A novel esterase from Ralstonia sp. M1: Gene cloning, sequencing, high-level expression and characterization
Quyen. D. T Dao. T. T Nguyen. S. L. T
Protein Expression and Purification ; 2007 [Vol.51] Pages:133-140
Abstract
A newly isolated gene from Ralstonia sp. M1, encoding an esterase, was cloned in Escherichia coli and its nucleotide sequence determined. The 1.6kb insert revealed one complete open reading frame, predicted to encode an esterase (320 aa, 34.1kDa) with a pI of 9.86. EstR contained a putative oxyanion hole H36 G37, a conserved pentapeptide G103 HSLG107 and a conserved catalytic His265
and Asp237. The EstR sequence shared 64–70 and 44–48% identity with the hydrolases/acyltransferases from Burkholderia strains and from Ralstonia strains, respectively, 44 and 38% identity with the lactone-speciWc esterase from Pseudomonas Xuorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41mg/g wet cells. The Ni–NTA-puriWed esterase EstR showed an optimal activity in the temperature range 60–65°C and pH range 7.5–9.0 towards p-nitrophenyl caproate. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine. Metal ions showed slight effect on esterase activity. The inhibitor phenylmethanesulfonyl Xuoride inhibited strongly the esterase. Triton X-45 induced the activation of EstR, but other detergents slightly to strongly decreased or completely inhibited. Among tested p-NP esters, caproate was the most preferential substrate of this esterase.
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