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Automated filter paper assay for determination of cellulase activity

Automated filter paper assay for determination of cellulase activity,10.1385/ABAB:107:1-3:689,Applied Biochemistry and Biotechnology,Stephen R. Decker

Automated filter paper assay for determination of cellulase activity   (Citations: 23)
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Recent developments in molecular breeding and directed evolution have promised great developments in industrial enzymes as demonstrated by exponential improvements in β-lactamase and green fluorescent protein (GFP). Detection of and screening for improved enzymes are relatively easy if the target enzyme is expressible in a suitable high-throughput screening host and a clearly defined and usable screen or selection is available, as with GFP and β-lactamase. Fungal cellulases, however, are difficult to measure and have limited expressibility in heterologous hosts. Furthermore, traditional cellulase assays are tedious and time-consuming. Multiple enzyme components, an insoluble substrate, and generally slow reaction rates have plagued cellulase researchers interested in creating cellulase mixtures with increased activities and/or enhanced biochemical properties. Although the International Union of Pure and Applied Chemists standard measure of cellulase activity, the filter paper assay (FPA), can be reproduced in most laboratories with some effort, this method has long been recognized for its complexity and susceptibility to operator error. Our current automated FPA method is based on a Cyberlabs C400 robotics deck equipped with customized incubation, reagent storage, and plate-reading capabilities that allow rapid evaluation of cellulases acting on cellulose and has a maximum throughput of 84 enzyme samples per day when performing the automated FPA.
Journal: Applied Biochemistry and Biotechnology - APPL BIOCHEM BIOTECH , vol. 107, no. 1, pp. 689-703, 2003
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    • ...The colorimetric assay based on 3,5-dinitrosalicylic acid (DNS) for estimating total reducing sugars was employed in a microplate format [44-47]...

    Brian C Kinget al. Arsenal of plant cell wall degrading enzymes reflects host preference ...

    • ...Manual [8,9] and semi-automated [10] microplatebased methods using artificial substrates have been described already, and the new generation of manual [11] and partially automated [12] microplate methods can use micronized biomass to assess these enzymatic activities...
    • ...Several of these methods make use of 3,5-dinitrosalicylic acid (DNS) [8,10,11] to assay the reducing sugars released by the enzymes because DNS assay is particularly suited to the microplate format...
    • ...This assay, based on the 3,5-dinitrosalicylic acid (DNS) method [8,10,11,16], was performed as described in Figure 1. The DNS reagent (5 g DNS and 150 g sodium potassium tartrate dissolved in 0.5 L of 0.4 N sodium hydroxide) was stored in the dark at room temperature...

    David Navarroet al. Automated assay for screening the enzymatic release of reducing sugars...

    • ...However, the filter paper assay requires considerable effort and is error-prone [72,73]...
    • ...a-Cellulose is normally used for total cellulase activity measurements [7,72]...
    • ...In addition, a-cellulose can be used in automated screening platforms [72] as suspensions of a-cellulose particles can be handled by pipetting...

    Gernot Jägeret al. Practical screening of purified cellobiohydrolases and endoglucanases ...

    • ...Filter paper has long been used as a standard substrate in cellulase and biomass conversion research and one of the earliest reports of an automated biomass assay technique used filter paper as the substrate [13]...
    • ...In 2006, Berlin and coworkers reported making paper handsheets from ethanol organosolv pulped yellow poplar and dispensing it into microtiter plates as disks generated using a paper punch, similar to the method by Decker and coworkers for a filter paper-based HTP assay [9, 13]...
    • ...The first successful attempt at applying this scale to biomass hydrolysis was the adaptation of the IUPAC standard filter paper assay for measuring cellulase activity to a 96-well plate format [13, 33]...

    Stephen R. Deckeret al. High-Throughput Screening Techniques for Biomass Conversion

    • ...The conventional International Union for Pure and Applied Chemistry (IUPAC) filter paper assay (FPA) method for measuring hydrolytic activity has been automated by Decker et al. (2003) to reduce operator error and adapted to use small reaction volumes by Xiao et al. (2004)...
    • ...The DNS reducing sugar assay was previously adapted to utilize a miniaturized 96-well microplate system (Decker et al., 2003; Xiao et al., 2004, 2005)...

    Brian C. Kinget al. An optimized microplate assay system for quantitative evaluation of pl...

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