Abstract
BackgroundIn industries lipolytic reactions occur in insensitive conditions such as high temperature thus novel stout esterases with unique properties are attracts to the industrial application. Protein engineering is the tool to obtain desirable characters of enzymes. A novel esterase gene was isolated from South China Sea and subjected to a random mutagenesis and site directed mutagenesis for higher activity and thermo-stability compared to wild type.ResultsA novel esterase showed the highest hydrolytic activity against p-nitrophenyl acetate (pNPA, C2) and the optimal activity at 40 °C and pH 8.5. It was a cold-adapted enzyme and retained approximately 40% of its maximum activity at 0 °C. A mutant, with higher activity and thermo-stability was obtained by random mutagenesis. Kinetic analysis indicated that the mutant Val29Ala/Tyr193Cys shown 43.5% decrease in Km, 2.6-fold increase in Kcat, and 4.7-fold increase in Kcat/Km relative to the wild type. Single mutants V29A and Y193C were constructed and their kinetic parameters were measured. The results showed that the values of Km, Kcat, and Kcat/Km of V29A were similar to those of the wild type while Y193C showed 52.7% decrease in Km, 2.7-fold increase in Kcat, and 5.6-fold increase in Kcat/Km compared with the wild type. The 3-D structure and docking analysis revealed that the replacement of Tyr by Cys could enlarge the binding pocket. Moreover Y193C also showed a better thermo-stability for the reason its higher hydrophobicity and retained 67% relative activity after incubation for 3 h at 50 °C.ConclusionsThe superior quality of modified esterase suggested it has great potential application in extreme conditions and the mutational work recommended that important information for the study of esterase structure and function.
Highlights
In industries lipolytic reactions occur in insensitive conditions such as high temperature novel stout esterases with unique properties are attracts to the industrial application
Esterases with novel properties have a more and broad application prospect: coldadapted esterases are applied to the industrial reaction at low temperature and benefit energy conservation [6,7,8]; salt-tolerant esterases are suitable for the reactions under
Gao et al Microb Cell Fact (2018) 17:45 a high salt concentration [9,10,11]; organic solvent-tolerant esterases/lipases are necessary for the substrates which are insoluble to water and the trans-esterification reaction in biodiesel production [5, 12, 13]
Summary
In industries lipolytic reactions occur in insensitive conditions such as high temperature novel stout esterases with unique properties are attracts to the industrial application. Esterases have been widely used in food production, detergent products, pharmaceuticals, perfumes, degradation of pollutants and the synthesis of optically pure compounds [4, 5] owing to their broad array of substrate specificity and versatility in the reactions they catalyze [3]. Gao et al Microb Cell Fact (2018) 17:45 a high salt concentration [9,10,11]; organic solvent-tolerant esterases/lipases are necessary for the substrates which are insoluble to water and the trans-esterification reaction in biodiesel production [5, 12, 13]. All information of mutations can facilitate the discovery of an esterase with more application value
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