Cellulases and Their Applications

5 Cellulase Complex of Ruminococcus and a New Mechanism for Cellulose Degradation J. M . L E A T H E R W O O D

Downloaded by UNIV OF BATH on July 3, 2016 | http://pubs.acs.org Publication Date: June 1, 1969 | doi: 10.1021/ba-1969-0095.ch005

Nutritional Biochemistry Section, Department of Ainmal Science, North Carolina State University, Raleigh, N . C. 27607

Three variants of Ruminococcus albus were designated on the basis of their clearing of the cellulose around the colonies on cellulose roll tubes. Beta-colonies do not form significant clear zones while gamma- and alpha-colonies form a sharp clear zone and a diffuse clear zone, respectively.

A sub-

stance diffusing from a beta-colony interacts with a substance from either an alpha- or gamma-colony to form an enzyme complex that appears to act as a single entity. This apparent protein-protein

interaction is discussed with re-

spect to its formation, degradation, and adsorption to cellulose. A new mechanism of cellulose degradation is proposed that involves an affinity factor combined with a hydrolytic factor to form a complete cellulase.

A n apparent protein-protein interaction has been observed i n cultures of Ruminococcus that results in the formation of an enzyme complex that degrades cellulose. O n the basis of these and other observations, we propose a new mechanism for cellulose degradation that involves a single cellulase complex. Formation of a Cellulase Complex Ruminococcus albus strain 7, (1), was grown on cellulose roll tubes according to the Hungate technique ( 2 ) . Either Avicel ( F M C Corporation, Marcus Hook, Pa.) or balled filter paper was used as the cellulose source. Three milliliters of melted cellulose-medium at 45 °C. i n 16 X 150 mm. tubes were inoculated with the bacteria under a gas phase of 95% C 0 and 5 % H . The tubes were subsequently stoppered and rolled in a tray of ice in order to form a film of agar medium around the 2

2

53 Hajny and Reese; Cellulases and Their Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1969.

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CELLULASES A N D THEIR APPLICATIONS


inside surface of the tubes. Three variants of colony-types based on the clearing of celluose due to the cellulolytic activity of the microorganism were observed after approximately three days of incubation at 37 °C. Beta-colonies do not form a significant clear zone around the colony, whereas gamma- and alpha-colonies form a sharp clear zone and a diffuse clear zone, respectively. Reversion to other colony-types occurs at approximately 1 to 5 % , much too high for mutation. The reason for the three variants is unknown. A n unusual phenomenon was observed when either the alpha- or gamma-variants were grown along with the beta-variant i n cellulose roll tubes at proper dilutions. A substance diffusing from a beta-colony interacts with a substance from an alpha-colony to form an enzyme complex that appears to act as a single entity (Figure 1). If a substance diffusing from a colony has a zone of equal concentration i n the pattern of a circle around the colony, the theoretical pattern for clear-zone formation can be predicted for either the formation of a single complex or a two-enzyme reaction and activation of a pre-enzyme. F o r either a two-enzyme reaction or the activation of a pre-enzyme, the area of clearing should follow the pattern of overlapping circles with centers of the circles represented by the two colonies. However, the interaction of two components to form a single complex that has affinity to cellulose would be i n the pattern of a narrow area of clearing with the outer perimeter of clearing only becoming wider as complete cellulose hydrolysis occurs. As can be seen i n Figure 1 the distance of diffusion and the rate of diffusion are approximately equal for the two substances. O n this basis we believe the reaction is that of two different proteins interacting to form a complete cellulase. W h e n the complete cellulase is formed, it no longer diffuses i n the presence of cellulose. The clearing of cellulose because of the complete cellulase is usually more effective than occurs around alpha-colonies. W h e n complete cellulase is bound to cellulose, apparently it is active and is not bound i n an inactive, nonspecific affinity. A New Mechanism for Cellulose Degradation W e propose a new mechanism for cellulose degradation that is based on the formation of the cellulase complex and is compatible with the results reported i n the scientific literature. A n affinity factor and a hydrolytic factor are necessary for the formation of a complete cellulase which can hydrolyze native cellulose to cellobiose (Figure 2 ) . The amount of affinity factor ( x A ) that combines with hydrolytic factor ( y H ) probably is not x = y = 1. This new mechanism does not require a separate enzyme for the formation of reactive cellulose. The natural

Hajny and Reese; Cellulases and Their Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1969.

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Cellulose Complex of Ruminococcus

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formation of the complete cellulase complex should occur at the cell level. However, one should be able to separate the component factors of the complete cellulase by physical and chemical techniques. The separate hydrolytic factor can hydrolyze soluble cellulose derivatives. However, to effectively hydrolyze insoluble cellulose, the hydrolytic factor must be held i n position on the insoluble cellulose by the affinity factor.

