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- Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate = Cellulose binding domain 기능의 고속 분석
- Hyeon Dong Kim; Su-Lim Choi; Haseong Kim; Jung Hoon Sohn; Seung Goo Lee
- Bibliographic Citation
- Biotechnology and Bioprocess Engineering, vol. 18, no. 3, pp. 575-580
- Publication Year
- Cellulose-binding domain (CBD) enriches cellulolytic enzymes on cellulosic surfaces and contributes to the catalytic efficiency by increasing enzyme-substrate complex formations. Thus, high affinity CBDs are essential for the development of efficient cellulose-degrading enzymes. Here, we present a microtiter plate-based assay system to measure the binding affinity of CBDs to cellulose. The assay uses a periplasmic alkaline phosphatase (AP) as a fusion reporter and its activity is detected using a fluorogenic substrate, 4-methylumbelliferyl phosphate. Lignocellulose discs of 6 mm in diameter were used as substrates in 96-well plate. As a result, the enzyme-linked assay detected the binding of CBDs on the cellulosic discs in a highly sensitive manner, detecting from 0.05 to 1.0 μg/mL of APCBD proteins, which is several hundred times more sensitive than conventional protein measurements. The proposed method was applied to compare the binding affinity of different CBDs from Cellulomonas fimi to lignocellulose discs.
- alkaline phosphatasecellulasecellulose-binding domainenzyme-linked assayperiplasmic expression
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- Synthetic Biology and Bioengineering Research Institute > Synthetic Biology Research Center > 1. Journal Articles
Synthetic Biology and Bioengineering Research Institute > 1. Journal Articles
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