KMID : 0545120140240111559
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Journal of Microbiology and Biotechnology 2014 Volume.24 No. 11 p.1559 ~ p.1565
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Effective Microwell Plate-Based Screening Method for Microbes Producing Cellulase and Xylanase and Its Application
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Jennifer Jooyoun Kim
Kwon Young-Kyung Kim Ji-Hyung Heo Soo-Jin Lee Young-Deuk Lee Su-Jin Shim Won-Bo Jung Won-Kyo Hyun Jung-Ho Kwon Kae-Kyoung Kang Do-Hyung Oh Chul-Hong
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Abstract
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Cellulase and xylanase are main hydrolysis enzymes for the degradation of cellulosic and hemicellulosic biomass, respectively. In this study, our aim was to develop and test the efficacy of a rapid, high-throughput method to screen hydrolytic-enzyme-producing microbes. To accomplish this, we modified the 3,5-dinitrosalicylic acid (DNS) method for microwell plate-based screening. Targeted microbial samples were initially cultured on agar plates with both cellulose and xylan as substrates. Then, isolated colonies were subcultured in broth media containing yeast extract and either cellulose or xylan. The supernatants of the culture broth were tested with our modified DNS screening method in a 96-microwell plate, with a 200 ¥ìl total reaction volume. In addition, the stability and reliability of glucose and xylose standards, which were used to determine the enzymatic activity, were studied at 100¨¬C for different time intervals in a dry oven. It was concluded that the minimum incubation time required for stable color development of the standard solution is 20 min. With this technique, we successfully screened 21 and 31 cellulase- and xylanase-producing strains, respectively, in a single experimental trial. Among the identified strains, 19 showed both cellulose and xylan hydrolyzing activities. These microbes can be applied to bioethanol production from cellulosic and hemicellulosic biomass.
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KEYWORD
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bacteria, fungi, screening, cellulase, xylanase, 3, 5-dinitrosalicylic acid
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