Figure 1. Cellulase complex formation as the result of substances diffusing from two colony-types on cellulose roll tubes. The arc or line of clearing occurring between the colonies is because of degradation of the cellulose Several observations related to this phenomenon have been made on the cellulase of Ruminococcus albus strain 7. Crystalline cellulose is degraded by this organism. Cell-free preparations are active in decreasing the turbidity of cellulose suspensions. The amount of hydrolytic activity against soluble cellulose derivatives is not different for the three variants. T w o major cellulolytic components have been separated by


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G-100 Sephadex gel filtration. The small component, with molecular weight of 41,000, has been purified. The larger component, which is excluded on G-100 Sephadex, can be disassociated to an active hydrolytic enzyme of the size of the small component by treatment with /?-mercaptoethanol. The large component has a strong affinity for cellulose, whereas the small component is not significantly adsorbed to cellulose. However, cellulase in these cultures prior to any purification step is adsorbed strongly to cellulose. In addition, the proportion of hydrolytic factor that is adsorbed to cellulose decreases from over 90% in a young culture to less than 50% in the late stationary phase while the hydrolytic factor continues to increase. This is attributed to a natural degradation of the complex which results i n a free, non-adsorbable hydrolytic factor. This mechanism could be unique for these anaerobic bacteria, however, the major findings reported in the literature are compatible with this proposal. " C i enzyme" (3, 4) requires the addition of C , a "hydrolytic factor," in order to effectively demonstrate activity. It may be that the C i component is an "affinity factor" that binds the "hydrolytic factor," C , to the insoluble cellulose. x

x

AFFINITY FACTOR (xA)

COMPLETE CELLULASE (AxHy)

HYDROLYTIC FACTOR/ (yH)

Figure 2. A new mechanism for cellulose degradation proposes the combination of affinity factor and hydrolytic factor to form a complete cellulase complex which can hydrolyze native cellulose to cellobiose This new mechanism has been considered as an explanation for the general phenomena of resistance, extent, and nature of cellulose hydrolysis. Some speculations of these matters with respect to the new mechanism are: ( A ) differences in the rate of degradation of resistant celluloses may be caused by the difference in numbers and accessibility of binding sites for the affinity factor, ( B ) microorganisms could cause a decrease in the rate of cellulose degradation by producing proteins that bind sites in an inactive manner. This could account for large quantities of cellulosic residue remaining when reacting with certain microorganisms and enzyme preparations, and ( C ) the effect of fragmentation with little change in crystallinity could result if the binding sites are more prevalent in spe-


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cific areas. Once bound, complete cellulase could affect complete hydrolysis of the local area. This would result in a rapid loss of fiber strength with very little weight loss and production of reducing substances. Acknowledgment This research was supported in part by P H S research grant U I 00572 and N S F research grant G B 7405.


Literature

Cited

(1) Bryant, M . P., Small, N., Bouma, C., Robinson, I. M . , J. Bacteriol. 76, 529 (1958). (2) Hungate, R. E., Bacteriol. Rev. 14, 1 (1950). (3) Mandels, M . , Reese, E. T., Develop. Ind. Microbiol. 5, 5 (1964). (4) Selby, K., Maitland, C. C., Biochem. J. 104, 716 (1967). R E C E I V E D October 14,

1968.

Discussion K. Selby: " W e have thought of this mechanism too, and to test it we tried to see whether the addition of C i would modify or increase the adsorption of C . W e did this on Avicel and we found that it didn't affect C adsorption. So, I am a little bit puzzled as to how the mechanism of fixation that Leatherwood is suggesting comes about. If I understand you correctly, your argument is based on the shape of the clear zone between the colonies." J. M. Leatherwood: "That is a major consideration. In Figure 1A we have a curved fine because of interaction among several colonies while Figure I B has some lines with less curvature. If two separate activities were involved the lines would be wider and shaped like the overlap of two circles." Selby: "The thing that is puzzling me a bit is the shape of this clearance zone and the fact that there is an uncleared zone between it and the colony producing C i and C . What you are suggesting presumably is that there is excess C i produced by this colony which diffuses out and that something is happening when it reaches C from these other colonies, which are not producing C i . This is the basic assumption, right? W o u l d you accept this as a possibility, that diffusing out with this C i , and behind it is glucose, arising from the degradation of the cellulose in your plate? This glucose could inhibit solubilization, which might account for the shape of the zone. In other words, there is an area, beyond the clearance zone immediately round the colony, in which there is sufficient x

x

x

x


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glucose to inhibit solubilization. From this is coming a supply of C i and the shape of the trailing edge may be determined by the inhibiting glucose. I submit that this hypothesis could account for the shape observed." Leatherwood: "We put in 0.03% cellobiose with the cellulose in the media and the concentration of cellobiose decreases from this, so that I don't think simple inhibition by carbohydrate can be the explanation."


