Cellulase assay

Unclassified strains gifts from Dr. J. H. Walsh, Department of Biochemistry, University of Manchester, Institute of Technology. Cellulase assays (21) were carried out for 7 days at 37°C with 1 mL of cell-free culture filtrate. Ci added amounted to 180-200 fig protein. 

The IUPAC cellulase assay has significant limitations and merely serves as the best-... 

Sharrock, K. R., Cellulase assay methods A review. J. Biochem. Biophys. Methods 1988, 17, 81-106. 

Table I. Types of Cellulose Used as Substrates for Various Cellulase Assays...

Assays for endo-l,4- -glucanase [EC 3.2.1.4] (i.e., CMCase) and saccharifying cellulase (i.e., international filter paper U, IFPU) activities partially followed the methods recommended in the 1987 lUPAC report (65). When even undiluted enzyme samples fail to give the required glucose yield under prescribed assay conditions, the lUPAC committee recommends a less precise method. In the current study, cellulase activities in digester extracts were so low that the CMCU could only be defined as follows one CMC unit of activity was that amount of enzyme required to liberate one Hg glucose from CMC in 60 min. 

Besides his fundamental research in the carbohydrate field, the functions of Courtois as the head of a hospital laboratory for many years led him to publish a number of papers dealing with clinical chemistry, among which may be cited determination of ethyl alcohol, proteins, acidic phosphatases, and trehalase in blood determination of the basic groups of proteins by phytic acid study of the phytosoluble glycoproteins in biological fluids and identification and determination of scyllitol in urine. Under the aegis of the International Pharmaceutical Federation, he participated in the standardization of the methods proposed for the assay of such enzymes as cellulases and hemicellulases. 

Assay of Endocellulase Activity. Cellulase is an enzyme complex a synergistic action between the components is required for a complete hydrolysis of the insoluble cellulose. There is no consensus about the substrate to be used for the cellulase activity measurements. 

All assays contained the same number of Cx (CM-cellulase) units of activity 26) and 200 /xg of Ci protein. Neither C nor Cx from either organism showed a significant capacity for solubilizing cotton fiber when acting in isolation. 

All assays (21) contained C 180 / g protein, 1000 units of Cx (CM-cellulose) activity (26), and 100 units of /3-glucosidase where necessary, /3-glucosidase isolated from the cellulase of F. solani (21) was added. The a-celluloses were the residues left after delignification and extraction with 18% (w/v) NaOH of oat straw, birch wood, and ryegrass. 

The categories of substrates which are used for assays of cellulase enzymes are shown in Table I. The use of crystalline, insoluble forms of cellulose as substrates makes assays difficult and has led to such trivial names as Avicelase activity. These assays are useful as indications of the capacity of an enzyme system to degrade native cellulose and indicate the presence of CBH enzyme which cannot be assayed in the presence of endoglucanases or / -glucosidase. The susceptibility to enzymatic attack generally increases with the hydration of the polymer chains that accom-... 

Overall crude cellulase activity was measured using Remazol brilliant blue acid-swollen cellulose (cellulose-azure, Calbiochem.) (22). The assay consists of combining 40 mg cellulose azure, 1.0 mL of lOOmM sodium citrate buffer (pH 4.8), and 1.0 mL of enzyme solution and... 

Table II. Summary of Various Cellulase Activity Assays Incu- bation Volume Time Temp Product ...

Figure 6. pH-activity profile of cellulolytic enzyme activities from Thermoactinomyces sp. under assay conditions. (Q) CM-cellulase, incubation time 10 min (A) Avicelase, incubation time 20 min (O) /3-glu-cosidase, incubation time 30 min. 

Cell walls, total membrane-bound components, and ribosomes were separated and assayed for cellulase activity to study the subcellular localization of the enzymes as follows. Segments (approx. 5 g fresh wt) were ground in two volumes of extraction medium containing 0.4M sucrose (ribonuclease-free), 5mM Mg acetate, lOmM Tris-HCl (pH 7.5 at 22°C), 20mM KC1 and 5mM / -mercaptoethanol. The brei was filtered and the filtrate centrifuged at 500 Xg for 20 min. The post-500 Xg supernatant was fractionated essentially as previously described (28). Aliquots (7 mL) of the supernatant were layered on a discontinuous gradient composed of 2 mL 70% (w/v) sucrose and 3 mL 15% (w/v) sucrose both in lOmM Tris-HCl (pH 7.5 at 22°C), lOmM KC1, 2.5mM Mg acetate and ImM / -mercaptoethanol. The tubes were centrifuged at... 

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