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Antibiotic D i s k s d n Improvement in the Filter Paper Assay for Celluhe *

INTRODUCTION A recent surge of interest in the large scale saccharification of cellulose to glucose by the use of microbial enzymes has been spurred by both the dwindling supply of fossil fuels and the potential utilization of waste cellulosic materials. The efficient degradation of native cellulose is dependent upon the synergistic action of at least four different enzymes; cellobiohydrolase, endo-p- 1,Cglucanase, p- 1,4-exoglucanase, and cellobia~e.'-~ order to assay the combined action of these enzymes, a In filter paper substrate has been recommended (50 mg strip of Whatman No. 1 filter paper).' However, the cutting and curling of these filter paper strips is tedious and time consuming. In addition, the susceptibility of this filter paper strip to degradation by the cellulases is great, resulting in a high degree of fiber production and necessitating laborious dilutions. The purpose of this paper is to describe a modification of this filter paper assay that eliminates many of these problems and allows direct interconversion of units obtained employing either method. MATERIALS AND METHODS The enzyme preparations used in this study were obtained from Trichoderma viride QM 94147and T. viride mutant NG-14.' The fungi were grown in shake flasks on modified Vogels containing 1% Whatman CC41 crystalline cellulose as a carbon source. At nine days the clarified culture filtrates were the source of enzymes and contained 0.9 (QM 9414) and 1.9 (NG-14) mg/ml protein as measured by the Folin method" on the 5% trichloroacetate acid precipitable material. The filter paper assay was carried out by the method of Mandels' using 1 x 6 cm (50 mg) Whatman No. 1 filter paper strips as a substrate. The disk assay utilized 4 in. filter paper antibiotic assay disks 740E (Schleicher and Schuell, Keene, N.H.), in place of the 50 mg strips. The enzymes were diluted in 0.05M citrate buffer, pH 4.8, with a final volume in the assay of 1.5 ml. All assays were carried out in 18 mm test tubes at 50C (stationary water bath) for 1 hr. Free reducing sugar was quantitated by the dinitrosalicylic (DNS) acid method" expressed as glucose equivalents. Filter paper units or disk units are calculated as defined by Mandels,' i.e., a filter paper 2 unit is expressed as $ glucose/min based on the release of 2 mg reducing sugarihr under the defined assay conditions. This eliminates artifically high assay results merely owing to the initial rapid degradation of small amounts of amorphous cellulose in the paper. Two mg glucose/hr is equivalent to 0.185 IUB units (pmoV min). RESULTS AND DISCUSSION A comparison of the free reducing sugar released from either filter paper or disk as a function of enzyme concentration for QM 9414 and mutant NG-14 is shown in Figure 1. The filter paper units for the two preparations are 2.76 units/ml (NG-14)

* Journal paper of the New Jersey Agricultural Experiment Station, Rutgers, The State University of New Jersey, New Brunswick, N.J. 08903
Biotechnology and Bioengineering, Vol. XX, F'p. 297-300 (1978) 0006 3592l78/OO2O.0297$01.OO

@ 1978 John Wiley & Sons, Inc.





06 .
ENZYME (rnl)


Fig. 1. Comparison of reducing sugar released from 1 x 6 cm filter paper strips and 4 in. antibiotic assay disks by NG-14 and QM 9414 enzyme preparations. and 0.37 units/ml (QM W14), respectively. The assay disks are more resistant to cellulase degradatian by both culture filtrates, but the relative rate of release of the reducing sugar shows similar patterns for the two substrates. This suggests that the filter paper strips and the antibiotic disks are comparatively compatible substrates in terms of attack by the cellulase complex enzymes. The semilog plot' of the data in Figure 1 is shown in Figure 2 for the NG-14 enzyme preparation. The dilution of enzyme necessary to release 2 mg glucose from the filter paper strips is 0.067 ml. This is equivalent to 2.76 filter paper (FP) units/ ml. The corresponding dilution using the disks as a substrate is 0.185 ml and the resulting units are I.O/ml. A linear correlation exists within the 2 mg glucose equivalent range between the units of filter paper activity and the units of disk activity. A conversion factor of 2.8 can be calculated and employed to interconvert the units for comparative purposes. Because the disks are more resistant to cellulase attack, lower, more easily handled, dilutions may be assayed.





