Enzyme Assay Filter Paper Activity

Filter paper activity, which describes the overall cellulolytic activity of an enzyme preparation, was determined by the method of Mandels et al. (16). A 1 x 6 cm strip of Whatman no.l filter paper (Hillsboro, OR), which equals 50 mg of cellulose, served as the substrate and was added to the sample solution containing 0.5 mL of appropriate diluted enzyme (supernatant of culture broth) and 1.0 mL of 0.05 M citrate buffer (pH 4.8). After 60 min of incubation at 50°C, the hydrolysis was terminated by the addition of 3 mL of DNS solution, and the mixture was further assayed for reducing sugar content by the DNS method. One international filter paper unit (FPU) was defined as the amount of enzyme that releases 1 pmol of glucose/min under the assay conditions. Activities were reported as FPU/milliliter. 

Cellulase activity of the samples was determined as filter paper activity (FPA) expressed in filter paper units (FPU) using Mandels procedure (15), and (3-glucosidase activity was assayed using 4-nitrophenyl-(3-D-glucopyranoside substrate according to Berghem and Petterson s (16) method. All samples were analyzed in triplicate and the mean values were calculated. The relative standard deviation of enzyme activity measurements was always below 5%. 

Multifect GC from Genencor, Rochester, NY. The enzyme solutions were characterized by the following activities endo-l,4-(3-xylanase, (3-xylosidase, acetylesterase, a-L-arabinofuranosidase, and filter paper activity (FPase, which describes the overall cellulolytic activity). The dilutions were calculated to provide 100 U of xylanase/g of total xylose in the reaction medium. Hydrolysis reactions were stopped by boiling for 5 min to inactivate enzymes and clarified by centrifugation prior to analysis. A blank OCL sample was assayed identically to enzyme-treated OCL using water instead of the enzyme solution. Control experiments were carried out for each enzyme, in which buffer replaced the OCL. All the hydrolysis trials were performed in duplicate. 

The filter paper activity of the enzyme samples was determined at 50 °C according to standardized NREL filter paper assay [11]. Carboxymethylcellulase (CMCase endoglucanase) and Avicelase (exoglucanase) activity was determined by measuring the release of reducing... 

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. 

A great advantage of spectrophotometric assays is that they can be carried out in microtiter plates or as filter paper assays, thus allowing a high sample throughput coupled with small sample volumes. Such systems were used for example for the screening of epoxide hydrolases [34]. A classical example of activity tests is for amylolytic enzymes where starch agar plates are stained with iodine after a certain reaction time. The radius of clear spots is a measure of the reaction rate [35]. 

The purified proteins were assayed for enzyme activity on filter paper, swollen cellulose, BMCC, and CMC following the procedures stated by Irwin et al (5). 

Methods for the assay of hexosaminidase A and total hexosaminidase activities in dried blood spots on filter paper offer considerable advantages for screening (Chamoles et al., 2002). The deficient activity of the lysosomal enzymes hexosaminidase A and total hexosaminidase (hexosaminidase A plus B) are usually measured in plasma or extracts of leukocytes. To tubes containing a 3-mm-diameter blood spot, elution liquid and substrate solution were added. After incubation at 37°C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. The method was proven reliable even after storage for up to 38 months at room temperature. For total hexosaminidase, the substrate was 4-methyl-umbelliferyl-2-acetamido-... 

Release of Cellulolytic Enzymes. Culture medium was filtered from 4-day-old cultures of D. zeae. The mycelial mats were rinsed with distilled water, then stripped from the filter paper and fragmented in a minimum of distilled water in a semi-micro Waring Blendor cup. Portions of the mycelial suspension were pipeted into solutions of the compounds being tested, and the flasks were shaken in the dark for 20 minutes. The suspensions were then filtered, and the filtrates were assayed for enzyme activity. 

Cellulase is a complex of enzymes showing various types of activities. Cellulose substrates include highly resistant crystalline forms such as cotton, various types of microcrystalline cellulose such as Avicel and hydrocellulose, sulfite pulps such as Solka Floe, as well as filter paper and cotton fabrics. More susceptible substrates include swollen or reprecipitated cellulose, cellophane, and ball-milled cellulose. Most susceptible are the soluble derivatives (of low D.S.) such as carboxymethylcellulose and cellulose sulfate. It is not surprising that there are many assay methods to detect or measure cellulase (9). These methods differ markedly in sensitivity, and in cellulase components detected, depending on the substrate used, the effect measured, and the duration and conditions of... 

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