Cellulases and Their Applications - American Chemical Society

4 The Purification and Properties of the C 1

Component of the Cellulase Complex

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K E I T H SELBY Shirley Institute, Didsbury, Manchester M20 8RX, England

Certain

cellulolytic

fungi yield

cell-free filtrates

capable

of extensive degradation of highly ordered forms of cellulosic material

These filtrates have been shown to contain

a so-catted C -component, which, although essential for this 1

type of activity, is virtually without action when freed from the other (C ) components. C -components, with very similar x

1

properties, have been isolated from Trichoderma viride and from Penicillium funiculosum.

The powerful

synergistic

action on cotton previously found between the C and C 1

x

components of T . viride was also displayed by those from P. funiculosum; cross-synergism has also been demonstrated. An attempt has been made to explain the role of C in the 1

solubilization of cotton.

V U T h e n Reese, Siu, and Levinson (24) first suggested the existence of a Ci-component i n the cellulase system, they d i d so because certain microorganisms could grow on media containing cellulose as sole carbon source whereas others required more easily assimilable forms of carbon, such as glucose or carboxymethylcellulose. It was suggested, therefore, that although a l l "cellulase" systems probably contained enzymes called C capable of digesting easily accessible forms or derivatives of cellulose, only those that contained a Ci-component could utilize highly ordered forms of cellulose, as found, for example, i n cotton hairs and i n some other vegetable fibres, against which attack is principally influenced by the supra molecular structure of the substrate. It was further suggested that C i acted as a chain-separating enzyme, which at the time Siu, as he said " i n semi-jest" (29, 30), referred to as a "hydrogen bondase"; the Ci-component was thus thought of as an enzyme whose presence was required before the depolymerizing enzymes ( C ) could start to break down the cellulose chain. Ten years after this theory was postulated x

x

34 In Cellulases and Their Applications; Hajny, G., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1969.


4.

SELBY

C

±

Component of the Cellulose Complex

35

there had been little further progress because no cell-free filtrates had been isolated with sufficient activity towards highly ordered substrates to suggest that they contained a significant amount of the Ci-component. A hopeful sign came from the discovery (26) that filtrates from Myrothecium verucaria, suitably manipulated, could produce 30% solubilization of cotton, and this was the stage that had been reached at the time of the last A C S symposium on cellulases held in Washington i n 1962 (22). It subsequently became apparent from the high solubilizations of cotton obtained by single treatments with filtrates from Trichoderma viride (10) that this would be a better organism to use in the search for Ci-component. Fortunately, at this time, there were developments in the techniques for the fractionation of macromolecules; at a 1967 A C S 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 i n 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. Over the last five years, the results of many fractionation studies on cellulases have been published; the greatest number of these has been on enzymes from Trichoderma viride (and T. koningii). The reason for this interest must surely be the demonstration by several groups of workers i n 1964 and 1965 ( 2, 5, 8, 10, 16, 19) that filtrates from this fungus contained the Ci-component. During this period the importance of a discovery reported by Gilligan and Reese 10 years earlier (3), which at the time seems to have attracted little attention, was realized. Fractionation of a filtrate from T. viride on calcium phosphate gel had yielded two components between which synergistic action was found. Because this action was tested only on a swollen and partially degraded cellulosic substrate (Walseth cellulose) its potential importance i n relation to Ci-action was not appreciated until Mandels and Reese (10) showed that this synergism could best be demonstrated on cotton, with the obvious corollary that C i must be involved. O n the basis of fractionation studies made at this time, Ci-type enzyme was envisaged as the only component capable on its own of solubilizing highly ordered forms of cellulose; cotton was used by Mandels and Reese (JO) and i n some of Flora's work (2) and crystalline hydrocellulose (an acid-hydrolyzed cotton) by L i , Flora, and K i n g (8). A d d i tion of C to C i caused about threefold increases in solubilization. Contemporary work at the Shirley Institute centered on rigorous fractionation of the cellulase system of T. viride into components displaying single x

In Cellulases and Their Applications; Hajny, G., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1969.

36

CELLULASES A N D THEIR APPLICATIONS


enzyme function, which seemed imperative i n view of the demonstrable synergism between C i and other components of the cellulase system. A n account of this work has already been published (28), so that only a brief description of the purification procedure w i l l be given here. A typical fractionation of a filtrate from T. viride on Sephadex G-75 is shown in Figure 1. A low molecular-weight carboxymethylcellulase, which apparently d i d not participate i n the solubilization of cotton, was removed at this stage. Although the other components were unresolved, there was

H 0-6

a 4

0-4

1 > o-io

1

\*A.

