Cellulase Adsorption

The first step in cellulose hydrolysis is the adsorption of the enzyme onto the cellulose. The rate of adsorption depends on the viscosity and agitation of the system. In a dilute, well mixed system, adsorption equilibrium is established in about 5 min. The adsorption equilibrium is described empirically by a Langmuir Adsorption isotherm [31]  

For a natural substrate like ground cellulose or fruit pulp, of order 

10 mgg L Chemically pretreated cellulose such as paper pulp can have much higher P values. Higher dosages of cellulase than P have little effect on the rate of reaction. The adsorption behavior does not follow a reversible Langmuir profile, as the enzyme is very difficult to wash off its substrate. A Langmuir isotherm implies reversible adsorption. 

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Kadam, K. and Knutsen, J. (2001), Saccharification Experiments 6-9 Characterization of Cellulase Adsorption onto Pretreated Corn Stover, National Renewable Energy Laboratory, Golden, CO. 

Conventionally, cellulase adsorption onto (or accessibility for) cellulose, such as that of T. reesei cellulases onto amorphous/crystalline/lingo-cellulose, has been investigated by Langmuir-type isotherms 20, 34). They measure overall cellulase protein adsorption, assuming a uniform cellulose surface. 

Langmuir-type isotherms, governed by an adsorption constant and a capability No, are widely used to measure the adsorption of cellulase onto cellulose. The parameter No reflects the portion of s ace cellobiosyUattice units accessible for cellulase adsorption, regardless whether or not the adsorption is followed by hydrolysis. In contrast to the static No, is kinetic and measures those productive cellulase adsorptions that can lead to subsequent p-1,4 bond cleavage. 

For a given cellulase, AvicePs ( ) was less than PASC s (j), suggesting less productive cellulase adsorption on more crystalline cellulose surface. With crystalline cellulose substrate, it could be more difficult for the cellulase to thread a cellulose chain into its active site clefl/tunnel, since more inter-chain H bonds would need to be broken in a highly cr3 talline region than in an amorphous/chain end region. 

Lu, Y., B. Yang, D. Gregg, J. Saddler and S. Mansfield (2002). Cellulase adsorption and an evaluation of enzyme recycle during hydrolysis of steam-exploded softwood residues. Applied Biochemistry and Biotechnology 98-100(1) 641-654. 

Since protein adsorption to an anion exchange resin is reversible and does not constitute a classical immobilization, the ability of the resins to retain activity under various conditions must be determined. Macrosorb KAX DEAE bound -D-glucosidase was tested with solutions of primary interest for their final application. Several batch washes of a 1% w/v slurry were required to ensure complete equilibrium elution for a given concentration, as determined from the absence of pNPG units in subsequent washes. Several salt solutions of typical fermentation media components were tested. These included 3 mM to 50 mM solutions of MgSO, KHgPO, NaQ, and sodium acetate. Also, incubations with cellulase solutions were tested to determine if the proteins present in a cellulose hydrolysis would displace the -D-glucosidase. Both of these displacement studies were carried out at 22°C and 40 C. 

These results demonstrate several useful properties of the C. fimi CBDs for enzyme immobilization and purification. Presumably, the CBDs of other bacterial cellulases (Figure 3) can be used in similar ways. It remains to be seen whether these differ significantly in their affinity or capacity for adsorption if so, certain CBDs may be preferable for specific purposes. 

The study of cellulases has progressed considerably in the present decade. Recombinant DNA techniques have been applied and protein-chemical and structural studies have provided new insights. Crystallization of the first cellulases has succeeded recently and detailed structural information may be expected soon. It is hoped that this will give a further incentive to studying the intricate reaction mechanism of these enzymes and their substrate interactions (adsorptions). The important synergy phenomena certainly need a more systematic approach and new techniques should be applied in this area. 

Our chromophoric substrates proved to be valuable in the study of several aspects of the enzymology of these cellulases. A rapid and specific method for purification (affinity chromatography) has been developed. Following our collaboration with several groups, new insights into the domain arrangement and tertiary structures of two cellulases were obtained. Contributions to the elucidation of the synergistic action (adsorption-hydrolysis) of these enzymes were achieved. 

