Study of cellulases

The results of multi stage enzyme treatment suggests that further study of cellulase action is fully warranted. One hopeful area would be to decrease the hydrolysis time and enzyme concentration, and increase the number of stages. Another approach would be to remove the sugars by some other means, such as dialysis or fermentation (20). 

Native or processed cellulose (e.g., cotton, Avicel, filter paper) and its soluble derivatives (e.g., CMC, HEC3) are substrates most often used in the study of cellulases. The classification based on the use of these substrates (l,4-/ -D-glucan cellobiohydrolases (CBH), exo-cellulases, Avicelases and... 

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. 

Nojiri, M., and Kondo, T. 1999. Kinetic studies of cellulase for water-soluble cellulose derivates. In Ohmiya, K., Hayashi, K., Sakka, K., Kobayashi, Y., Karita, S., and Kimura, T. (Eds.), Genetics, biochemistry and ecology of cellulose degradation (pp. 199-206). Tokyo Uni Publishers Co. 

In order to avoid the complications arising from the use of CMC samples of too low and too high DS values, samples in the range 0.8 < DS < 1.0 were recommended for comparative studies of cellulase activity. To make comparisons of absolute activity determinations meaningful it was necessary to determine the numerical values of the quotient c and the Staudinger exponent x for the CMC samples studied. With CMC samples in the mentioned DS range the following conclusions can be drawn from measurements with four different cellulases ... 

While there has been much progress in the study of cellulases, the applications of such technology have been limited by a lack of economical pretreatment of the lignocellulose. Without such pretreatment hydrolysis is slow and incomplete. The value of enhanced enzymatic attack on lignocellulose is not limited to the production of sugar and chemicals. The same procedures would be applicable to the increased digestibility of coarse fodder by ruminants. 

A wild strain Aspergillus terreus isolated from wood drifted ashore in India (Araujo and D Souza, 1980) exhibits an increased production of 3-glucosidase as compared with other known producers of cellulases. Therefore, in our studies of cellulases we concentrated on this organism. We first determined the effect of cultivation conditions on the properties and production of individual enzymes of the cellulolytic complex (D Souza and... 

A sugar fluoride monomer was fmther used for the synthesis of thio-oHgosaccharides, which can be an inhibitor of cellulase and are useful for the mechanistic study of cellulase function [143]. Hemithiocello-oUgomers from tetraose to tetradecaose were obtained via enzymatic polymerization using 4-thio-/8-cellobiosyl fluoride as monomer by cellulase catalyst [144]. 

Irwin, D. C., Spezio, M., Walker, L P. and Wilson, D. B. (1993). Activity studies of eight purified cellulases specificity, synergism, and binding domain effects. Blotechnol Bioeng 42,1002-1013. 

Figures 1 and 2 show positive correlation, although of varying degrees of goodness of fit, between the specific activities of endo ucanase, exoglucanase, and -glucosidase activities with the cellulose composition and degradation in digester feed, respectively. These data indicate a direct relationship between the cellulose content in the feed and cellulase enzyme complex production by the digester consortia. In this system, as in many others studied, cellulose is an effective inducer of cellulase secretion (67,68),...

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... 

A literature survey indicated that very little work has been done to produce an optimal cellulase system as described above. Here, we used solid-state fermentation (SSF) to achieve this objective. SSF processes, such as the "koji" process, have been used extensively for amylase production on wheat bran in Japan its application was extended to cellulase production on wheat bran and Ugnocellulosic materials by Toyama (13), Since then, wheat bran has become an important substrate for producing various products by SSF (14-20), In this study, we tested various lignocellulosic substrates for the production of cellulase and )3-glucosidase from T, reesei QMY-1 by SSF. 

Cost sensitivity studies have shown that the successful commercialization of cellulase-based processes, such as the conversion of cellulose to fermentable sugars, is highly dependent on the cost of enzyme production (i). Because fungal -D-glucosidase (EC 3.2.1.21) is the most labile enzyme in this system under process conditions (2), and k to efficient saccharification of cellulose, this enzyme was targeted for application of stabilization technology, both through chemical modification and immobilization to solid supports. 

Selective Production of Xylanases by Cellulolytic Microorganisms. Until recently there was little information on common or separate genetic control of cellulase and xylanase synthesis in microorganisms (60). Studies on this subject were complicated by the fact that numerous microbial ceUulases and xylanases are non-specific with respect to cellulose and xylan as substrates. As could be expected from a comparison of both polysaccharide structures, non-specificity is more frequently observed with cel-lulases, because their substrate binding sites can easily accommodate substrate using an unsubstituted p-(l 4)-linked chain of D-xylopyranosyl units. 