Biochemical Journal

Figure 1. Fractionation of a culture filtrate from T. viride on Sephadex G-75 (2)


4.

SELBY

C Component of the Cellulase Complex

37

t


20

3

W

20

30 Fraction no

40

50

60

Biochemical Journal

Figure 2. Separation of the C and C -components from T. viride cellulase by chromatography on DEAESephadex (2) r

x

evidence for the existence of separate peaks of cellulase, carboxymethylcellulase, cellobiase, and material absorbing at 280 n.m. Moreover, the positions of the peaks suggested that the "cellulase" activity could result from synergism between the material absorbing at 280 n.m. and the carboxymethylcellulase and/or cellobiase. The optically dense component, believed to be C i , was separated from cellobiase and carboxymethylcellulase by gradient elution on DEAE-Sephadex (Figure 2 ) ; the carboxymethylcellulase and cellobiase were subsequently resolved on SE-Sephadex at p H 5.1 (Figure 3 ) . These studies had thus yielded a carboxymethylcellulase with no activity towards cellobiose, a cellobiase with no activity towards carboxy-


38

C E L L U L A S E S A N D THEIR

\ 1

fa i

/

J L-a

' Downloaded by PENNSYLVANIA STATE UNIV on June 8, 2012 | http://pubs.acs.org Publication Date: June 1, 1969 | doi: 10.1021/ba-1969-0095.ch004

APPLICATIONS

Biochemical Journal

Figure 3. Separation of the carboxymethylcellulose and cellobiase of the effraction of T. viride cellulase on SE-Sephadex (2) methyloellulose and a Ci-component, obtained from the fractionation on DEAE-Sephadex, which was inactive towards both cellobiose and carboxymethylcellulose and had virtually no activity towards cotton. H o w ever, when the Ci-component was recombined with either the unresolved C or appropriate mixtures of the separated cellobiase and carboxymethylcellulase, the activity towards cotton was restored completely. A l l possible recombinations of the three components were made i n the proportions in which they were originally present i n the culture filtrate. The results are shown in Table I from which the following points can be appreciated: x

(1) (2) (3) limited; cotton.

C i is virtually without activity in the absence of C . There is no synergism between the components of C . Synergism between C and the individual components of C is all three must be present to account for the activity towards x

x

t

x

Clearly the property, ascribed (2, 10) to the Ci-component of T. viride cellulase, of being able, on its own, to solubilize cotton and crystalline hydrocellulose, is lost when the C i is highly purified; this property is possessed only by mixtures of C i and C acting i n synergism. In the x



4.

SELBY

C

t


39

separation on DEAE-Sephadex reported by Mandels and Reese (10), employed without prior removal of protease and low molecular-weight carboxymethylcellulase, the relative positions of the A , and C components were similar to those of the cellobiase, carboxymethylcellulase, and Ci-components respectively (Figure 2). The major difference was that their C-component had lost less activity towards cotton on purification than had our Ci-component, so that the synergism was less marked; from their Figures 6 and 9 the enhancement of activity on recombination would seem to be about five-fold. Subsequently, Flora (2) purified the "hydrocellulase" ( C i ) from T. viride by adsorption on Avicel, followed by ion-exchange chromatography on a carboxylic acid resin. His "hydrocellulase" was still capable of causing extensive weight loss of native cotton fiber, however, and, although synergism was demonstrated with some of the other components separated on Avicel, the maximum effect obtained represented only a threefold enhancement of activity. Table I Component

Relative Cellulase Activity (% )

Original solution Ci C Cj + C

100 1 5 102

CMC-ase Cellobiase CMC-ase + cellobiase C + CMC-ase C + cellobiase C + CMC-ase + cellobiase

4

O

"*

u

-30

-10 •20

0

Figure 5. Existence of a C component in P. funiculosum cellulose, shown by isoelectric focusing. The column had a volume of 110 ml. and the pH gradient was 3-6. The Crcomponent was measured by a zone-clearance technique in a plate assay involving solubilization of cellulose suspended in agar r

The C i - and C -components from T. viride and P. funiculosum also exhibited cross-synergism; for example the C i - from P. funiculosum could act almost as effectively on cotton i n synergism with the C from T. viride as with its own C (Figure 7). This Ci-preparation has been used to confer the ability to solubilize highly ordered forms of cellulose on certain "cellulase" preparations, which, although apparently deficient i n ( V component, contain other useful polysaccharases. x

x

x

The Ci-components appear to have all the characteristics of an enzyme, although its precise function has not been identified. Both samples are probably glycoproteins with a carbohydrate: protein ratio of about 1:1, apparent molecular weights by gel-filtration of about 57,000, and isoelectric p H values of 4.05. Their behavior on exposure to adverse conditions of p H was similar (Figure 8), but the C i from P. funiculosum displayed greater thermal stability than the C i from T. viride (Figure 9 ) .



