Microplate-Based Filter Paper Assay to Measure Total Cellulase Activity Zhizhuang Xiao, Reginald Storms, Adrian Tsang Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal H4B 1R6 Quebec, Canada; telephone: (514) 848-2424 extension 3405; fax: (514) 848-4504; e-mail: tsang @vax2.concordia.ca Received 18 February 2004; accepted 29 July 2004 Published online 30 September 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20286 Abstract: The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample num- bers. To achieve this, we reduced the enzymatic reaction volume to 60 Al from the 1.5 ml used in the IUPAC meth- od. The modified 60-Al format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Tricho- derma reesei and Aspergillus species determined with our 60-Al format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-Al format FPA is quantitative and highly reproducible. Moreover, the addition of excess h-glucosidase increased the sensitivity of the assay by up to 60%. B 2004 Wiley Periodicals, Inc. Keywords: filter paper assay; total cellulase activity; h- glucosidase; DNS; high throughput screening INTRODUCTION Cellulose is an insoluble glucose polymer linked with h-1, 4-glycosidic bonds. The complete hydrolysis of cellulose into glucose requires the synergistic action of several cel- lulases, which include endoglucanase (EC 3.2.1.4), h-1, 4- cellobiohydrolases (EC 3.2.1.91), and h-glucosidase (EC 3.2.1.21) activities. Cellulases are useful in many industrial applications. They are used to convert cellulosic biomass to glucose, which is further fermented into ethanol (Galbe and Zacchi, 2002; Sun and Cheng, 2002). The addition of cellulases in detergents brightens the color of cotton textiles, softens the fabric, and improves the removal of particulates (Niehaus et al., 1999). Other applications of cellulases include im- provement of the nutritional value of cellulosic materials and forage crops, enhancement of pulp quality, and in- creased digestibility of organic matter with high fiber con- tent (Garcia et al., 2002; Bowman et al., 2002). The cellu- lase market is expected to expand dramatically particularly as the industrial scale conversion of cellulosic materials to ethanol is implemented (Cherry and Fidantsef, 2003). A variety of strategies can be employed to obtain ef- fective cellulases for industrial application. For example, different naturally produced cellulolytic enzymes can be produced and tested in combinations to create potent syn- ergistic systems that hydrolyze cellulose in concert. Strat- egies that utilize directed evolution such as random muta- genesis by error-prone PCR and DNA shuffling provide promising methods to create novel and improved enzymes (Chen and Arnold, 1993; Stemmer, 1994). Both strategies, but particularly the latter, involve the identification of en- zymes with improved characteristics within a large back- ground of wildtype, neutral, and deleterious mutants. An effective high-throughput assay is therefore needed to screen for cellulases with improved characteristics. Since the Commission on Biotechnology of The Inter- national Union of Pure and Applied Chemistry (IUPAC) proposed a number of standard procedures for the measure- ment of cellulase activities in 1984, the filter paper assay (FPA) has been widely used to measure the total activity of cellulases (Wood and Bhat, 1988). Filter paper is used as the standard substrate because it is readily available, inexpensive, and assays performed with it reliably provide reproducible results (Coward-Kelly et al., 2003). This assay is performed so that 0.5 ml of diluted enzymes releases about 2.0 mg of glucose equivalents in 60 min, as deter- mined by the dinitrosalicylic acid (DNS) reducing sugar assay (Miller, 1959; Wood and Bhat, 1988). However, the IUPAC filter paper assay is time-con- suming, labor-intensive, and requires large amounts of re- agents. It is also difficult to obtain adequate sensitivity and reproducibility when characterizing new cellulase mixtures using this method. Factors that affect sensitivity and repro- ducibility often result from the fact that most natural cel- lulase complexes tend to have a shortage of cellobiase or h-glucosidase activity (Coward-Kelly et al., 2003; Breuil et al., 1986). Most recently, Coward-Kelly et al. (2003) B 2004 Wiley Periodicals, Inc. Correspondence to: Adrian Tsang Contract grant sponsors: Strategic Projects grant from the Natural Sciences and Engineering Research Council of Canada, Genome Que ´bec, and Genome Canada