Molecular cloning, purification and biochemical characterization of a novel pyrethroid-hydrolyzing carboxylesterase gene from Ochrobactrum anthropi YZ-1

• Published in: Journal of hazardous materials Vol. 221-222; pp. 206 - 212
• Main Authors: Zhai, Yi, Li, Kang, Song, Jinlong, Shi, Yanhua, Yan, Yanchun
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 30-06-2012
• Subjects: activated sludge; Amino Acid Sequence; amino acid sequences; Amino acids; Base Sequence; Biodegradation; Carboxylesterase; Carboxylesterase - chemistry; Carboxylesterase - genetics; Chromatography, Gas; Cloning; Cloning, Molecular; Degradation; DNA Primers; enzyme activity; Enzymes; Escherichia coli; Escherichia coli - genetics; Esterases; Genes; Genes, Bacterial; genomic libraries; Genomic library; Hydrolysis; molecular cloning; Molecular Sequence Data; Ochrobactrum anthropi; Ochrobactrum anthropi - enzymology; Ochrobactrum anthropi - genetics; Ochrobactrum anthropi - isolation & purification; open reading frames; pesticides; phylogeny; Polymerase Chain Reaction; pyrethrins; Pyrethrins - metabolism; Pyrethroids; sequence homology; Sequence Homology, Amino Acid; Strain; Substrate Specificity; temperature; Biodegradation; Pyrethroids; Genomic library; Carboxylesterase
• ISSN: 0304-3894; 1873-3336
• DOI: 10.1016/j.jhazmat.2012.04.031

► Strain YZ-1 capable of degrading pyrethroids was isolated and identified. ► The genomic library of strain YZ-1 was constructed successfully. ► The cloned pytZ was a new pyrethroid-degrading gene. ► PytZ displayed broad substrate specificity, high enzyme activity and stability. ► It provided a favorable choice for the biodegradation of pyrethroids residues.
Strain YZ-1 was isolated from activated sludge and identified as Ochrobactrum anthropi. This strain was capable of degrading pyrethroids pesticides, suggesting the presence of degrading enzymes. In the present study, a novel esterase gene pytZ was cloned from the genomic library of YZ-1 successfully. The pytZ contained an open reading frame of 606bp encoding a pyrethroid-hydrolyzing carboxylesterase. Deduced amino acid sequence showed moderate identities (39–59%) with most homologous carboxylesterase, except a putative carboxylesterase from O. anthropi ATCC 49188 with the highest identity of 85%. Phylogenetic analysis revealed that PytZ belonged to esterase VI family. The gene pytZ showed no any sequence similarity with reported pyrethroid-hydrolyzing genes and was a new pyrethroid-degrading gene. PytZ was expressed in Escherichia coli BL21 (DE3) and purified using Ni-NTA Fast Start. PytZ was able to degrade various pyrethroids. The optimal temperature and pH were 35°C and 7.5. This enzyme was very stable over a wide range of temperature and pH. No cofactors were required for enzyme activity. Broad substrate specificity, high enzyme activity, and the favorable stability make the PytZ a potential candidate for the detoxification of pyrethroid residues in biotechnological application.