A novel esterase from Ralstonia sp. M1: gene cloning, sequencing, high-level expression and characterization.
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 H36G37, a conserved pentapeptide G103HSLG107 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-specific esterase from Pseudomonas fluorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41mg/g wet cells. The Ni-NTA-purified esterase EstR showed an optimal activity in the temperature range 60-65 degrees 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 fluoride 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.
Quyen
Dinh Thi
DT
Institute of Biotechnology, Vietnamese Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr. Caugiay, 10600 Hanoi, Viet Nam. quyen@ibt.ac.vn
Dao
Thi Tuyet
TT
Thanh Nguyen
Sy Le
SL
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D004950:Esterases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D043367:Ralstonia / Q000201:enzymology* / Q000235:genetics; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D003902:Detergents; D012997:Solvents; D004950:Esterases