Filter paper cellulase units

International filter paper cellulase activity unit. hiang, M. Linden, J.C. Mohagheghi, A. Grohmann, K. Himmel, M.E. AppL Microbiol TechnoL, submitted. 

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. 

In a typical enzymatic hydrolysis of a 5% suspension of ball-milled newsprint, a sugar syrup containing 1.6% glucose, 1.4% cellobiose, and 0.2% xylose is readily obtained. Figure 3 is illustrative of kinetic results obtained with T. viride cellulase at 3.5-filter-paper-units/mL (FP units/ mL) strength in a 5% suspension of ball-milled newsprint. 

Frequently we use filter paper as a substrate for the measurement of cellulase activity because it is well defined and yields reproducible results. The activity (IU) we obtain with filter paper is referred to as filter paper unit (FPU). The experimental procedure to measure the FPU of cellulase (Mandels et al., 1976) is as follows ... 

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%. 

The cellulase and P-glucosidase activities were 30 filter paper units/g of DM substrate ... 

Enzymes were supplied by Novozyme (US Office Franklinton, NC) and included cellulase (Celluclast 1.5 L 48 international filter paper units [IFPU]/mL), (3-glucosidase (Novozym 188 66.8 x 103 IU/mL), and glucoamylase (Novozyme AMG300L). Sugars were purchased from Sigma (St. Louis, MO), and all other chemical and media reagents were from Fisher (Fairview, NJ). 

The hemicellulose-degrading enzymes (Ultraflo L) and the cellulases (Celluclast 1.5L) were kindly donated by Novozymes A/S. Celluclast 1.5L had a filter paper activity of 80 filter paper units/mL, determined according to the procedure of Mandels (8-9). For the present study the most important side activities of Ultraflo L were endo-l,4-P-xylanase, P-xylo-sidase, and a-arabinofuranosidase (10). 

Commercial cellulase and P-glucosidase (Novo Nordisk, Bagvaerd, Denmark) supplied from Novozymes Korea were used. A mixture of Celluclast (80 IU or international filter paper units [IFPU]/mL) and Novozym 188 (792 cellobiase units [CBU]/mL) was used with a ratio of 4 IU of Celluclast/CBU of Novozym to alleviate end-product inhibition by cellobiose. 

The enzyme preparations were provided by Novo Nordisk. Cellulase (Celluclast) possessed 115 filter paper units (FPU)/mL P-glucosidase (Novozym 188) had 570 cellobiose units (CBU)/mL. 

For a cellulosic material such as wood chips or crop waste, cellulase enzymes cannot penetrate the structure and make glucose. A pretreatment is required to destroy the fiber structure and allow cellulase access to the substrate. Pretreatment is typically carried out at 180-250 °C for a few seconds to a few minutes in 0.5% to 2% sulfuric acid. The resulting material is of a muddy texture. An aqueous slurry of pretreated cellulose is made at 5% to 15% sohds. Cellulase enzymes are added at a concentration of 5 to 25 filter paper units per gram of cellulose. The slurry is stirred, and the enzymatic hydrolysis is carried out for 4 to 7 days. At this point, most of the cellulose is converted to glucose, and the unhydrolyzed residue consists primarily of lignin. 

SSF of RPS with fungal cellulase and a thermotolerant yeast, Kluyveromyces marxianus, was used to convert cellulose fibers of RPS samples to ethanol by Lark et al. [ 112]. The cellulase loading was 8 filter paper units (FPU)/g dry RPS. About 32 and 35 g/1 of ethanol were produced from 180 and 190 g/1 dry materials, respectively, after 72 h of incubation. This indicates that at least 72 % of the cellulose in the RPS was converted into ethanol. During incubation, the thick slurry of RPS was liquefied within 24 h resulting in the reduction of the waterholding capacity of RPS to 30%-35% of the original. 

In one experiment, for example (Figure 9), 10 liters of medium (1.0% Solka Floe, 0.05% proteose peptone) in the 15-liter culture vessel was inoculated with a 100 ml. shake flask culture. The culture was allowed to grow for six days to reach a high cellulase value. Two liters of the culture was then harvested, and two liters of fresh nutrient pumped in from the nutrient reservoir to keep the volume 10 liters. Harvesting and replacement with fresh medium continued on a regular basis, with fresh nutrient reservoirs added, and the harvest vessel replaced as required. Solka Floe remained at 1.0% but peptone level was varied from 0.01% to 0.2%. Tween 80 at 0.1% was included after day 12. The maximum filter paper activity was 1.86, and maximum Cx 86 units/ml. 

Fig. 1 Cellulase (Filter Paper Unit=FPU) (a) and Xylanase (b) and activities produced by H. jecorina after growth on 230°C PBHW-pretreated filled square) and untreated filled triangle) Tifton 85 bermudagras. Standard deviations are presented by error bars...

Two commercials enzymes, Celluclast 1.5-L and Novozyme 188 (Sigma, St Louis, MO, USA), were used for enzymatic hydrolysis. Celluclast 1.5-L contained 98.2 FPU/ml of total cellulase P-glucosidase activity of Novozyme-188 was 540 unit/ml. One FPU is defined as the enzyme amount that releases 1 p.mol of glucose equivalents from Whatman no. 1 filter paper in 1 min. One unit of 3-glucosidase activity is defined as the enzyme amount that converts 1 imol of cellubiose to 2 pmol of glucose in 1 min [17],... 

Readers wishing to convert from cellulase loading in milligram protein per gram solids to filter paper units per gram solids may note that the specific activity of T. reesei cellulase preparations is about 0.6 filter paper units mg protein. 

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