Microplate-Based Filter Paper Assayto Measure Total Cellulase ActivityZhizhuang Xiao, Reginald Storms, Adrian TsangCentre ...
supplemented cellobiase into a cellulase system with low        Filter Paper Assay Protocolslevels of h-glucosidase activi...
where FPU/ml is the determined cellulose activity in the                    glucoside as the substrate. A 20-Al aliquot of...
Determination of Cellulase Activity Using Standard                        reagents were adjusted proportionally to that of...
Table II. Effects of reaction volumes on the measurement of cellu-                 Table IV. Comparison of Trichoderma C-2...
Table V. Comparison of Aspergillus cellulase C-2605 activity using the         format is a robust assay that can rapidly a...
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  1. 1. Microplate-Based Filter Paper Assayto Measure Total Cellulase ActivityZhizhuang Xiao, Reginald Storms, Adrian TsangCentre 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.caReceived 18 February 2004; accepted 29 July 2004Published online 30 September 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20286Abstract: The standard filter paper assay (FPA) published creased digestibility of organic matter with high fiber con-by the International Union of Pure and Applied Chemistry tent (Garcia et al., 2002; Bowman et al., 2002). The cellu-(IUPAC) is widely used to determine total cellulase activity. lase market is expected to expand dramatically particularlyHowever, the IUPAC method is not suitable for the parallelanalyses of large sample numbers. We describe here a as the industrial scale conversion of cellulosic materials tomicroplate-based method for assaying large sample num- ethanol is implemented (Cherry and Fidantsef, 2003).bers. To achieve this, we reduced the enzymatic reaction A variety of strategies can be employed to obtain ef-volume to 60 Al from the 1.5 ml used in the IUPAC meth- fective cellulases for industrial application. For example,od. The modified 60-Al format FPA can be carried out in different naturally produced cellulolytic enzymes can be96-well assay plates. Statistical analyses showed that thecellulase activities of commercial cellulases from Tricho- produced and tested in combinations to create potent syn-derma reesei and Aspergillus species determined with our ergistic systems that hydrolyze cellulose in concert. Strat-60-Al format FPA were not significantly different from the egies that utilize directed evolution such as random muta-activities measured with the standard FPA. Our results genesis by error-prone PCR and DNA shuffling providealso indicate that the 60-Al format FPA is quantitative promising methods to create novel and improved enzymesand highly reproducible. Moreover, the addition of excessh-glucosidase increased the sensitivity of the assay by up (Chen and Arnold, 1993; Stemmer, 1994). Both strategies,to 60%. B 2004 Wiley Periodicals, Inc. but particularly the latter, involve the identification of en-Keywords: filter paper assay; total cellulase activity; h- zymes with improved characteristics within a large back-glucosidase; DNS; high throughput screening ground of wildtype, neutral, and deleterious mutants. An effective high-throughput assay is therefore needed to screen for cellulases with improved characteristics.INTRODUCTION Since the Commission on Biotechnology of The Inter-Cellulose is an insoluble glucose polymer linked with h-1, national Union of Pure and Applied Chemistry (IUPAC)4-glycosidic bonds. The complete hydrolysis of cellulose proposed a number of standard procedures for the measure-into glucose requires the synergistic action of several cel- ment of cellulase activities in 1984, the filter paper assaylulases, which include endoglucanase (EC 3.2.1.4), h-1, 4- (FPA) has been widely used to measure the total activity ofcellobiohydrolases (EC 3.2.1.91), and h-glucosidase (EC cellulases (Wood and Bhat, 1988). Filter paper is used as3.2.1.21) activities. the standard substrate because it is readily available, Cellulases are useful in many industrial applications. inexpensive, and assays performed with it reliably provideThey are used to convert cellulosic biomass to glucose, reproducible results (Coward-Kelly et al., 2003). This assaywhich is further fermented into ethanol (Galbe and Zacchi, is performed so that 0.5 ml of diluted enzymes releases2002; Sun and Cheng, 2002). The addition of cellulases in about 2.0 mg of glucose equivalents in 60 min, as deter-detergents brightens the color of cotton textiles, softens the mined by the dinitrosalicylic acid (DNS) reducing sugarfabric, and improves the removal of particulates (Niehaus assay (Miller, 1959; Wood and Bhat, 1988).et al., 1999). Other applications of cellulases include im- However, the IUPAC filter paper assay is time-con-provement of the nutritional value of cellulosic materials suming, labor-intensive, and requires large amounts of re-and forage crops, enhancement of pulp quality, and in- 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- Correspondence to: Adrian Tsang Contract grant sponsors: Strategic Projects grant from the Natural lulase complexes tend to have a shortage of cellobiaseSciences and Engineering Research Council of Canada, Genome Quebec, ´ or h-glucosidase activity (Coward-Kelly et al., 2003; Breuiland Genome Canada et al., 1986). Most recently, Coward-Kelly et al. (2003)B 2004 Wiley Periodicals, Inc.
