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.
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

Objective To observe the effect of antibacterial dressing tamponade combined with seal negative pressure drainage in the treatment of necrotizing fasciitis .Methods 52 patients with necrotizing fasciitis were selected . After full scavenging of fasciitis and necrotic tissue,then filled the whole wound with a kind of dressings with antibacterial properties-Nano silver antiseptic dressing,and a negative pressure drainage tube was placed at the bottom of the wound.Then the wound was closed with a transparent paste ,and the air was cut off,in order to make the antibacterial dressing could not only anti bacteria ,but also play a role in the growth of the wound ,and at the same time,the tissue leachate in the wound was drained through the negative pressure tube in time ,so as to accelerate the wound healing and reduce the use of antibiotics .Results Among the 52 cases,the rest were cured except for 1 case of individual cause of abandonment of treatment died of toxic shock .,the average hospitalization time was (29.0 ±15.3)days,days of using antimicrobial was (3.8 ±1.6)days,90% cases stopped using antibiotics within 1 week.Conclusion Antibacterial dressing tamponade combined with seal negative pressure drainage in the treatment of necrotizing fasciitis can reduce the switching frequency,reduced antibiotic using intensity,shorten the healing time,it is a kind of very good method.