Celluclast

Although most cellulase enzyme complexes, including Celluclast, contain xylanase activity (data not shown), the amount of released xylose after enzymatic hydrolysis was negligible, especially compared with the amount of released xylose after pretreatment. 

Three enzymatic mixtures with predominantly cellulase and xylanase activity were used for the saccharification tests. To hydrolyse the cellulose, the mixtures Celluclast 1.5L and Novozym 188 from Novo Nordisk were... 

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). 

Steam treatment of an industrial process stream, denoted starch-free wheat fiber, was investigated to improve the formation of monomeric sugars in subsequent enzymatic hydrolysis for further bioconversion into ethanol. The solid fraction in the process stream, derived from a combined starch and ethanol factory, was rich in arabinose (21.1%), xylose (30.1%), and glucose (18.6%), in the form of polysaccharides. Various conditions of steam pretreatment (170-220°C for 5-30 min) were evaluated, and their effect was assessed by enzymatic hydrolysis with 2 g of Celluclast + Ultraflo mixture/ 100 g of starch-free fiber (SFF) slurry at 5% dry matter (DM). The highest overall sugar yield for the combined steam pretreatment and enzymatic hydrolysis, 52 g/100 g of DM of SFF, corresponding to 74% of the theoretical, was achieved with pretreatment at 190°C for 10 min followed by enzymatic hydrolysis. 

Determined by acid hydrolysis with 1% F12S04 at 130°C for 40 min followed by enzymatic hydrolysis with 2 g of Celluclast + Ultraflo/ 100 g of slurry (5% DM). 

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). 

The DM content of the steam-pretreated SFF was adjusted to 5% and the pH set to 5.0 with NaOH. Enzymatic hydrolysis was performed using a Celluclast + Ultraflo mixture (1 1) at a ratio of 2 g of enzyme/100 g of slurry (10). Hydrolysis was carried out for 72 h in 100-mL shake flasks maintained at 50°C and shaken at 200 rpm in a laboratory rotary shaker-incubator (LSR/L-V Adolf Kiihner AG) for 72 h. Samples were withdrawn after 0,2, 4.5, 7.5,11,14, 24,31, 38,48, 60, and 72 h for analysis of monosaccharides. Direct enzymatic hydrolysis of a 5% DM SFF slurry was also performed as a reference to evaluate the effect of steam pretreatment on the yield. 

When 0.5 g of Celluclast + Ultraflo/100 g of SFF was used, a total yield of 38.4 g of sugars/100 g of SFF, corresponding to 55% of the theoretical, was obtained. When only Celluclast was used the total sugar yield was reduced to 33.5 g/100 g of SFF. The materials pretreated at 220°C for 5 min and at 170°C for 15 min showed curves similar to those presented in Fig. 5 although the yields were slightly lower than those obtained with the material pretreated at 190°C for 10 min. 

Direct enzymatic hydrolysis with the supplementation of Ultraflo to the Celluclast resulted in a significant increase in yield compared with when Celluclast alone was used (Fig. 6). The maximum sugar yield for direct enzymatic hydrolysis, 36.6 g/100 g, corresponding to 52% of the... 

SP, steam pretreatment EH, enzymatic hydrolysis C, celluclast U, ultraflo. 

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. 

Table 1 shows the main activities present in the various reaction mixtures using Celluclast 1.5L, Novozym 342, Viscozyme L, Pentopan 500BG, Pulpzyme HC, Multifect Xylanase, and Multifect GC. Although most endoxylanases present in the commercial preparations exhibit optimal activity near 50°C, hydrolysis assays were carried out at a lower temperature (35°C) to increase enzyme stability and to reduce the possibility of inactivation of other enzymatic activities having unknown stability profiles. 

Enzymatic posthydrolysis enabled monosaccharide recoveries in the range usually attained for other feedstocks to be obtain (17,18). The higher recoveries were obtained with Celluclast 1.5L and Viscozyme L, with arabinose recoveries close to 75%, xylose recoveries of 63%, and close to total glucose recovery. Furthermore, no additional production of microbial inhibitors occurs during the enzymatic step, with the exception of acetic acid. However, the long incubation time and catalyst costs may constitute important constraints for the near-term implementation of an enzyme-based conversion process to the conversion of OCL to monosaccharides. 

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. 

All hydrolysis experiments were conducted with a combination of cellulase (Celluclast) and P-glucosidase (Novozym 188) at a ratio of 1 2... 

Celluclast. [Novo Nordisk] Cellulase enzyme for breakdown of cellulosic material for prod, of fermentable sugar and for municipal cellulosic waste treatment. 

The pretreatment conditions for the dilute H2SO4 acid-pretreated com stover used in this study have been reported elsewhere [19]. Acid-pretreated com stover (12.5% total solids) was neutralized with NH4OH and hydrolyzed with Celluclast 1.5 L (15 filter paper units/g total solids [TS]) and Novozyme 188 (16.8 cellobiase units/g TS) at 50 °C and pH 5.0 for 48 h. The hydrolyzate was neutralized to pH 6.0 and filter sterilized. 

Acknowledgements We thank Novozymes A/S for providing enzymes Celluclast and Novozyme 188. We further thank Thomas Andersen and Gitte Hinz-Berg from BioCentrum for excellent technical help. 

The hydrolysis tests were performed at 50 °C in a rotary shaker at 200 rpm, and samples were taken regularly for sugar analysis. In all hydrolysis experiments including the SSF, enzymes consisted of a mixture of Celluclast 1.5 1 and Novozyml88 (both provided by Novozymes A/S, Bagsvaerd Denmark) in a volume ratio of 3 1. 

Acknowledgment We thank Novozymes A/S, Bagsvaerd, Denmark, for supplying the enzymes Celluclast and Novozyme 188, and Thomas Andersen from BioCentram for the technical help. 

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],... 

One sample (Lignin III redigest) was digested a second time with a commercial enzyme preparation, Celluclast (Novo Industry, Denmark), at 50 C for 2 days. The isolated lignin suspension was stored at 4°C until... 

Brown rot wood was prepared from birch wood which had been decayed by Polyporus Betulinum and digested with Celluclast without milling pretreatment. 

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