Selby: "I still am puzzled by what this mechanism implies, in view of the fact that we could not alter the adsorption of either component by adding the other." Leatherwood: "Probably the classical protein-protein interaction is an antigen antibody reaction, in which two proteins interact to form a complex. A n d it is well known that excess concentration of one of these can interfere with the precipitin reaction. So, whenever you test affinity factors and hydrolytic factors, you must test at various levels. This would be one reason for overlooking the interaction. W e have found an increase in adsorption of hydrolytic factor, by addition of it to solutions that had what we considered excess affinity factor." K. E. Eriksson: "I w i l l tell you something about the fractionation of the extra-cellular enzymes from Chrysosporium lignorum. W e have grown this fungus on a very large scale on cellulose as the sole carbon source, The cell-free culture solution was precipitated by ammonium sulfate, dialyzed and fractionated on a polyacrylamide P 150 column. W e obtained two protein peaks from the fractionation. In the first peak we had aryl-/?-glucosidase activity which also contains a very slight activity against cellobiose. I w i l l not discuss this enzyme here, but I can tell you that we have obtained about the same fractionation picture from the culture solution of Stereum sanguinolentum. From this fraction we were later able to get three different aryl-/3-glucosidases; some of them also showed cellobiase activity. These results w i l l soon be published in Archives of Biochemistry and Biophysics. But now I w i l l discuss the Chrysosporium lignorum enzymes. The peak obtained on the Sephadex G-75 column containing activity against carboxymethylcellulose was fractionated on a Sephadex A 50 column. The fractionation was obtained by a discontinuous increase of the buffer concentration. F r o m this fractionation three C M C a s e peaks were obtained. The enzymes in these peaks differed in polysaccharide concentration. The first peak contained 15%, the second 10% and the third 7% of carbohydrate. W e have been able to analyze the sugar component and found only mannose and traces of galactose. These certainly are not artifacts, since the organism was grown on pure cellulose. The three enzyme fractions did the same work—e.g., hydrolyzing cellodextrins. Cellopentaose was, for instance, hydrolyzed down to cellobiose



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and glucose. There was, however, one difference between the peaks. If a cellopentaose solution (1%) was incubated with the three different fractions, we obtained a difference in transglucosidation activity. The first peak of transglucosidase activity was so strong that insoluble cellodextins precipitated from the solution. The second enzyme fraction d i d the same work but to a slighter extent, and with the third enzyme solution we d i d not obtain any precipitation at all. W e had found that a concentrated unfractionated culture solution hydrolyzed cotton cellulose at a reasonable rate. W h e n a cotton fiber suspension was incubated with the different C M C a s e peaks (equal numbers of cellulase units) the hydrolyses of the cotton fibers were much lower than for the nonfractionated culture. The number of C M C a s e units were the same in both cases. The fractionation diagram from the Sephadex A 50 column contained a protein peak which had no C M C a s e activity. W e thought that this might be the C i enzyme. Our next experiment was to take each of the three C M C a s e fractions, mix them with the same amount of the solution from the protein peak, and test their activity against cotton cellulose. B y doing so we found that the rate of hydrolysis of the cotton cellulose was increased about 10 times. W e l l , we think this must be the C i enzyme. W e are now trying to purify and characterize it. W e have already found that when we incubate a solution of cellohexaose with the enzyme, the result of the hydrolysis is cellobiose and nothing but cellobiose. Our opinion concerning the C i enzyme, if this is it, of course, is that it is an enzyme acting endwise splitting off cellobiose units. In our opinion this also makes sense. W e think that the reason for a synergistic effect between a random cellulase and this endwise-acting cellulase is the following. A random-acting cellulase ( C M C a s e , C ) hydrolyzes a /M,4-glucosidic linkage. The chances that this bond w i l l remain broken are not very good because of steric factors. Hydrogen bonds w i l l put the glucose in position so that the /M,4-glucosidic bond can be formed again. This w i l l not happen in the presence of endwiseacting enzyme. W h e n this enzyme has a free cellulose chain-end to work with, it will cut off cellobiose units, and the cellobiose w i l l go into solution. x

Since the enzyme which we consider the C i enzyme has not been completely purified yet, I w i l l not stress our results too much, but I think the possibility for the mechanism of breakdown of cellulose I have postulated here makes sense, and I hope that it soon w i l l be possible for us to confirm these results." E. T . Reese: "I would like to say that we have worked with a related fungus, published under a different name. This is Chrysosporium pruinosum (Gilman and Abbott) Carmichael, which we have been calling Sporotrichum pruinoides Q M 826. Its cellulase system is much like that of D r . Erikksson's C . lignorum, in having relatively good C i activity."


Cellulases and Their Applications

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