  2. 2. supplemented cellobiase into a cellulase system with low Filter Paper Assay Protocolslevels of h-glucosidase activity to increase the production All the enzyme activity values presented are averages ob-of reducing sugar, thereby increased the sensitivity and tained from assays performed in triplicate. The standardreproducibility of the FPA. Decker et al. (2003) replaced FPA assays were performed essentially as described pre-the traditional filter paper strips with filter paper disks, viously (Miller, 1959; Wood and Bhat, 1988). Diluted en-Solka-Floc, SigmaCell-20, and Avicel PH101 to suit as- zyme, in 0.5 ml volume, was added to 25-ml test tubessay automation. However, the enzyme values detected with containing a 1 Â 6 cm paper strip in 1.0 ml of 50 mM NaActhese assays differed from the standard filter paper method buffer, pH 4.8. After 60 min of incubation in a 50jC wa-by 4% to 60%. In the cellulase assay presented here, fil- ter bath, 3.0 ml of DNS reagent was added to each tube,ter paper was retained as the substrate. However, the re- followed by vigorous boiling for 5 min. Finally, after theaction volume was reduced so that cellulase assays could colored solution was diluted with 20 ml of H2O, 200-Albe conducted in a 96-well microplate format suitable for samples were transferred to a 96-well flat bottom platehigh-throughput screening. Furthermore, we show that sup- (Corning, Corning, NY) and the absorbance at 540 nm wasplementing cellulase assays with excess h-glucosidase in- measured with a plate reader (Power Wave HT Bio-TEKcreased assay sensitivity. Instruments, Winooski, Vermont). The amount of enzyme used for an assay should produce about 2.0 mg of glucose equivalents in 60 min of incubation at 50jC (Wood andMATERIALS AND METHODS Bhat, 1988). The 96-Al microplate-based assay was performed as fol-Enzymes and Chemicals lows. A 32-Al aliquot of diluted enzyme was added into wells containing a 7-mm diameter filter paper disk andCellulase C-2730 from Trichoderma reesei, Cellulase C- 64 Al of 50 mM NaAc buffer, pH 4.8. After 60 min of2605 from Aspergillus species, C-6105 Novozyme 188 incubation at 50jC, 50 Al of each reaction was transferred(h-glucosidase) from Aspergillus niger, and all chemicals into the corresponding well of a second 96-well PCR platewere purchased from Sigma-Aldrich (St. Louis, MO). containing 100 Al of DNS and incubated at 95jC for 5 min. All incubations were performed in a temperature cycler (Perkin-Elmer Applied Biosystems 9700 PCR system, Nor-Substrates walk, CT). Following color development, a 36-Al aliquot ofWhatman No. 1 filter paper was cut into 1.0 Â 6.0 cm strips each sample was transferred to the wells of a flat-bottom,with a paper cutter for the standard 1.5 mL FPA and into 96-well plate containing 160 Al of H2O and the absorbancecircles with diameter of 7.0 mm with an office paper punch at 540 nm was measured in a plate reader. Relative to thefor the 96-well microplate assays. amount of glucose equivalents expected to be produced in the standard FPA, the amount of enzyme used in the 96-Al FPA should release about 128 Ag of glucose equivalents inReaction Volume of FPA in 96-Well Plate each well.The ratio of the reaction volume to the area of the filter The 60-Al format assay was carried out as follows. Apaper strip used in the standard IUPAC FPA was main- 20-Al aliquot of diluted enzyme was added into wells con-tained for our initial assessment of the 96-well assay plate- taining 40 Al of 50 mM NaAc buffer and a filter paper disk.based method. In the standard FPA, 0.5 ml of enzyme was After 60 min of incubation at 50jC, 120 Al of DNS wasadded into 1.0 ml of 50 mM NaAc buffer, pH 4.8, con- added into each reaction and incubated at 95jC for 5 min.taining a filter paper strip (1 Â 6 cm = 600 mm2), while the Finally, a 36-Al aliquot of each sample was transferred to7-mm diameter paper disk, whose area is 38.5 mm2, was the wells of a flat-bottom plate containing 160 Al of H2O,used in the 96-well plate assay. Accordingly, the reaction and the absorbance at 540 nm was measured.volume for assays done in 96-well plates