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Title 

Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate

 

Cellulose binding domain 기능의 고속 분석

Authors 

Hyeon Dong KimSu-Lim ChoiHaseong KimJung Hoon SohnSeung Goo Lee

Publisher 

Springer Verlag (Germany)

Issue Date 

2013

Citation 

Biotechnology and Bioprocess Engineering, vol. 18, no. 3, pp. 575-580

Keywords 

alkaline phosphatasecellulasecellulose-binding domainenzyme-linked assayperiplasmic expression

Abstract 

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.

ISSN 

1226-8372

Link 

http://dx.doi.org/10.1007/s12257-013-0242-3

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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