esterase, was isolated from the genomic library. [•] The highest sequence identity of Est12 was only 77% with a hypothetical esterase. [•] Est12 was cold-active and remained 41% maximum activity at 0°C. [•] Esterase activity was significantly increased by high salinity. [ABSTRACT FROM AUTHOR]