was calculated as(1.0 ml buffer + 0.5 ml enzyme) H 600 mm2 Â 38.5 mm2 =0.096 ml = 96 Al. Calculation of Filter Paper Units (FPU) Under the IUPAC recommended assay conditions, oneDetermination of Evaporative Loss FPU is defined as an average of one Amole of glucose equivalents released per min in the assay reaction. FPU/mlTo determine the evaporative loss of the buffer during was calculated using the following formula:60 min of enzymatic action at 50jC, 96 Al of 50 mM NaAcbuffer, pH 4.8, was added into each well of a 96-well PCR A540 sample plate (UltiDent Scientific, St. Laurent, Quebec, Canada). FPU ml ¼ ð5:55 lmole mgÞ A540 =mg standardThe net weight of the buffer in the plate was measured before and after incubation at 50jC. Then the evaporative 1 1 Âloss of buffer was determined. 60 min Xml XIAO ET AL.: MICROPLATE-BASED CELLULASE ASSAY 833
  3. 3. where FPU/ml is the determined cellulose activity in the glucoside as the substrate. A 20-Al aliquot of varyingsuitably diluted sample; A540 sample is the absorbance amounts of diluted cellulase enzyme was mixed with 40 Alobtained from the DNS assay for each cellulase assay; of 1.0 CBU/ml Novozyme 188 in 50 mM NaAc, pH 4.8.A540/mg standard is the absorbance for 1 mg of glucose as Different ratios of h-glucosidase units (or cellobiase units,derived from the glucose standard curve (for example, the CBU) to FPUs were supplemented into the assay. Thecurve in Figure 1A was used to calculate the A540/mg assay without the addition of h-glucosidase was used asstandard used for the standard FPA assays); 5.55 Amole/ the control.mg is the number of Amoles of glucose in 1 mg; 60 minis the assay incubation time, and X ml is the volume ofsuitably diluted cellulase that was assayed. In the standard, RESULTS AND DISCUSSION96-Al and 60-Al FPA assays, X ml is 0.5 ml, 0.032 ml and0.02 ml, respectively. Modification of the Standard Filter Paper Assay Initially, we wanted to use the same ratio of reactionSupplement of h-Glucosidase volume to filter paper area for the microplate-based assayh-Glucosidase activity was determined as described pre- as is used in the standard IUPAC FPA. For this, we beganviously (Wood and Bhat, 1988) using p-nitrophenyl-h-D- by determining the weight of the 1 Â 6 cm filter paper strips used in the standard FPA and the 7-mm circles to be used in the microplate assay. The average weight of 100 Whatman No. 1 filter paper strips was 52.3 F 1.3 mg, with 2.5% of coefficient of variation, whereas the average weight of 100 filter paper circles was 3.42 F 0.28 mg, with 8.2% of coefficient of variation. Since the average weight of the filter paper substrate is directly proportional to its area, we initially used 96 Al reaction volumes for assays done in 96-well assay plates. This enabled us to maintain the same ratio of substrate, enzyme, buffer, DNS reagent, and water for the 96-Al format assay as we used in the standard FPA. In the standard FPA, all components are added into one 25-ml test tube, where the enzymatic hy- drolysis, the color development with DNS reagent, and dilution of the colored solution take place before the ab- sorbance is measured. In the 96-Al FPA, due to the well capacity, it was necessary to sequentially transfer the re- action products into two other microplates to develop color and then measure the absorbance at 540 nm. Reducing Evaporative Loss Previous results found that there was about 45% evap- orative loss of the water from the wells of a 96-well plate after 60 min of incubation at 50jC without a lid on the plate (Decker et al., 2003). In order to minimize evaporation during incubation we incubated the 96-well PCR plate in a temperature cycler, which has a built-in 105jC hot lid to prevent evaporation. A plastic mat was also used to cover the PCR plate. The combination of hot lid and plastic mat reduced the evaporative loss to only 4.0%. Glucose standard curves for the IUPAC method are generated using samples that are generally not subjected to the 50jC incubation step. To offset the effect of evap- orative loss on enzyme activity estimates, the glucose stan-Figure 1. Standard DNS curves for measuring the release of glucoseduring FPAs. The absorbance obtained with different amounts of glucose dard curve was plotted using a series of glucose standardswas measured using a plate reader. The best-fit lines are shown. A: Curve that were also subjected to incubation in the temperaturefor the standard FPA. B: Curve for the 96-Al FPA. cycler at 50jC for 60 min.834 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 88, NO. 7, DECEMBER 30, 2004