44

CELLULASES A N D THEIR APPLICATIONS

20

30 Fraction no.

Figure 6. Separation of the C component and cellobiase of P. funiculosum cellulose on Sephadex G-75 at pH 4.5 r

Although the thermal stability of the cellulase complex from T. viride is limited by that of the Ci-component, this is not so for P. funiculosum, i n which the Ci-component is the most stable and the carboxymethylcellulase the most labile. Increased thermostability of the whole cellulase complex can thus be obtained by adding T. viride carboxymethylcellulase to the P. funiculosum system. The role of the Ci-component i n the cellulase complex is still not clear. Opinions have been expressed from time to time concerning the possible identity of C i with the swelling factor discovered by Marsh and his coworkers (11,12,13,14), for both features are presumed to be involved in the early stages of attack on cotton and swelling may reasonably be considered to be a necessary pre-requisite of hydrolytic degradation. However, Youatt (35), Nisizawa, Suzuki, and Nisizawa (15) and, very recently, W o o d (34) have produced evidence supporting the original suggestion of Reese and Gilligan (23), that S-factor activity is a property of one of the C -components and not of C i . x

In his original concept, Siu (29) had suggested that rupture of hydrogen bonds might form an essential part of C i action. W h e n K i n g (6) reported fragmentation of hydrocellulose particles on exposure to a partially purified cellulase from T. viride and attributed this action to "a cellulase component not previously recognized as being involved in cellulose breakdown" it seemed that this might be a manifestation of "hydrogen bondase" action. King showed that when hydrocellulose par-


Cj Component of the Cellulase Complex


50

Relativt concentration

Figure 7. Cross synergism of the C component of P. funiculosum cellulase with its own C^-component and with the C -component of T. viride cellulase. A relative concentration of 1.0 is here used to express the concentration of components that, when recombined, produce a solubilization equivalent to that caused by a culture filtrate from P. funiculosum diluted three fold r

x

100

10

Figure 8. Effect of pH on the stability (at 40°C.) of the C components of P. funiculosum and T. viride cellulases. Each sample of Cj was kept for 4 hr. at 40°C. at the pH shown. The pH was then changed to 4.5 and residual C activity measured after addition of the C,-components r

r



46

C E L L U L A S E S A N D THEIR APPLICATIONS

FViod of htating, hour

Figure 9. Thermal stability of the C^components of P. funiculosum and T. viride cellulases at pH 4.5 tides were exposed to cellulase there was a very rapid fragmentation of each into at least 1500 smaller particles. It is known that acid hydrolysis of cotton produces crystallites that aggregate to form particles of about 5 p. diameter ( I ) , which suggested that the fragmentation step might be regarded simply as disaggregation or peptization; particle-size measurements, reported subsequently ( 9 ) , reinforced this view. The writer feels, however, that it is not possible from this evidence to suggest that similar disaggregation precedes attack on the cotton hair, for the fibrillar bundles in cotton cannot be held to resemble the simple aggregates used b y K i n g and would be less likely to disaggregate simply by adsorbing protein; the accessibility of the fibrillar bundles to protein molecules ought to be much lower. Nevertheless, similar particles, about the size of individual crystallites—ca. 60 X 600 A.—are formed when cotton is treated with whole culture filtrate. Evidence that disaggregation caused by adsorption of C i is not an essential prerequisite for the degradation of cotton comes from comparison of rate of adsorption with rate of degradation. The rate of adsorption of C -free C i on crystalline hydrocellulose is much more rapid than on cotton; i n a typical experiment the adsorption of C i from solution by a sample of hydrocellulose was virtually complete i n less than one minute, whereas an equal weight of cotton had not adsorbed a measureable amount of C i after two hours. Residual C i i n each solution was measured by adding an excess of C and measuring solubilization (28). However, when each substrate was treated with C i in the presence of excess C , hydrocellulose was solubilized only twenty times as rapidly x

x

x


Library American Chemical Society 4.

