Abstract:

In enzyme engineering, screening for transformant containing accurate gene is labor-intensive and time-consuming. In this study, xylanase gene was cloned into pET20b to transform E. coli strain BL21(DE3) competent cells. 10 transformants were inoculated in 0.5ml LB for culturing overnight, 9.5ml LB was added and cultured for 2.5h. After induction for 5h, cells were harvested and lyzed by freezing-thawing procedure in a simplified lysis buffer. 7 transformants were regarded as positive for having activities closer to or higher than that of the positive control, thereafter, plasmid DNA was sequenced to confirm gene’s accuracy. Different from previous method screening transformant for gene, the present method screened transformant for enzyme activity, called activity-screening method. The preliminarily assayed activities were compared for finding out transformant having high activity. The procedure lasted for 2~3 days, having characteristic of high-throughput for assaying transformant’s enzyme activity in parallel.