  4. 4. Determination of Cellulase Activity Using Standard reagents were adjusted proportionally to that of the 96-AlFPA and 96-Ml FPA reactions. Figure 2 shows that the standard curves obtainedThe release of glucose equivalents was determined using for assays performed in both 60 Al and 135 Al reaction vol-a standard curve produced using glucose samples having umes were linear.concentrations of 0, 2, 4, 6, 8, 10 mg/ml in 50 mM NaAc For the 135-Al reactions, the measured enzyme activitybuffer, pH 4.8. The absorbance at 540 nm versus glucose was 20% lower than that of 96-Al FPA. However, when theamount in the reaction was plotted using the standard for- reaction volume was reduced to 60 Al, the determinedmat (25-ml glass tubes) and microplate format methods enzyme activity was not significantly different from that(Fig. 1). The data indicate that both methods accurately obtained with the 96-Al FPA (Table II). These results sug-measure the quantity of glucose over the concentration gest that the amount of substrate was rate-limiting whererange tested. the reaction volume was 135 Al. Hence, increasing the To initiate a comparison of the standard assay and our amount of enzyme used, while keeping the substrate quan-modified assay, the C-2730 cellulase was diluted 100-fold tity constant, resulted in reduced assay sensitivity. On thein 50 mM NaAc buffer (pH 4.8) and divided into 30 sam- other hand, decreasing the amount of enzyme used by aboutples. These 30 cellulase samples were separated into two 40%, the 60-Al reaction did not adversely affect the abilityequal groups, one of which was assayed using the standardFPA and the other with the 96-Al FPA. The results obtainedare presented in Table I. A t-test performed on the datashowed that there was no significant difference at the 95%confidence level between the units of enzyme activity de-termined with the standard FPA and the 96-Al FPA. Our96-Al FPA carried out in 96-well PCR plates thereforeaccurately measured cellulase activity levels.Effects of Different Reaction Volumes on FPAWhen 7.0-mm diameter filter paper disks are used as thesubstrate, a reaction volume of 96 Al maintains the sameratio of the reaction volume to the amount of substrate as isused in the standard IUPAC assay. We next examined theeffect of different reaction volumes on the sensitivity of theassay. Using the 7-mm diameter filter paper disks assubstrates, we performed the assays in reaction volumes of60 Al and 135 Al. The volumes of enzyme and otherTable I. Trichoderma cellulase C-2730 activity determined using thestandard FPA and the 96-Al FPA.Sample Standard FPA (FPU/ml) 96-Al FPA (FPU/ml) 1 45.6 45.3 2 44.2 43.6 3 50.2 44.7 4 43.2 42.8 5 52.5 61.8 6 42.6 41.8 7 51.0 41.7 8 42.6 39.5 9 42.4 43.510 41.9 61.611 43.0 57.512 44.5 60.113 43.3 48.114 45.2 47.215 48.1 45.8Average 45.4 F 3.3 48.3 F 7.5 Figure 2. Standard DNS curves for measuring the release of glucose in the 60-Al FPA and the 135-Al FPA. The absorbance obtained with dif- T-test: The data obtained from the 96-Al FPA were compared to those ferent amounts of glucose was measured using a plate reader at 540 nm.obtained from the regular standard FPA. The calculated t-value is 1.32, The best-fit lines are shown. A: Curve for the 135-Al FPA. B: Curve forwhich is below the critical t-value of 2.05 at P = 0.05 and DF = 28. the 60-Al FPA. XIAO ET AL.: MICROPLATE-BASED CELLULASE ASSAY 835
