title: Cloning, expression and mutation of a triazophos hydrolase gene from Burkholderia sp. SZL-1.
abstract: Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C-50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research.
authors.affiliation: Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Tongwei Road 6, Nanjing, 210095, PR China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Tongwei Road 6, Nanjing, 210095, PR China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.affiliation: Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Tongwei Road 6, Nanjing, 210095, PR China.
authors.forename: Su-Hui
authors.initials: SH
authors.lastname: Guo
authors.affiliation: Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Tongwei Road 6, Nanjing, 210095, PR China.
authors.forename: Ming-Gen
authors.initials: MG
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Tongwei Road 6, Nanjing, 210095, PR China.
authors.forename: Meng-Jun
authors.initials: MJ
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Tongwei Road 6, Nanjing, 210095, PR China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Hong
authors.affiliation: Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Tongwei Road 6, Nanjing, 210095, PR China huangxing@njau.edu.cn.
authors.forename: Xing
authors.initials: X
authors.lastname: Huang
mesh_terms: D001673:Biodegradation, Environmental; D019117:Burkholderia / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D052918:Environmental Restoration and Remediation; D015870:Gene Expression; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D063086:Organothiophosphates / Q000378:metabolism*; D012988:Soil Microbiology; D014230:Triazoles / Q000378:metabolism*
chemical_list: D063086:Organothiophosphates; D014230:Triazoles; C012584:triazophos; D006867:Hydrolases
keywords: Burkholderia sp. SZL-1; mutation; trhA; triazophos