Abstract
Thermostable enzymes have the potential as the biocatalyst for industrial applications. To compare the relationship of enzymatic thermostability, the moderately thermophilic and mesophilic bacteria were utilized to explore the properties of esterases. By using the shotgun libraries of mesophilic Thalassomonas agarivorans, and Aeromonas sp., and moderately thermophilic Ralstonia sp., esterases-encoding Lip20, Lip4 and LipRT for α/β-hydrolase fold were cloned, sequenced, and characterized. According to the recombinant proteins overexpressed by Escherichia coli, these results indicated that Lip20, Lip4 and LipRT preferred to hydrolyze short-length p-nitrophenyl (p-NP) esters. The optimal temperature required for the activity of Lip20, Lip4 and LipRT was 30, 40 and 60°C, respectively, corresponding to the trend of bacterial growth temperature. Even at low temperature, cold-adapted Lip4 from Aeromonas sp. revealed well enzymatic activity. In addition, after 60 min incubation between 40-60°C, over 92% residual activity can be retained by the thermostable analysis of LipRT from Ralstonia sp.. Inspecting the predicted structures and amino acid composition, we found that the high helix content was exhibited in LipRT. Also, high frequency residues of Val, Phe and Arg for increasing hydrophobic and salt-bridge interactions were observed. These factors could improve LipRT thermal stabilization and lead to become more rigid.
Highlights
The worldwide demand for enzymes is increasing, and expenditure on enzymes is expected to reach $6.9 billion by 2017
They were identified as Aeromonas sp., Ralstonia sp. and T. agarivorans
Three clones from T. agarivorans, Aeromonas sp. and Ralstonia sp. libraries produced significantly clear zones, indicating that they were capable of producing esterases, and were selected for shotgun sequencing
Summary
The worldwide demand for enzymes is increasing, and expenditure on enzymes is expected to reach $6.9 billion by 2017. Lipolytic enzymes account for 21% of the worldwide industrial enzyme market and are the third largest commercialized enzymes after proteases and carbohydrases [1]. They are widely present in animals, plants, and microorganisms. Lipolytic enzymes are used in various industrial areas, such as food, bioremediation, pharmaceuticals, detergents, biodiesel production, and waste treatment [2]. The development of high-yield production and thermostable lipolytic has attracted considerable attention. A considerable number of studies have been made on the thermostability by structural comparison of enzymes between the different optimal temperatures [3]. According to the different amino acid composition between thermophilic and mesophilic proteins, thermostable features can provide guidelines for the design of thermo-tolerance proteins [4]
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