Characterization of cellulases

Our early studies dealt with characterization of cellulase from Clostridium thermocellum (4, 5), the first described thermoanaerobe. More recently, we have characterized the saccharidases in three new non-cellulolytic thermoanaerobic species (6-12). Table II compares the general properties of thermophilic saccharidases identified in C. thermosulfurogenes strain 4B (6), C. thermohydrosulfuricum strain 39E (7), and Thermoanaerohacter strain B6A (13). It is worth noting here that... 

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

By using both enzymes in combination, the yield of crystalline D-glucose is increased.100 The use of cellulosic wastes for the production of glucose is of commercial interest, and in consequence much research work has been done on the characterization of cellulases. In particular, the cellulase system of the fungus T. ressei has the full complement of enzymes required to degrade crystalline cellulose. This and other fungal extracellular cellulases are commercially available.101... 

In an excellent chapter under the headline Criteria for Characterization of Cellulases, Whitaker (55) has reviewed the results of work on this subject up to 1961. This chapter dealt with works comprising characterization of cellulases both as enzymes and as proteins. As far as known to the present author no essentially new methods have been presented since Whitakers review concerning characterization of cellulases as enzymes. Characterization of the Ci enzyme has failed so far though a considerable amount of work has been carried out lately on the Ci enzyme from two species of Trichoderma, namely T. viride and T. koningi. 

On the other hand a lot of work with new methods has been carried out concerning characterization of cellulases as proteins. The isoelectric focusing works by Ahlgren et al. (I, 2) have already been described. The cellulase, the protein structure of which is best characterized, is the cellulase from P. notatum. The work carried out by Pettersson et al. (9,... 

Hideno A, Inoue H, Tsukahara K, Yano S, Eang X, Endo T, Sawayama S. (2011). Production and characterization of cellulases and hemicellulases hy Acremonium cellulolyticus using rice straw subjected to various pretreatments as the carhon source. Enzyme Microb Technol, 48, 162-168. 

Kluepfel D, Shareck E, Mondou E, Morosoli R (1986) Characterization of cellulase and xylanase activities of Streptomyces lividans. Appl Microbiol Biotechnol 24 230-234... 

The classification of cellulase sequences into domains allows the further recognition of distinct sequences that characterize each family. To make these sequences evident, one must use multiple sequence alignments of the members of each individual family. We have concentrated on the Microhispora bispora endoglucanase, and present some initial multiple sequence alignments of the catalytic and binding domain families of which it is a member. 

The objective of the current work was to characterize carefully the dynamics of cellulase production and metabolic activity following cellulose addition in a batch cultivation of the strain T. reesei Rut-C30. Cells were initially grown on glucose as the carbon source, and after its depletion, cellulose was added. Since it is difficult to follow the growth directly after addition of a solid substrate, on-line measurements of C02 evolution were used to follow the metabolic activity of the cells. Frequent samples were also taken to measure enzyme activity and sugar concentrations. 

Sheir-Neiss, G. and Montenecourt, B. S., Characterization of the secreted cellulases of Trichoderma reesei wild type and mutants during controlled fermentations. Appl. Microbiol. Biotechnol. 1984, 20, 46-53. 

With the example provided by the characterization of these chemically and enzymologically pure cellulases, we decided to purify and describe the enzyme components of a cellulolytic organism, Trichoderma viride. Brief descriptions of the properties of partially purified exo-fi (1 4)glucanase (20), Ci or hydrocellulase (48), and endo-fi-(l 4)-... 

Work in several laboratories (22, 27, 55, 56) has shown a pattern of cellulase action in cellulolytic organisms which requires at least one of a set of three closely related enzymes in order to hydrolyze crystalline cellulose effectively. These enzymes often possess little ability to degrade either CM-cellulose (as measured viscosimetrically) or crystalline cellulose. Nevertheless they are characterized by the capacity to cleave swollen cellulose or cellooligosaccharides almost entirely to cellobiose by virtue of their / -( - 4)glucan cellobiohydrolase activity. Recognition of this pattern has been difficult because prior to this report the three enzymes had not been purified and characterized apart from contaminating enzyme activity. 

The fact that cellulase enzymes act on an insoluble substrate, cellulose, moves the kinetics outside Michaelis-Menten on several counts. First of all, the enzyme can be adsorbed to the substrate or imadsorbed, but only the adsorbed enzyme acts on the cellulose. Even more puzzHng is the substrate concentration. Do we count the entire substrate, or just that in close contact with the enzyme Clearly, we have to start from first principles in characterizing the cellulase/cel-lulose system. 

From the results of cellulase digestion and the characterization of a number of oligosaccharides, an improved partial structure for the runner bean cell wall xyloglucan was proposed (O Neill and Selvendran, 1985b). It would appear that the xyloglucan does not contain a simple repeating unit but is composed of a block-type structure that is commonly encountered in plant polysaccharides (Stephen, 1983). 

Fortunately, at this time, there were developments in the techniques for the fractionation of macromolecules at a 1967 ACS Symposium, a review of the existing fractionation procedures for cellulases (4) revealed that, although notable successes had been achieved, better methods for the purification and characterization of the enzyme components were needed. Towards the end of 1961, developments in the production of gel-filtration media and of ion-exchange forms of the same materials made possible the application of these techniques to the separation of the components of the cellulase system. 

Discrimination of Cellulase Components A, B, and C. The physical and chemical properties as well as substrate specificities of the highly purified cellulases of Ps. fluorescens have been characterized and are summarized in Table IV. Cellulase A is different from Cellulase B in the mobility on zone electrophoresis and in the pattern of Sephadex G-25 chromatography, but similar in substrate specificity toward several reduced and nonreduced cello-oligosaccharides. On the other hand, Cellulase C is different from A in the pattern of DEAE-Sephadex chromatography and the substrate specificity, and from B in all respects. These characteristics of each cellulase component are therefore different enough to be used as criteria to discriminate one from the other. 

Lienqueo ME, Salgado JC, Asenjo JA (1999) An expert system for selection of protein purification processes experimental validation. J Chem Technol Biotechnol 74(3) 293-299 Liu J, Xi W (2006) Purification and characterization of a bifunctional enzyme with chitosanase and cellulase activity from commercial ceUulase. Biochem Eng J 30(l) 82-87 Liu LC, Prokopakis GJ, Asenjo JA (1988) Optimization of enzymatic lysis of yeast. Biotechnol Bioeng 32(9) 1113-1127... 

M. Nojiri and T. Kondo, Application of regioselectively substituted methylcel-luloses to characterize the reaction mechanism of cellulase, Macromolecules, 29 (1996) 2392-2395. 

Cavedon, K., Leschine, S. B., and Parola, E. C. (1990). Characterization of the extracellular cellulases from a mesophlllc Clostridium (strain C7),... 

Li, J., Y. M. Du, and H. B. Liang. 2006a. Low molecular weight water-soluhle chitosans Preparation with the aid of cellulase, characterization, and solubihty. J. Appl. Polym. Sci. 102 1098-1105. 

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