  5. 5. Table II. Effects of reaction volumes on the measurement of cellu- Table IV. Comparison of Trichoderma C-2730 activity measured usinglase activity. the standard FPA and the 60-Al FPA. 96-Al FPA 135-Al FPA 60-Al FPA Sample Standard FPA (FPU/ml) 60-Al FPA (FPU/ml)Sample (FPU/ml) (FPU/ml) (FPU/ml) 1 45.6 44.41 58.0 42.2 51.7 2 44.2 49.72 58.8 41.0 54.5 3 50.2 45.03 48.5 45.3 48.6 4 43.2 52.64 50.4 45.9 48.5 5 52.5 52.65 51.0 48.9 52.4 6 42.6 48.56 51.5 40.4 47.6 7 51.0 41.37 55.9 40.5 56.3 8 42.6 44.78 51.6 30.1 46.0 9 42.4 44.9Average 53.2 F 3.6 41.8 F 5.2 50.7 F 3.4 10 41.9 45.9 11 43.0 43.9 T-test: The data obtained from the 96-Al FPA were compared in- 12 44.5 45.3dependently with data obtained from the 135-Al FPA and the 60-Al PFA. 13 43.3 46.4The t-value for 35-Al FPA is calculated as 4.75, which is above the critical 14 45.2 48.1t-value of 2.14 at P = 0.05 and DF = 14. However, the t-value for the 60-Al 15 48.1 52.2FPA is 1.35, which is below the critical t-value. Average 45.4 F 3.3 47.0 F 3.5 T-test: calculated t-value = 1.27, which is below the critical t-value of 2.05 at P = 0.05 and DF = 28.of the assay to accurately determine the amount of cellulaseactivity present. the variation in results. We used filter paper disks with an60-ML FPA Is Highly Reproducible average weight of 3.4 mg, while those used by Decker et al.In the 96-Al format FPA, we had to transfer a portion of the (2003) had an average weight of 2.65 mg. Since FPA isreaction mixture to a second 96-well plate for the color based on a nonlinear reaction and the activity estimates aredevelopment step. The 60-Al format assay can accommo- influenced by the size of the filter paper, the use of differentdate the 120 Al of DNS reagent required to perform the filter paper disks as substrate may lead to variation incolor development step in the same 96-well plate without results. Moreover, the use of a higher temperature andthe necessity of transferring the reaction mixtures to a fresh incubation time in color development, 98jC for 10 min bymicroplate. Table III showed the comparison of the com- Decker et al. (2003) versus 95jC for 5 min in ourponents of the filter paper assays performed in 96-well procedure, may have exaggerated the enzyme activity mea-plates and standard FPA. sured by their miniaturized assay. Recently, Decker et al. (2003) reported a miniature-scale To verify the utility of the 60-Al assays, we performedFPA. However, the detectable filter paper activities were assays in this format along with the standard FPA. Table IV56% higher than that obtained with standard FPA (Decker shows that the determined enzyme activity with the 60-Alet al.). We are not clear why their miniaturized FPA yielded FPA was comparable to that obtained with the standarddifferent results from ours and from the standard FPA. Two FPA. Another commercial cellulase preparation, C-2605discernable differences in the procedures may contribute to from Aspergillus species, was also assayed using the 60-Al Table III. Comparison of the components of the filter paper assays in different reaction volumes. Standard FPA 96-Al FPA 60-Al FPA 2 2 Filter paper substrate 600 mm (52 mg) 38.5 mm (3.4 mg) 38.5 mm2 (3.4 mg) Buffer volume 1.0 ml 64 Al 40 Al Enzyme (or 0.5 ml 32 Al 20 Al glucose standard volume) Enzyme reaction at Test tubes in 96-well microplates 96-well microplates 50jC for 60 min waterbath in temperature in temperature cycler cycler Volume of DNS 3.0 ml 50 Al reaction mixture + 120 Al 100 Al DNS Color development 100jC for 5 min 95jC for 5 min 95jC for 5 min Amount of H2O 20 ml 160 Al to 36 Al of 160 Al to 36 Al of added prior to the reaction/DNS the reaction/DNS measurement mixture mixture836 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 88, NO. 7, DECEMBER 30, 2004