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Fig. 2. Semilog plot of free reducing sugar released from 1 X 6 cm filter paper strips and 4 in. antibiotic assay disks as a function of NG-14 enzyme concentration. The disks are made of especially purified cellulose (95% a-cellulose) and thus have a higher degree of crystallinity than routine filter paper. This, and the inherent nature of the disks leads to several advantages of the disk substrate over the filter paper strip, which are summarized in Appendix I. They are elaborated on below: 1) There is no tedious cutting involved. The disks are commercially available at a low cost. The average weight of the disks is the 46-48 mg, c.f. approximately 50 mg for the filter paper strip. 2) There is no curling involved. The filter paper assay requires that the 1 x 6 cm strips be uniformly curled in the bottom of the tube to allow homogeneous access of the substrate to the enzyme. This is the most technically fastidious procedure of the assay, as more often than not, the paper strip will remain stuck on the side of the test tube above the reactants. It takes considerable mechanical agitation to dislodge such stuck strips. In contrast, the 4 in. antibiotic disks readily and perfectly fit in the bottom of an 18 mm tube. 3) The disks give a lower background in the free reducing sugar assay. 4) As the disk cellulose is more crystalline, the disks are more resistant to enzymatic digestion, and fiber production is reduced. This eliminates clogging of the flow cell and other turbidometric problems in the spectrophotometric quantitation of the reducing sugar. 5) The increased resistance eliminates tedious dilutions of the enzymes. As hyperenzyme producing strains of cellulolytic microorganisms will most probably be employed in commercial cellulase fermentations, more resistant assay substrates will be important. 6) Since a linear correlation exists between the filter paper units and the disk units, a simple conversion factor of 2.8 can be employed to compare the two activities. The substitution of the filter paper antibiotic disks for the 1 x 6 cm filter paper strips considerably simplifies the assay for the cellulase complex enzymes and



should prove useful for monitoring cellulase fermentations and screening for hyperproducing cellulolytic microorganisms.
APPENDIX I Advantages o Disks over Filter Paper Strips : f
1) No cutting involved; average weight 46-48 mg; 95% a-cellulose. 2) No curling in the bottom of tube. 3) Lower background in DNS assay for reducing sugar. 4) Reduced fiber production : eliminates flow cell clogs. . 5 ) Substrate more resistant : eliminates tedious dilutions. . 6) Conversion factor between FP units and disk units.

This research was supported by funds from the New Jersey Agricultural Expenment Station and by the Energy Research and Development Administration grant No. ERDA 4-4P18-2539.
1. E. K. Gum and D. R. Brown, Biochim. Biophys. Acta, 446, 371 (1976). 2. L. E. R. Berghem, L. G. Pettersson, and U. Axio-Fredriksson, Eur. J. Biochem., 61, 621 (1976). 3. T. M. Wood and S . I. McCrae, Biochem. J., 128, 1183 (1972). 4. K.-E. Ericksson and B. Pettersson, Eur. J. Biochem., 51, 193 (1979. 5. K.-E. Ericksson and B. Pettersson, Eur. J. Biochem., 51, 213 (1975). 6. M. Mandels, R. Andreotti, and C. Roche, in Enzymatic Conversion of

Cellulosic Materials: Technology and Applications, E. L. Gaden, M. H. Mandels, E. T. Reese, L. A. Spano, Eds., Wiley, New York, 1976, p. 21. 7. M. Mandels, in Cellulase as a Chemical and Energy Resource, C. R. Wilke, Ed., Wiley, New York, 1975, p. 81. 8. B. S. Montenecourt and D. E. Eveleigh,Appl. Environ. Microbiol., In Press. 9. H. J. Vogel, Microbiol. Gen. Bull., 13, 42 (1956). 10. B. S. Montenecourt and D. E. Eveleigh, Appl. Environ. Microbiol., 33, 178
(1977). 1 1 . 0. H. Lowry, N. J. Rosebrough, A. L. Fan, and R. J. Randall, J . Biol. Chem., 193, 165 (1951). 12. G. L. Miller, Anal. Chem., 31, 426 (1%9).

BLAND MONTENECOURT S. DOUGLAS EVELEIGH E. Department of Biochemistry and Microbiology Cook College, Rutgers The State University of New Jersey New Brunswick, New Jersey 08903 G . KEITH ELMUND JOHN PARCELLS Department of Microbiology Colorado State University Fort Collins. Colorado 80522 Accepted for Publication July I 1, 1977