Gene Cloning, Protein Expression And Characterization Of Cold-Active And Salt-Tolerant Esterases From Psychrobacter Sp.

Esterase （EC3.1.1.X） is one of the most valuable industry biocatalysts and able to hydrolyze the esters into alcohols and acids, or to catalyze the reverse reaction to produce various esters. So it is widely studied and applied in the fields of food, pharmaceuticals, agriculture, and chemical industries. Cold-active enzymes could exhibit high catalytic efficiency under low temperatures and are not stable under elevated temperatures, which could meet some special industrial process requirements （low temperature） and achieve the goal of highly efficient catalysis, energy reservation, and the synthesis of thermolabile products.In this study, we screened and isolated two psychrophilic strains, namely Psychrobacter sp. and Psychrobacter pacificensis, that were proved to be most active in producing cold-active esterases. By constructing their genomic libraries in Escherichia coli DH5a and library screening through the Luria-Bertani agar plate containing the esterase substrate tributyrin, we identified two positive recombinant clones able to form clear halos. After clone sequencing and bioinformatics analysis, two esterase-encoding genes were identified:the gene est12from Psychrobacter sp. and the gene est10from Psychrobacter pacificensis. The gene est10（Genbank accession no. KC291403） contained an open reading frame of672bp that encoded a protein of223amino acids with an estimated molecular mass of24,647Da and the protein Est10shared the highest similarity （92%） with a caboxylesterase from Psychrobacter sp. PAMC2119（NCBI reference sequence WP₀10199455.1）; The gene est12（Genbank accession no. KF313138） contained an open reading frame of990bp and encoded a protein of329amino acids with an estimated molecular mass of35,150Da and the protein Est12shared the highest similarity （77%） with a caboxylesterase from Psychrobacter sp. PAMC211（NCBI reference sequence WP₀10200623.1）.Protein Est10and Est12were induced, expressed and purified in Escherichia coli BL21（DE3）, and their enzymatic properties were characterized. Est10showed the highest activity against the short-chain substratee p-nitrophenyl butyrate （C4） among the tested p-nitrophenyl esters （C2-C16）, and no activity to p-nitrophenyl palmitate （C16）. Est10exhibited the optimal activity around25℃and pH7.5, and maintained as high as55.0%of its maximum activity at0℃, indicating its cold-adaptation. Est10was fairly stable under room temperatures, retaining more than80%of its original activity after incubation at40℃for2h. It was slightly activated at a low concentration （1mmol/L） of Zn2+, Mg2+, Ba2+, Ca2+, Cu2+, Fe3+, urea and EDTA, but was inhibited by DTT and totally inactivated by PMSF. Interestingly, increased salinity considerably stimulated Est10activity （up to143.2%of original activity at2mol/L NaC1） and stability （up to126.4%after incubation with5mol/L NaCl for6.5h）, proving its salt-tolerance.0.05%and0.1%Tween20, Tween80, Triton X-100and CHAPS increased the activity and stability of Est10while SDS, CTAB had the opposite effect. Est10was quite active after incubation with several30%organic solvents （methanol, DMSO, ethanediol） but exhibited little activity with30%isopropanol, ethanol, n-butanol and acetonitrile.Phylogenetic analysis suggested that the protein belonged to a new lipase/esterase family. Substrate specificity study showed that Est12preferred short-chain p-nitrophenyl esters and was most active towards p-nitrophenyl butyrate. Est12displayed the optimal activity at pH7.5and35℃, and remained41%activity at0℃while being unstable at temperatures above40℃, indicating its cold-adaptation. Besides, Estl2was a salt-tolerant esterase as4.5mol/L NaCl significantly declined Km from0.069to0.033mmol/L and increased kcat from4.20to9.21s-1, resulting in the increased catalytic efficiency kcat/Km from60.72to276.31s-1·mM-1. The enzyme activity was also quite stable after24h incubation in0-4.5mol/L NaCl solutions. In addition, Est12was very active and stable in the presence of several detergents and organic solvents.The two new cold-active, salt-tolerant esterases with certain tolerance to detergents and organic solvents, may be of potential application value in some industry process requiring extreme conditions （low temperature, high-salinity and organic synthesis）.