High throughput screening atrazine chlorohydrolase mutants with enhanced activity through Haematococcus pluvialis expression system.
- Author:
Huizhuan WANG
1
;
Xiwen CHEN
;
Xiaohua HAO
;
Defu CHEN
Author Information
1. Laboratory of Molecular Genetics, College of Life Sciences, Nankai University, Tianjin 300071, China.
- Publication Type:Journal Article
- MeSH:
Amidohydrolases;
genetics;
Atrazine;
metabolism;
Bacterial Proteins;
genetics;
Biodegradation, Environmental;
Chlorophyta;
genetics;
metabolism;
Herbicides;
metabolism;
High-Throughput Screening Assays;
Hydrolases;
biosynthesis;
genetics;
Mutagenesis, Insertional;
Pseudomonas;
enzymology;
genetics
- From:
Chinese Journal of Biotechnology
2011;27(4):620-628
- CountryChina
- Language:Chinese
-
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.