A pilot study for large scale production of cellulase from Triohoderma reesei has been developed at the U.S. Army Natick Development Center (Fig. 7 (20)). In March 1977, enzyme produc-... 

Chromophoric substrates were also used as tools in the study of the binding of several cellulase components to their natural substrates (such as Avicel). This is illustrated here in the investigation of the synergy in binding of CBH I and CBH II from Trichoderma reesei onto Avicel. The enzymes were differentiated with CNPL (see above), which was a substrate only for CBH I (core I). Thus, the amount of CBH II adsorbed when a mixture of both enzymes was added, either simultaneously or sequentionally, to Avicel was calculated from the amount of CBH I bound (activity measurements with CNPL) subtracted from the values for total protein binding (280 nm absorbance reading). The results obtained from these experiments are summarized as follows ... 

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. 

One of the favored organisms for study of cellulolysis by Trichoderma is T. reesei. Consequently, many mutant strains which hyperproduce cellulase have been obtained by treatment with ultraviolet light, gamma irradiation, the linear accelerator, diethyl sulphate and N-methyl-N -nitro-N-nitroso-guanidine (7). Whereas much of the study of T. reesei has been with cellulose as substrate, it is relevant to consider the other fractions of natural lignocelluloses hemicellulose and holocellulose (the combined cellulose and hemicellulose fraction). 

The most complete studies of (1—>4) thiooiigosaccharides were achieved in the cellobiose and maltose series with cellulases and a-amylases of various origins. 

Besides his fundamental research in the carbohydrate field, the functions of Courtois as the head of a hospital laboratory for many years led him to publish a number of papers dealing with clinical chemistry, among which may be cited determination of ethyl alcohol, proteins, acidic phosphatases, and trehalase in blood determination of the basic groups of proteins by phytic acid study of the phytosoluble glycoproteins in biological fluids and identification and determination of scyllitol in urine. Under the aegis of the International Pharmaceutical Federation, he participated in the standardization of the methods proposed for the assay of such enzymes as cellulases and hemicellulases. 

Hphe study of the synergism shown by enzymes of the cellulase complex in solubilizing highly ordered native cellulose has now progressed to a stage where it is possible to state with some certainty the principal enzyme components involved. Not everyone agrees, however. 

Tt is a widely recognized fact that true cellulolytic microorganisms A produce three basic cellulase components IS), and that these enzyme components act in concert to hydrolyze crystalline cellulose to glucose (6). Many research laboratories have undertaken the task to purify cellulose components from various cellulolytic microorganisms and to study the mechanisms of cellulose hydrolysis. Much information has accumulated concerning the mode of action of cellulose hydrolysis since Reese et al. first proposed the Ci-C concept (7). In spite of this, however, conflicting reports still flourish concerning the composition of the "cellulase complex, the multiplicity of cellulase components, the biosynthesis of cellulose, and the mechanisms of cellulose hydrolysis. 

T. reesei is a useful experimental organism for studying regulation of extracellular protein biosynthesis. When grown in a medium in which an exogeneous inducer serves as the major or sole carbon source, T. reesei, synthesizes and secretes a cellololytic enzyme into the medium. Similarly, the extracellular cellulase is produced upon limitation of the carbon source and limitation of the utilization of the carbon source (31). Presently, there appears to be little data in the literature concerning regulation of cellulase biosynthesis. 

More experiments are needed before any conclusions can be made. It appears that the isolation and study of a mutant which synthesizes only one cellulolytic enzyme component is needed if headway is to be made on determining the nature of regulatory control of cellulase biosynthesis. 

Sprucewood holocellulose was treated with an endo-p-1,4-mannanase isolated from Aspergillus niger and an endo-/3-1,4-xylanase, two avicelases, and a cellobiohydrolase C isolated from Trichoderma viride. The mannanase hydrolyzed about a quarter of the mannan in 2-3 days without xylan or cellulose degradation. The xylanase hydrolyzed about half the xylan with 10% mannan solubilization. The three cellulases hydrolyzed up to 45% of the cellulose and 20% of the xylan, accompanied by 40-70% solubilization of the mannan. Combined xylanase-mannanase treatment hydrolyzed about half the xylan and mannan. Addition of mannanase to to cellulose-treated samples increased the degradation of the cellulose and mannan. Micromorphological studies of the variously treated specimens revealed a loss of substances in P/Slf T, and adjacent zones of S2 of the tracheid wall. 

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