DEVELOPMENT AND APPLICATION OF AN EFFECTIVE SCREENING METHOD BASED ON A MICROWELL PLATE FOR MICROBES PRODUCING CELLULASE AND XYLANASE

Abstract

In this study, we developed an efficient screening method for microbes secreting cellulase and xylanase by applying the modified 3,5-dinitrosalicylic acid (DNS) method on a 96 microwell plate. Seawater and groundwater-based medium was used to culture marine and terrestrial microbial samples, respectively. Initial culture was carried out on agar plates containing both cellulose and xylan as substrates. Then pure colonies were isolated and subcultured in broth mediumcontaining cellulose and xylan substrate as well as yeast extract. The supernatants of broth culture were tested with our modified DNS screening method in a 96 microwell-plate, total reaction volume being 200 μl. In addition, stability and reliability of glucose and xylose standards, which are used to determine the enzymatic activity, at reaction temperature of 100 oC in dry oven was studied for different time intervals. It was concluded that the minimum incubation timerequired for stable color development is 20 min. With this novel method, we successfully screened 21 cellulase producing microbes and 31 xylanase-producing microbes from total of 116 selected strains in a single experimental trial. Among the strains, 19 of them showed both cellulase and xylanase activity.to culture marine and terrestrial microbial samples, respectively. Initial culture was carried out on agar plates containing both cellulose and xylan as substrates. Then pure colonies were isolated and subcultured in broth mediumcontaining cellulose and xylan substrate as well as yeast extract. The supernatants of broth culture were tested with our modified DNS screening method in a 96 microwell-plate, total reaction volume being 200 μl. In addition, stability and reliability of glucose and xylose standards, which are used to determine the enzymatic activity, at reaction temperature of 100 oC in dry oven was studied for different time intervals. It was concluded that the minimum incubation timerequired for stable color development is 20 min. With this novel method, we successfully screened 21 cellulase producing microbes and 31 xylanase-producing microbes from total of 116 selected strains in a single experimental trial. Among the strains, 19 of them showed both cellulase and xylanase activity.