If one assumes that a Langmuir-type adsorption isotherm can be used to relate the concentration of adsorbed cellulase on the cellulose to the free cellulase in solution, i.e.,... 

E°ads — saturation concentration of cellulase/gram of cellulose K = no. adsorption sites/gram of cellulose rj = no. adsorption sites/unit surface area R = mean mass radius/cellulose particle n — no. cellulose particles/gram of cellulose... 

Azevedo, H., Bishop, D., and Cavaco-Paulo, A. 2002a. Possibilities for Recycling Cellulases After Use in Cotton Processing. Part I Effects of End-Product Inhibition, Thermal and Mechanical Deactivation, and Cellulase Depletion by Adsorption. Appl. Biochem. Biotechnol., 101, 61-75. 

Gusakov, A. V., Sinitsyn, A. P., Markov, A. V., Sinitsyna, O. A., Ankudimova, N. V., and Berlin, A. G. 2001. Study of protein adsorption on indigo particles confirms the existence of enzyme-indigo interaction sites in cellulase molecules. J. Biotechnol., 87, 83-90. 

Cellobiase. Figure 2 outlines the general purification steps used to isolate a pure cellobiase (named for its function in the cellulase complex) and three forms of the hydrocellulase. In purifying cellobiase it was expedient to replace the adsorption or affinity column with a batch separation on DEAE-Sephadex A-50 and to complete the purification with cation exchange chromatography on SP-Sephadex (49, 50). Table IV is a summary of the purification of the cellobiase and co-purification of... 

Enzymes, immobiiized, are attached to a solid support by adsorption or chemical binding or mechanical entrapment in the pores of a gel structure, yet retain most of their catalytic powers -ase is a suffix identifying that the substance is an enzyme. The main part of the name describes the nature of the chemical reaction that can be catalyzed, as in cellulase, an enzyme that catalyzes the decomposition of cellulose... 

Similar adsorption data have also been reported for the adsorption isotherms of many compounds in various systems. For example, the adsorption data of several jS-blockers, particularly those of propranolol acquired in a 1 to 7000 relative concentration range, on an immobilized cellulase. Cel 7A, fit very well to the bi-Langmuir model, as illustrated in Figure 3.13 [47,55]). The enantioselective site was identified as a pair of amino acid residues in the tunnel formed by the main chain of the protein. The parameters of the isotherm depend on the pH as illustrated in Figure 3.14. A bi-Langmuir model was also found to accoimt well for the separation of pairs of enantiomers on polymers molecularly imprinted with one of the enantiomers [56]. Note, however, that there are also many systems in which the adsorption isotherms of enantiomers are not accounted for by a bi-Langmuir model showing that enantioselectivity is often achieved by a complex... 

Gilkes N, Jervis E, Henrissat B, Tekant B, Miller RC Jr, Warren RAJ, Kilburn DG (1992) The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose. Journal of Biological Chemistry 267 6743 - 6749... 

Fornstedt,T, Goetmar, G., Andersson, M., Guiochon, G. Dependence on the Mobile-Phase pH of the Adsorption Behavior of Propranolol Enantiomers on a Cellulase Protein Used as the Chiral Selector, /. Am. Chem. Soc., 1999,121, 1164-1174. 

Specific Adsorption on Different Cellulose Columns. Purification of cellulolytic enzymes, including the Ci enzyme, has been carried out by Li et al. (32) by specific adsorption. By passing the crude enzyme through a column of Avicel the Ci fraction was retained by the column while 95% of the cellulase activity could be eluted. The cellulase fraction was then further purified by passing a column of alkali-swollen cellulose where the cellulase was retained. The enzyme could, however, be eluted by buffer in a high yield from the column. The adsorption on an alkali-swollen cellulose column was then repeated. The yield of enzyme obtained with the specific adsorption method is surprisingly high if compared with the yield on Sephadex and on polyacrylamide gel columns. 

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