SELBY

47

C Component of the Cellulase Complex t

as cotton. The rate of adsorption of C i was unaffected by the presence of C and that of C was unaffected by the presence of C i . If, in preparation for the attack by C , the function of C i is to produce gross structural changes i n the cotton hair without release of material into solution, such changes should be accompanied b y a fall i n strength, particularly wet strength. Losses of wet strength for given small losses of weight produced by C i , C , and mixtures of C i and C , suitably diluted to reduce their activity, were compared (Figure 10) and showed that, on the contrary, rapid reduction of wet strength was more a property of C than C i . x

x

x

x

x


x

Loss in vwight %

Figure 10. Comparison of losses of wet strength and of weight caused by the prolonged action on cotton of the C and C -components of T. viride cellulase. Comparative data are given for the action of a dilute solution of the recombined components r

x

Failure to detect any function that C i alone could perform, that was not equally a property of C or of mixtures of the enzymes led to the conclusion that both enzymes must be present on the substrate simultaneously when contributing to the solubilization of highly ordered forms of cellulose. Attempts were made, therefore, to interpret the action of C i in terms of its contribution to the synergism displayed b y the cellulase complex. The function of C i is not to aid adsorption of C on cellulose, since the rates of adsorption of both carboxymethylcellulase and cellobiase are unaffected by the presence of C i . Furthermore, the low adsorption of C i x

x


48

CELLULASES AND THEIR APPLICATIONS


Figure 11.

Possible mechanism for synergistic action of C and C on cellulose microfibril

t

x

by cotton, already mentioned, is not increased in the presence of C . A further possible explanation hinges on new theories concerning the supramolecular structure of cotton (33), i n which the fiber is considered to consist of completely crystalline elementary microfibrils, each containing about 100 cellulose chains. It is reasonable to assume that the regular array of molecular chains w i l l be disturbed at intervals b y the occurrence of chain ends, as shown diagrammatically in Figure 11. The accompanying disturbance i n hydrogen bonding between chains in the vicinity of the chain end may be insufficient to enable C , acting alone, to split off soluble sugars, but when both components are present, a single bondrupture by C might allow the hydrogen bonding to be further disturbed by C i with consequent loosening of a short length of surface chain, which might then be susceptible to more extensive attack by C . Chain ends occur at intervals of about 200 glucose units along the microfibril, which would explain both the occurrence of D . P . 200 fragments and the difficulty in detecting the effect of C i or C , acting alone, by measurements of swelling, loss of weight, or loss of wet strength. This theory obviously borrows features from the "hydrogen bondase" concept (29), but contains the added notion of a very limited number of sensitive sites to explain the difficulty in detecting the influence on the cotton hair of the purified components of the cellulase system when acting alone. x

x

x

x

x

Acknowledgment I wish to thank Glaxo Laboratories, L t d . for their financial support of part of this work. Literature

Cited

(1) Battista, O. A., Smith, P. A., Ind. Eng. Chem. 5 4 (9), 20 (1962). (2) Flora, R. M . , Ph.D. Thesis, Virginia Polytechnic Institute; Ann Arbor, Mich.: University Microfilms (65-2041) (1965). (3) Gilligan, W., Reese, E. T., Can. J. Microbiol. 1, 90 (1954).


4.

SELBY

(4)

(5) (6) (7)

(8)


(9) (10)

(11) (12) (13) (14) (15) (16)

(17)

(18)

(19)

(20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33)

