Cellulolytic extracellular

The strains were cultured on Mandels medium + 1% citrus pectin for 5 days and the enzymatic activities of culture filtrates were determined on three substrates citrus pectin, polygalacturonic acid and filter paper, (a) extracellular proteins are in p.g/ml. (b) p>ectinolytic activities on pectin (PC) and on polygalacturonic acid (TO) and Pectin esterase (PE) are in units/ml. (c) total cellulolytic activity (filter paper, fp) are in mg of liberated reducing sugars/ml. 

H. Murata, J. L. McEvoy, A. Chatterjee, A. Collmer, A. K. Chatterjee. Molecular cloning of an aepA gene that activates production of extracellular pectolytic, cellulolytic, and proteolytic enzymes in Erwinia carotovora subsp. carotovora. Mol. Plant Micr. Interact. 4 239 (1991). 

No doubt the cost of xylanolytic enzymes will be one of the factors determining their application in the pulp and paper industry as well as in other areas. Economically feasible xylanase production can be achieved in paper mills employing xylanase-positive transformants of common industrially used microorganisms that are capable of utilizing inexpensive carbon sources originating there. A substantial improvement in the production of xylanolytic systems can be expeaed from mutants of non-cellulolytic microorganisms that are resistant to catabolic repression. Such mutants usually exhibit hyperproduction of extracellular enzymes. 

This chapter deals with three aspects of the cellulolytic enzyme system of Thermoactinomyces sp. the location of the CM-cellulase, Avicelase, and / -glucosidase (cellobiase) activities in the culture, the multiplicity of the extracellular enzyme system, and the stability of the different activities as a function of pH, temperature, and time. The results are discussed with reference to saccharification of cellulosic materials. 

Extracellular Cellulolytic Activities. The appearance of the CM-cellulase activity in a culture of Thermoactinomyces grown on 1% microcrystalline cellulose is shown in Figure 2. The extracellular CM-cellulase activity approached a maximum of 14-16 mg reducing sugar (RS) mL-1 min"1 within 18-24 hr. The Avicelase activity of the culture filtrate developed simultaneously with the CM-cellulase activity and amounted to 3 mg RS mL"1 hr"1. The extracellular protein concentration reached 1.7 mg/mL in the stationary phase (6). 

The CM-cellulase activity of the solids fraction shows a skewed curve over the period of 4-24 hr with a maximum of 3 mg RS mL"1 min"1 around 8 hr, at which point it makes up about 50% of the activity in the whole culture broth (Figure 2). No activity could be detected in the solids fraction in the late stationary growth phase. Within experimental error, the CMC activity of the culture filtrate plus that of the culture solids equals the activity of the whole broth. Similarly, it was found for Thermoactinomyces, strain MJ0r, grown on 0.5% microcrystalline cellulose, that there was a lag before an appearance of extracellular cellulolytic activity, as compared with the activity in the whole culture broth (4). In a culture of Thermoactinomyces, strain YX, the CM-cellulase activity can be desorbed readily by washing the solids fraction with water. These wash fractions also show Avicelase activity (6). This result, and the fact... 

Figure 5 also shows two 10-hr samples, 10a and 10b. Sample 10a was stored in solution at 4°C for one week, while sample 10b was stored frozen and then thawed immediately before application to the polyacrylamide gel. Both samples show the same protein band pattern. If proteolytic enzymes in the culture filtrate had acted on and partially degraded the extracellular proteins, a different band pattern would have been expected. Thus no product-precursor relationship appeared to exist between the various extracellular proteins in a culture filtrate of Thermoactinomyces. Moreover, it seems as if this organism produces at least three different extracellular cellulolytic enzymes simultaneously. 

The extracellular cellulolytic activities, CM-cellulase and Avicelase, are stable over 24 hr at 55°C in the pH range of 6.0-7.3, which suggests that no enzymatic activity would be lost in a saccharification under these conditions and that the enzymes would be recoverable. 

Fig. 1. Cellulolytic and xylanolytic activities and extracellular protein during time course of the cultivation of P. brasilianum IBT 20888. (A) Cellulolytic activities on cellulose (B) cellulolytic activities on xylan (C) xylanase activities on cellulose and (D) xylanolytic activities on xylan. ( ) BG ( ) CBH ( T ) EG ( O ) BX ( O ) AF ( V ) EX (+) extracellular protein ( —) cultivation on cellulose and (----) cultivation on xylan.

It should be emphasized that these phosphorolytic enzymes are intracellular. They are not found in the extracellular cellulase complex, and they are involved in the cellulolytic systems of relatively few microorganisms. 

Oince cellulose is insoluble high polymers under physiological condi- tions, cellulase which is destined to attack it has been expected to be an extracellular enzyme. In fact, most of cellulolytic microorganisms secrete some cellulase components into the culture medium, and almost all work on the cellulase have been performed using these extracellular components. In the cultures of cellulolytic bacteria, cellulases are not only found in their culture filtrates, but also are generally obtainable from the cells by treatment with autolytic agents—e.g., toluene (9, 19, 30). These facts indicate that at least certain components are existent within the bacterial cells and that their physiological function may be distinct from that of the extracellular components. 

The susceptibility of cellulose to enzymatic hydrolysis is determined largely by its accessibility to extracellular enzymes secreted by or bound on the surface of cellulolytic microorganisms. Direct physical contact between these enzymes and the cellulosic substrate molecules is an essential prerequisite to hydrolysis. Since cellulose is an insoluble and structurally complex substrate, this contact can be achieved only by diffusion of the enzymes from the organism into the complex structural matrix of the cellulose. Any structural feature that limits the accessibility of the cellulose to enzymes by diffusion within the fiber will diminish the susceptibility of the cellulose of that fiber to enzymatic degradation. In this review, the influence of eight such structural features have been discussed in detail. 

The accessibility of cellulose to the extracellular enzymes of cellulolytic microorganisms is determined in part by its distribution within the cell wall and the nature of the structural relationships among the various cell-wall constituents. These relationships are reviewed briefly. 

To appreciate fully the influence of the structural features of natural fibers on their susceptibility and resistance to enzymatic degradation, it is necessary to understand the relationship between cellulolytic microorganisms, their extracellular enzymes, and the fiber substrate itself. 

Size and Diffusibility of Cellulolytic Enzymes in Relation to the Capillary Structure of Cellulose. As discussed earlier, enzymatic degradation of cellulose requires that the cellulolytic and other extracellular enzymes of the organisms diffuse from the organism producing them to accessible surfaces on or in the walls of the fiber. This accessible surface is defined by the size, shape, and surface properties of the microscopic and submicroscopic capillaries within the fiber in relation to the size, shape, and diffusibility of the enzyme molecules themselves. The influence of these relationships on the susceptibility and resistance of cellulose to enzymatic hydrolysis has not been verified experimentally in natural fibers but the validity of the concepts that follow is demonstrated by the work of Stone, Scallan, Donefer, and Ahlgren (69). 

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