title: [High throughput screening atrazine chlorohydrolase mutants with enhanced activity through Haematococcus pluvialis expression system].
abstract: Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase. A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. AD1 was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type. The activities were 1.8-3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8-2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.
authors.affiliation: Laboratory of Molecular Genetics, College of Life Sciences, Nankai University, Tianjin 300071, China.
authors.forename: Huizhuan
authors.initials: H
authors.lastname: Wang
authors.forename: Xiwen
authors.initials: X
authors.lastname: Chen
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Hao
authors.forename: Defu
authors.initials: D
authors.lastname: Chen
mesh_terms: D000581:Amidohydrolases / Q000235:genetics*; D001280:Atrazine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D001673:Biodegradation, Environmental; D000460:Chlorophyta / Q000235:genetics / Q000378:metabolism*; D006540:Herbicides / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006867:Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D016254:Mutagenesis, Insertional; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D006540:Herbicides; D006867:Hydrolases; D000581:Amidohydrolases; C109894:AtzC protein, Pseudomonas; C100894:atrazine chlorohydrolase; D001280:Atrazine