Characterization of novel cold active and salt tolerant esterases with improved catalytic properties

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Esterase (EC 3.1.1.1) is a valuable industrial biocatalyst which hydrolyzes esters into alcohol and acid, or catalyzes ester formation through reverse reaction.Esterases show high enantion, regio-and stereo-selectivity towards its substrates.So this biocatalyst is studied widely and applied in different types of industrial applications such as in food,detergent, pharmaceuticals and chemical industries.Cold active esterases exhibit high catalytic efficiency at low temperature and could be energy saving and helpful for the synthesis of thermo labile compounds. Immobilization could increase the range of application of esterase through making it more stable to extreme conditions and benefits by reusability and fine chemical synthesis.Two novel esterase coding genes, EstLiu and EstH were cloned from the marine bacterium Zunongwangia profunda, overexpressed in E.coli BL21 (DE3) and purified by glutathione-S transferase (GST) affinity chromatography.The mature esterase EstLiu and EstH sequence encodes a protein of 273 and 547 amino acids residues, with a predicted molecular weight of 30 and 61.2 KDa.EstLiu contains the classical pentapeptidase motif from position 156 to 160 with the catalytic triad Ser158-Asp211-His243 and the conserved motif for the EstH is Gly 219-X-Ser 221-X-Gly 223 and the catalytic triads are Ser 221-Glu 337-His 456.Phylogenetic analysis showed EstLiu had no similarity with any of the established family of lipases/esterases, suggesting that it could be considered as a new family and EstH belongs to family Ⅶ.Both purified enzyme showed broad substrate specificity with the highest hydrolytic activity against p-nitrophenyl butyrate (C4). Though the optimal temperature 30℃ were same for both enzymes and quickened inactivation above 60℃, EstLiu showed remarkable activity (75%) at 0℃ and the optimal activity at pH 8.0 and EstH showed~50% of original activity at 0℃ and pH 8.5.Both of them were stable in high salt conditions (0-4.5 M NaCl) with good performance in the presence of organic solvents and detergents.To improve the characteristics and explore the possibilities for application of EstH, a new immobilization matrix, Fe3O4~cellulose nano-composite, was prepared where the magnetic nanoparticles of Fe3O4 were synthesized by co-precipitation of Fe2+ and Fe3+ ions in ammonia solution and Fe3O4~cellulose nano-eomposite was prepared by the solgel method and was characterized by Fourier Transform Infrared Spectroscopy (FTIR)and Scanning Electron Microscope (SEM). Compare with free form, interestingly the optimal temperature of immobilized EstH elevated to 35℃. It showed better temperature stability (48.5% compared to 22.40% at 50℃ after 30 min), prolonged half-life (32h compared to 18h), storage stability (~71% activity compared to~40% activity after 50 days of storage), pH tolerance (~73% activity at pH 4 and 10), and more importantly, reusability (~50% activity after 8 repetitive cycles of usage).Enzyme kinetics showed an increase in the Vmax (from 35.76 to 51.14μM/min) and Kcat (from 365 S-1 to 520 S-1) after immobilization onto nano-composite. The superior catalytic properties of EstLiu and immobilized EstH suggest their great potential in bioteehnology and industrial processes.
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