Enzyme cellulose

Fig. 1. Glucose concentration over time for semibatch saccharification experiments at 15% (w/w) initial insoluble solids concentration and 20 FPU/g of cellulose enzyme loading. Filtration was applied at two different times to remove glucose (vertical dashed lines). VF Only vacuum filtration was applied alone VF+UF ultrafiltration was also applied following vacuum filtration VF, Washed Vacuum filtration was applied and the filter cake was washed with water. Error bars represent averages 1 SD for two repeated experiments.

Fig. 3. Ethanol yield from pilot-scale fermentation of pretreated DG. Starch and cellulose in the pretreated DG slurry (10% [w/w]) were saccharified using glucoamylase and cellulose enzymes for 24 h prior to fermentation to ethanol using S. cerevisiae D5A yeast at 32°C, pH 5.0, and 75-rpm agitation.

The >3(1 — 4) linkage is particularly stable with respect to hydrolysis. Cellulose cannot be digested by mammals, but some insects (notably termites and wood-eating cockroaches), protozoans and fungi possess celluloses, enzymes that can hydrolyze the /3(1—>4) linkages. Ruminants, such as sheep and cattle, can digest cellulose because of the protozoans that live symbiotically in their digestive system. 

Enzymatic Hydrolysis. Saccharification of wood polysaccharides to sugars can be accomplished by enzymatic techniques instead of acid hydrolysis. The U.S. Army Natick Laboratories developed a method for conversion of cellulose to glucose with a cellulose enzyme from an active strain of the fungus Trichoderma viride. However, extensive pretreatment of wood is necessary before sufficient enzymatic hydrolysis will take place. 

It should be possible to improve the efficiency of the cellulose enzyme complex for hydrolyzing cellulose to glucose. The enzyme complex apparently contains decrystallizing and hydrolysis enzymes that work together to convert cellulose to glucose. Isolation of the specific enzymes and genetic engineering could provide a more efficient complex. 

For the enzymatic process, the feedstock is first pretreated with a dilute acid to break down the lignocellu-lose into lignin, hemicellulose, and cellulose. Enzymes called cellulases and xylanases are then used to break down the cellulose and hemicellulose fractions into six- and five- carbon sugars, respectively. Without the dilute acid treatment, the enzymes would not be able to come in efficient contact with the cellulose and hemicellulose. The sugars are then fermented to ethanol using organisms such as E. S. cerevisiaeP ... 

Cellulose Derivative Matrix. Four possible methods for preparing cellulosic, enzyme membranes are presented ... 

Cotton linters were swollen to an increasing extent by means of phosphoric acid of increasing concentration, Using a number of simple sugars plus a series of dextran molecules ranging in M.W. from 180 to 2,4 X 107 and diameter from 8 to 1600 A, it was possible to measure the pore volume and calculate the surface area within the swollen fibres accessible to all the molecules within this range. The substrates reacted with a commercial cellulose enzyme preparation, and the initial rate of reaction was compared with the accessibility of the substrate to molecules of various sizes. There was found to be a linear relationship between the initial reaction rate and the surface area within the cellulose gel which was accessible to a molecule of 40 A, diameter. 

Commercially produced Spezyme CP and Novozyme 188 were used for enzymatic hydrolysis. The cellulosic enzyme Spezyme CP, secreted by Trichoderma longibrachiatum. 

Fig. 6 Effect of Tween 20 on enzymatic hydrolysis of Avicel with 8% dry sohd loading at 15 FPU + 15 CBU/g cellulose enzyme loading...

Cellobiose is a disaccharide of two glucose molecules linked in a 3-1,4 bond produced from enzymatic hydrolysis of cellulose. Enzymes capable of breaking down cellobiose are... 

Because of the lack of homogeneous, purified, cellulose-degrading enzymes, having a variety of action patterns, that will act on the native, unmodified polysaccharide, it has not yet been possible to investigate the fine structure of cellulose in a manner analogous to that used for amylose. The situation is complicated by the insolubility of the substrate and its resistance to degradation by any single enzyme. A system of enzymes, the function of at least one component of which is at present unclear, is required. For this reason, it has not yet been possible to examine the fine structure of cellulose enzymically. 

Fig. 7. Lineweaver-Burk plots for no inhibition, glucose and cellobiose inhibition in <25 p cellulose, enzyme activity —2.0FP, temp. 50 C...

Le, K.D. et al., A streptavidin-cellulose-binding domain fusion protein that binds biotinylated proteins to cellulose. Enzyme Microb. Technol., 16,496,1994. 

An important process modification made for the enzymatic hydrolysis of biomass was the introduction of simultaneous saccharification and fermentation (SSF). In this process, cellulose, enzymes and fermenting microbes are combined, with the intention of reducing equipment and improving efficiency. 

Enzymes responsible for the hydrolysis of cellulose fractions of lignocellulosics to glucose perform sub-optimally mainly due to product and substrate inhibition . Therefore, keeping cellulose available to cellulases and product concentration to minimal levels can help maintain the stability and activity of cellulosic enzymes. 

Advantages of Genencor s biocatalytic conversion of biomass to value-added chemicals include, a) commercial viability, simplicity and economic feasibility b) prevention of product inhibition of cellulosic enzymes by concurrent conversion to bioproducts c) feasibility of quantitative conversion, d) elimination of byproducts e) higher productivity and yield on carbon J) production capacity enhancement. This biocatal) ic conversion concept is novel because a multienzyme process for converting renewable biomass to value-added commercial ingredients has not yet been commercially demonstrated. 

