Gene Cloning, Expression And Characterization Of Two Novel Esterases

Esterase is a multifunctional enzyme, which hydrolyzes the carboxyl ester, amid and thioester bonds of endogenous and exogemous compounds, and belongs to the family of serine hydrolase. Esterases from microorganisms could catalyze esterification, transesterification, interesterification and kinetic resolution of optically active compounds. These enzymes have many important biotechnology applications such as oil exploration, treatment of industrial waste water, food production, environment protection and medical biotechnology. Thermophilic esterases are the enzymes that catalyze the hydrolysis, synthesis and transfer reaction of esters under high temperature(optimum temperature≥60℃). Compared with mesophilic esterases, the thermophilic enzymes have many advantages in thermal stability, the resistance to denaturant and organic solvent and the catalytic rate, which make them to be the research focus in the filed of biocatalysis in recent years.In this paper, the two novel esterases EST10(Tm1160, from the thermophilic bacterium Thermotoga maritimd) and EstSt7(ST2026, from the hyperthermophilic archaeon Sulolobus tokodaii) were cloned, expressed and purified. The genes coding for esterase ESTIO and EstSt7were ampilified using PCR method, cloned into pET15b and designated to recombinant plasmids pET15b-Tm1160and pET15b-ST2026. The purified enzymes ESTIO and EstSt7were obtained after heat treatment, Ni-NTA affinity and Superdex-200gel filtration chromatography. The SDS-PAGE demonstrated that the relative molecular mass of the His6fusion enzyme EST10and EstSt7were37.2KDa and39.4KDa respectively.Gel filtration and chemical cross-linking experiments showed that the esterase EST10formed a dimmer in solution. Detailed studys on the enzymatic properties of the esterases EST10and EstSt7were carried out. The purified enzyme displayed optimal activity at70℃and pH5.0-5.5. The substrate specificity and kinetics indicated that the enzyme can hydrolyze p-nitrophenyl esters formed by short chain fatty acids (<C10) and the optimum substrate was p-nitrophnyl butyrate. EST10possessed high activity and extreme stability at high temperatures and low pHs. It had a half life of80min at90℃, and maintained about50%of its activity even after treatment for60min at70℃and pH4.5. In addition, the enzyme was stable in most organics (methanol, ethanol, DMF, acetone, isopropanol, isoamyl alcohol, chloroform), surfactants (Tween20, Tween80, Trtion-X100) and denaturants (SDS, DTT and urea).The metal ions Co2+、Cu2+and Fe2+had a slight inhibitory effect on the enzyme activity. EST10could hydrolyze ketoprofen ethyl ester to (S)-ketoprofen with high enantoselectivity, which made it to be a promising biocatalyst for industrial applications.Sequence alignment, phylogenetic analysis and comparison of the conserved motif reveal that esterase EstSt7(ST2026) should be grouped into a new bacterial lipase and esterase family, which was different from the current eight families of bacterial lipases and esterases. The optimum temperature of EstSt7was80℃and had a half-life of180min at90℃. Its activity maintained about80%of at pH8.0-10.0, with an optimum pH of9.0, which indicated it was an alkaline esterase. The hydrolysis kinetics and substrates specificity experiment demonstrated p-nitrophnyl butyrate was the optimum substrate. The enzyme showed high activity and stability against non-polar organic solvent (methanol, ethanol, propanol, acetone and chloroform) and surfactants (Tween20, Tween80and Trtion-X100). Most metal irons did not significantly affect the enzymatic activity while Cu2+, Fe2+and Zn2+could cause a drastic loss of the activity of EstSt7. SDS strongly inhibited its activity at both concentrations (1%and5%) and the enzyme activity was activated by5mM DTT (120%). The enzymatic activity can be greatly inhabited by the addition of chemical modifiers PMSF and DEPC, which demonstrated that Ser and His might be involved at the esterase catalytic site.