1. Abstract X-ray crystallography has confirmed that Non-Crystalline Cellulose( NCC) has highly amorphous character with less than 10% crystallinity. Because of the amorphous nature and open structure of NCC, large amount of soluble cello-oligosaccharides are formed along with cellobiose and glucose as hydrolysis products. Highly amorphous nature of NCC as substrate justify the basic assumption of pseudo first order reaction in activity determination of Endo-glucanase( Endo-G) where product( shorter chain cello-dextrin) release should only be a function of enzyme concentration. Here both HPLC and colorimetric methods have been used to quantify the sugars and reducing end group( In Cello-oligosaccharides and insoluble Cello-dextrin) respectively. Presently assay for Endo-G and Exo-G activity are done with two different substrate i.e. CMC and avicel respectively. The procedure described here is handy and less time consuming because single substrate is used and only one experiment is required for determination of relative activities of Endo-G and Exo-G( Exo-glucanase) for different Cellulases. Initial rate data for release of G1+G2 will give the indication of Exo-G activity and change in number of reducing end group will give the indication of Endo-G activity. Introduction It is known that Endo-glucanases are responsible to decrease the DP of Cellulose chain by acting on relatively amorphous region and Exo-glucanase works on chain ends. Non-Crystalline Cellulose has been prepared in our lab by acid treatment on Crystalline cotton. Structural changes expected in NCC with respect to Cotton are shown in fig.1. It is evident that NCC contains ample amount of attacking points for Endo-glucanase and Exo-glucanase while in cotton these domains are in much lesser quantity due to very high crystallinity and high DP. Due to peculiar structural characteristics, reaction of NCC with both the components of cellulase is expected to follow the first order kinetics with respect to enzyme during the initial phase when very low enzyme loadings are used as shown here: Enzyme Substrate Reaction: E + S P k dP =k[E][S] dt For pseudo first order Reaction with respect to enzyme when [S] >> [E] : E P k’ = k[S] dP = K’[E] dt Amorphous domain (Substrate for Endo-glucanase) Reducing Ends (Susbtrate for Exo-glucanase) Schematic Representation of Structural Differences between Crystalline Cotton & NCC Cotton NCC (Fig. 1) Materials and Methods Cellulase preparations namely Spezyme CP( From two different batches A: Lot No. 301- 00348-25 & B: Lot No.301-04075-054 ) and GC 220(Lot No.301-04232-162) are provided by Genencore. NCC has been prepared from cotton in our lab. As moisture content in NCC is approximately 85% and it is very sticky, it is not easy to disperse it by mixing only. Therefore ultrasonicator( Cole-Parmer, 750 Watt) has been used to make the 2% uniform solution of NCC in 0.05M citrate buffer. After sonication, continuous stirring is done till solution is taken for incubation. Capped glass tubes have been used to incubate the NCC solution and enzyme. Constant temperature shaking bath has been used for incubation. Vigorously boiling water bath is used for termination of reaction as well as after addition of DNS. DNS reagent has been prepared by the method as mentioned in NREL LAP-6( Biomass Program). BIO-TEK Synergy HT Spectrophotometer has been used to measure the absorbance. Procedure of Assay for simultaneous determination of Exo-glucanase and Endo-glucanase Activity 0.5ml 2% sonicated NCC Solution with Buffer Or 0.5ml Standard with different ratio of G1& G2 0.5ml of diluted Enzyme Solution Or 0.5ml citrate buffer solution Stop the reaction by boiling it for 5min. & Dilute it 3 times by cold DI water Stir the content and take 0.5ml sample containing uniform mixture liquid and solid Take liquid sample for finding the sugar concentration (From HPLC) Find the Glucose and Cellobiose concentration Add 3ml DNS reagent & boil for 5min., cool the content immediately, centrifuge it & measure the absorbance of liquid at 540nm Incubate at 50 o C for 30min. G1 (mg/0.5ml) G2 (mg/0.5ml) Absorbance(540nm) ExperimentalCalculated 000.081 0.500.1430.141 0.30.20.1310.130 0.20.30.1210.125 00.50.1180.115 Absorbance = 0.0805 + 0.1202*G1 + 0.0693*G2 Calibration Table Results Relative Activity ( Volume Basis)Exo-GlucanaseEndo-Glucanase Spezyme CP A1.000 Spezyme CP B1.1731.179 GC 2201.6861.553 Relative Activity (Protein Basis)Exo-GlucanaseEndo-Glucanase Spezyme CP A1.000 Spezyme CP B1.0111.016 GC 2200.9710.894 Discussion Basic assumption in this method is that both Endo-G and Exo-G are supersaturated with its respective substrates and only enzymes are limiting reagent in both set of enzyme-substrate reactions. Very low enzyme loadings should be used so that the extent of reaction is very low and the plot between the product release Vs. enzyme loading should be a straight line. This would eliminate any possibility of product inhibition. The largest source of error of this method is in the DNS reducing sugar measurement. The DNS method has an inherent problem with regard to the end point of reaction. Since rapid heating and cooling (Bang-Bang control) is difficult to realize, it induces large error in the final result of DNS sugar. This error is carried into cello-dextrin and eventually to Endo-G activity. Conclusion Relative activities of Exo-G and Endo-G for three cellulases obtained from this method are in good agreement with those measured by Avicel and CMC methods. Measurement of COS by HPLC requires careful procedures. There is a gross underestimation in direct analysis of COS. Accurate measurement is feasible only after secondary hydrolysis. References Ghose TK, Montenecourt B S, Eveleigh DE. Measurement of Cellulase activities. Pure Appl. Chemistry 1987, 59:257-268 Guillermo Coward-Kelly, Cateryna Aiello-Mazzari, Sehoon Kim, Cesar Granda, Mark Holtzapple. Suggested improvement to the standard Filter Paper Assay used to Measure Cellulase Activity. Biotechnology and Bioengineering, Vol.82,No.6,June 2003:745-749 Lee R. Lynd, Paul J. Weimer, Willem H. van Zyl, Isak S. Pretorius. Microbial Cellulose Utilization: Fundamentals and Biotechnology, Microbiology and Molecular Biology Reviews, Spt. 2002: 506-577 Cotton(77% Crystalline) α-Cellulose(65% Crystalline) NCC(8% Crystalline) Acknowledgement US Department of Energy for funding the project (US/DOE No. DE-PS36-00GO10482) Members of CAFI-II team