  6. 6. Table V. Comparison of Aspergillus cellulase C-2605 activity using the format is a robust assay that can rapidly and accurately mea-standard and the 60-Al FPA. sure the cellulase content of different enzyme preparations.Sample Standard FPA (FPU/ml) 60-Al FPA (FPU/ml) 1 4.29 4.10 CONCLUSIONS 2 4.07 4.26 3 4.35 3.95 We have developed a 60-Al format cellulase assay using 4 4.11 3.18 filter paper disks as the substrate. This highly reproducible 5 3.86 4.11 and accurate assay can be used to rapidly and easily assay 6 3.89 3.79 a large number of samples. It also reduces the amount 7 3.99 3.54 of chemicals and enzyme used by 25-fold relative to the 8 4.00 3.62 9 4.21 4.39 standard IUPAC method. As in the standard method, sup-10 4.10 4.01 plementing the enzyme with h-glucosidase dramatically11 4.21 3.85 increases assay sensitivity. The microplate-based assay de-12 3.97 4.13 veloped here therefore provides a useful technology plat-13 3.95 4.66 form for the discovery and characterization of cellulases for14 3.97 4.2815 3.87 3.62 industrial application.Average 4.06 F 0.15 3.97 F 0.38 We thank Dr. Xiao Zhang for comments and suggestions. T-test: calculated t-value = 0.86, which is below the critical t-value of2.05 at P = 0.05 and DF = 28. References Bowman GR, Beauchemin KA, Shelford JA. 2002. The proportion of theFPA to verify its reproducibility and accuracy (Table V). diet to which fibrolytic enzymes are added affects nutrient digestionStatistical analysis of the assay results showed that the 60-Al by lactating dairy cows. J Dairy Sci 85:3420 – 3429.format assay accurately measured the cellulase content of Breuil C, Mayers P, Saddler J. 1986. Substrate conditions that influence the assay used for determining h-glucosidase activity of cellulolyticthe C-2605 cellulase sample. microorganisms. Biotechnol Bioeng 28:1653 – 1656. Chen K, Arnold FH. 1993. Tuning the activity of an enzyme for un- usual environments: sequential random mutagenesis of subtilisin ESupplement of h-Glucosidase Increases the Cellulase for catalysis in dimethylformamide. Proc Natl Acad Sci U S A 90:Assay Sensitivity 5618 – 5622.Breuil et al. (1986) reported that most natural cellulase Cherry JR, Fidantsef AL. 2003. Directed evolution of industrial enzymes: an update. Curr Opin Biotechnol 14:438 – 443.complexes tend to have a shortage of h-glucosidase Coward-Kelly G, Aiello-Mazzari C, Kim S, Granda C, Holtzapple M.activity. By adding an excess amount of cellobiase (h- 2003. Suggested improvements to the standard filter paper assay usedglucosidase) into the cellulase Spezyme CP produced by to measure cellulase activity. Biotechnol Bioeng 82:745 – 749.Genecor, Coward-Kelly et al. (2003) showed that the filter Decker SR, Adney WS, Jennings E, Vinzant TB, Himmel ME. 2003.paper assay response was increased by 56%. These results Automated filter paper assay for determination of cellulase activity. Appl Biochem Biotechnol 105 – 108:689 – 703.suggested that extra h-glucosidase facilitated the conver- Galbe M, Zacchi G. 2002. A review of the production of ethanol fromsion of cellobiose into glucose. softwood. Appl Microbiol Biotechnol 59:618 – 628. We measured the amount of h-glucosidase activity in the Garcia O, Torres AL, Colom JF, Pastor F, Diaz P, Vidal T. 2002. Effect oftwo commercial cellulase preparations using p-nitrophenyl- cellulase-assisted refining on the properties of dried and never-driedh-D-glucoside as substrate. The ratio of h-glucosidase units eucalyptus pulp. Cellulose 9:115 – 125. Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of(cellobiase unit or CBU) to filter paper unit (FPU) for the reducing sugar. Anal Chem 31:426 – 428.Trichoderma reesei cellulase (C-2730) was about 1.0. No Niehaus F, Bertoldo C, Kaehler M, Antranikian G. 1999. Extremophiles asdetectable h-glucosidase activity was observed in the As- a source of novel enzymes for industrial application. Appl Microbiolpergillus cellulase (C-2605). We used the 60-Al format as- Biotechnol 51:711 – 729.say to evaluate the effect of addition of h-glucosidase on Stemmer WP. 1994. Rapid evolution of a protein in vitro by DNA shuf- fling. Nature 370:389 – 391.the FPA. The addition of excess h-glucosidase to the FPA Sun Y, Cheng J. 2002. Hydrolysis of lignocellulosic materials for ethanolincreased the amount of cellulase measured by 55% and production. Bioresour Technol 83:1 – 11.60%, respectively, with the C-2730 and C-2605 cellulase Wood TM, Bhat KM. 1988. Methods for measuring cellulose activities.preparations. These results further indicate that the 60-Al Methods Enzymol 160:87 – 112. XIAO ET AL.: MICROPLATE-BASED CELLULASE ASSAY 837

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