Screening，Expression And Characterization Of Esterases/Lipases

Esterases (EC3.1.1.1), which catalyze the hydrolysis and formation of esterbonds, are widespread in various different organisms, including animals, plants andmicroorganisms. They can participate in various reactions such as esterification,interesterification and kinetic resolution reaction. Microbial esterase is one of themost important industrial enzymes. Not only they do not need any co-factor inreaction systems, esterases also have other outstanding features, such as mild reactionconditions, simple method, high catalytic activity, excellent selectivity and theproduct is much easier to separate and recycle. Therefore, they can be widely used infood industry, medical industry, chemical industry, environmental protection andbiological protection and other fields. For high reaction activity under hightemperature and strong resistance to organic solvent, decontamination agent anddenaturing agent, thermophilic esterases are widely used in food, medicine, leather,oil extraction and waste disposal fields.Microorganisms are the main source of most industrial esterases. The traditionalmethod to obtain the desired esterase producing strains is screening microorganismfrom soil via optimization the culture condition for several rounds. The limitation ofthe method is that enzyme production is too low to reach the industrial scale. In recentyears, genetic engineering technologies such as the construction of recombinantplasmids and the selection of appropriate expression host are often taken to acquiremuch higher expression levels.In the present research, we selected a series of genes which encoding putativeEsterase/Lipase from Alcanivorax borkumensis SK2, Thermomonospora curvataDSM43183and Thermotoga naphthophila RUK-10according to the NCBI database.The target genes were amplified by PCR. The amplified DNA fragments were insertedinto pET plasmid resulting in the formation recombinant plasmids. The recombinantprotein was expressed in E. coli which transformed with recombinant plasmids.Finally, we screened out one Esterase/Lipase encoding gene through the measurement of enzyme activity. Then, the physical and chemical properties of the recombinantEsterase/Lipase was characterized in detail after purification, which lay thefoundation of the further application of the enzyme.Esterases ABO-2454and ABO-2108(from Alcanivorax borkumensis SK2),thermophilic esterases Tnap-0664and Tnap-1739(from Thermotoga naphthophilaRUK-10) were expressed in E. coli successfully. Thermophilic esterase Tnap-0664was screened out finally and purified by heat-treatment and Q-sepharosechromatography. Tnap-0664was a homotetramer with the subunit molecular mass of43.8kDa. The purification fold and recovery ratio were8.43and41.97%respectively.Our results showed that Tnap-0664had much higher hydrolysis activity for shortchain p-nitrophenol ester than longer analogues. The optimum temperature and pH oftanp-0664were above100oC (≥100oC) and9.0for the hydrolysis of p-NPC8(p-nitrophenyl caprylate) respectively. The half-life of the recombinant enzyme at70,80and90oC were150,120and20h respectively, and80%residual activity wasreserved in the pH range of8-11, suggesting that Tnap-0664had excellent thermaland pH stability. The enzyme activity improred by monovalent ions K+and Na+to111.73%and115.77%, and inhibited slightly by divalent ions Ni2+and Mg2+to73.85%and69.46%, while Ca2+, Mn2+, Fe2+, Zn2+and Cu2+exerted strong inhibitioneffect on Tnap-0664with the residual activity of19%,2.08%,2.44%,3.04%and0%,respectively. EDTA exerted an activation effect of112%, suggesting that Tnap-0664does not require mental ions to catalyze reactions. Acetic acid exerted a stronginhibition effect on enzyme activity, while other organic reagent inhibited enzymeactivity slightly. Km and kcat values of Tnap0664for the hydrolysis activity ofp-NPC8were76.5μM and182.50s-1respectively.