Key advantages of this simple and economical in-vitro biocatalytic process for biomass conversion to gluconate are a) prevention of product inhibition of cellulosic enzymes by concurrent conversion to gluconate, b) feasibility of quantitative conversion c) elimination of byproduct formation, d) higher productivity, e) increased production capacity, and f) higher yield on carbon. 

JP Weil, K. Westgate, K. Kohlman, and MR Ladisch, Cellulose Enzyme and Microbial Technology 1994, 16, 1002-1112. 

Enzymatic hydrolysis uses cellulose enzymes to perform hydrolysis of cellulose or hemicellulose under relative mild conditions (pH 4.8, 40-50 °C). The enzymatic methods avoid the use of corrosive acids. However, the hydrolysis reactions catalysed by enzymes are significantly slower than chemical hydrolysis, typically requiring days rather than minutes. 

Pseudomonas - mo-n9s [NL, fr. pseud- -h monad-, monos monad] (1903) n. A generic class of aerobic, mesophilic bacterium capable of releasing a variety of enzymes, including ceUulose-decomposing cellulose enzymes these enzymes are a factor in viscosity reduction of latex paints modified with ceUulosic thickener, and may... 

Keshk, S.M.A.S., Sameshima, K., 2006a. Influence of lignosulfonate on crystal structure of bacterial cellulose. Enzyme and Microbial Technology 40, 4—8. 

Materials Sargassum fusiforme (sampled from Shengsi island), cellulose (enzyme activity > 15 000 U/g, sinopharm Chemical Reagent Co., Ltd), trypsin (enzyme activity > 25 U/g, Sinopharm Chemical Reagent Co., Ltd). The other reagents are all analytically pure. 

Wilson, C.A. and Wood, T.M. (1992), Studies on the ceUulase of the rumen anaerobic fungus Neocallimastixfrontalis, with special references to the capacity of the enzyme to degrade crystalline cellulose , Enzyme Microbiol. Technol. 14 (4), 258-264. 

Fig. 10. Exchange reactions. A. Between AcCoA and H-CoA. The reaction mixture contained 0.01 ml of H-CoA solution (0.027 /imole), 0.07 ml of 4 X 10 M AcCoA (2.8 nioles) prepared in water, 0.10 ml of DEAE-cellulose enzyme fraction (30 lig protein) from a rapid inactivator human and 0.8M potassium borate buffer, pH 8.0 in a total volume of 0.27 ml at 27 °. Aliquots of 0.02 ml were removed and immediately mixed with 0.05 ml of 3 X 10" M DTNB dissolved in 0.05 M sodium acetate buffer, pH 5.0 (0.15 Mmoles) at 4° to stop the reactioa Aliquots of 0.01 ml were then removed from this mixture, and the Ac- H-CoA was separated from the H-CoA on DEAE-cellulose ion-exchange paper. The Ac- H-CoA area was cut out, placed in a counting vial, eluted with 0.02 ml of 0.6 N HCl and counted. Complete reaction mixture (o) mixture without AcCoA ( ) mixture with enzyme previously inactivated b y heating at 55° for 15 min (A). B. Between H-aniline and acetanilide. The reaction mixture contained 0.005 ml of H-aniline dissolved in water (115 mC/mmole, 0.0019 /tmole), 0.20 ml of 3 X lO M acetanilide dissolved in 0.1 M sodium pyrophosphate buffer, pH 8.0 (6.0 Mmoles), 0.10 ml of DEAE-cellulose enzyme fraction from a rapid inactivator human (30 Mg protein) and 0.10 M sodium pyrophosphate buffer, pH 8.0, in a total volume of 0.305 ml at 27°. Aliquots of 0.01 ml were removed, placed on CM-cellulose strips, and treated with 0.005 ml of acetone-absolute ethanol (1 1) to stop the reaction. The H-acetanilide was separated from the H-aniline by eluting the strips with glycine buffer at pH 2.8. The H-acetanilide area was cut out, placed in a count-...

Fig. 11. Formation and isolation of a [i- CJacetyl-N-acetyltransferase intermediate from a rapid inactivator rabbit liver. The DEAE-cellulose enzyme fraction (2.5 mg protein, specific activity 0.0395 /imole 2-acetylisonicotinic acid hydrazide formed per min per mg protein) was mixed with 16.4 nmoles of [i- C]acetyl-CoA (58.5 mC/mmole) and 0.01 M potassium phosphate buffer, pH 7.0, in a total volume of 0.40 ml. After 12 min of incubation at 27° the reaction mixture was cooled to 0°, placed on a Sepha-dex G-50 column (L5 cm X 18.5 cm) and eluted with 0.01 M potassium phosphate buffer, pH 7.0, at 0°. A flow rate of 1.0 ml/min was maintained and fractions of 1.0 ml each were collected over a 20-min period. The fractions were analyzed for radioactivity and for absorbance at 280 nm (A). The experiment was repeated with enzyme previously inactivated by heat at 55° for 15 min (B). In C, DEAE-cellulose enzyme fraction (2.5 mg protein, specific activity 0.0395 Ajmole 2-acetylisonicotinic acid hydra-...

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