C

t


49

Hashimoto, Y., Nisizawa, K., "Advances in Enzymic Hydrolysis of Cellulose and Related Materials," E . T. Reese, E d . , p. 93, Pergamon Press, London, 1963. Iwasaki, T., Hayashi, K., Funatsu, M . , J. Biochem. Tokyo 55, 209 (1964). King, K. W., Biochem. Biophys. Res. Comm. 24, 295 (1966). Koaze, Y., Yamada, Y., Ezawa, K., Goi, H . , Hara, T., "Proceedings of the Fourth Symposium on Cellulase and Related Enzymes," p. 8, Cellulase Association, Osaka Univ., Japan, 1964. L i , L. H . , Flora, R. M . , King, K. W . , Arch. Biochem. Biophys. 111, 439 (1965). Liu, T. H . , King, K. W., Arch. Biochem. Biophys. 120, 462 (1967). Mandels, M . , Reese, E . T., "Developments in Industrial Mycology," Vol. 5, p. 5, American Institute of Biological Sciences, Washington, D . C., 1964. Marsh, P. B., Plant Disease Reporter 37, 71 (1953). Marsh, P. B., Bollenbacher, K., Butler, M . L., Guthrie, L. R., Text. Res. J. 23, 878 (1953). Marsh, P. B., Merola, G. V., Simpson, M . E . , Text. Res. J. 23, 831 (1953). Marsh, P. B., Merola, G. V., Bollenbacher, K., Butler, M . L., Simpson, M . E . , Plant Disease Reporter 38, 106 (1954). Nisizawa, T., Suzuki, H . , Nisizawa, K., J. Ferment. Tech. 44, 659 (1966). Niwa, T., Kawamura, K., Nisizawa, K., "Proceedings of the Fifth Symposium on Cellulases and Related Enzymes," p. 44, Cellulase Association, Osaka Univ., Japan, 1965. Niwa, T., Okada, G., Ishikawa, T., Nisizawa, K., "Proceedings of the Fourth Symposium on Cellulase and Related Enzymes," p. 1, Cellulase Association, Osaka Univ., Japan, 1964. Ogawa, K., Toyama, N . , "Proceedings of the Fourth Symposium on Cellulase and Related Enzymes," p. 17, Cellulase Association, Osaka Univ., Japan, 1964. Ogawa, K., Toyama, N . , "Proceedings of the Fifth Symposium on Cellulase and Related Enzymes," p. 85, Cellulase Association, Osaka Univ., Japan, 1965. Okada, G., Niwa, T., Suzuki, H . , Nisizawa, K., J. Ferment. Tech. 44, 682 (1966). Okada, G., Nisizawa, K., Suzuki, H . , J. Biochem. (Tokyo) 63, 591 (1968). Reese, E . T., E d . , "Advances in Enzymic Hydrolysis of Cellulose and Related Materials," Pergamon Press, London, 1963. Reese, E . T., Gilligan, W . , Text. Res. J. 24, 663 (1954). Reese, E . T., Siu, R. G. H., Levinson, H . S., J. Bacteriol. 59, 485 (1950). Selby, K., "Advances in Enzymic Hydrolysis of Cellulose and Related Materials," E. T. Reese, Ed., p. 33, Pergamon Press, London, 1963. Selby, K., Maitland, C. C., Thompson, K. V. A., Biochem. J. 88, 288 (1963). Selby, K., Maitland, C. C., Biochem. J. 94, 578 (1965). Ibid., 104, 716 (1967). Siu, R. G. H . , "Microbial Decomposition of Cellulose," Reinhold, New York, 1951. Siu, R. G. H . , "Advances in Enzymic Hydrolysis of Cellulose and Related Materials," E . T. Reese, Ed., p. 257, Pergamon Press, London, 1963. Vesterberg, O., Svensson, H., Acta Chem. Scand. 20, 820 (1966). Wakabayashi, K., Kanda, T., Nisizawa, K., J. Ferment. Tech. 44, 669 (1966). Warwicker, J. O., Jeffries, R., Colbran, R. L . , Robinson, R. N., Shirley Inst. Pam. No. 93, Cotton Silk and Man-Made Fibres Research Association, Manchester, 1966.


50 (34) (35)

CELLULASES AND THEIR APPLICATIONS Wood, T. M., Biochem. J. 109, 2 1 7 ( 1 9 6 8 ) . Youatt, G., Text. Res. J. 32, 158 (1962).

RECEIVED October 21,

1968.


Discussion T . Cayle: " B y way of confirmation of D r . Selby's hypothesis of the opening in the fibril at which point the C i can enter, we have observed an increased rate of action on pulp being mechanically disrupted during the beating operation, in the presence of cellulase derived from Aspergillus niger. Presumably, the energy and grinding action imparted during beating provide additional openings in the fibril which permit greater entry for the C i component to act, thereby shortening beating time, and in some instances reducing tear strength of the finished paper." K. Selby: "Yes, we have done a similar thing in pulp from bagasse." K. E. Eriksson: "Have you investigated the activity of your C i enzyme after purification, against higher cellodextrins like cellopentose or cellohexose?" Selby: " N o , it is obvious from what D r . K i n g was saying earlier, that this is something that ought to be done. W e haven't, in fact, done it." J. M . Leatherwood: "I would like to ask about the adsorption of C i . Is C i adsorbed to hydrocellulose in an active or in an inert form?" Selby: "In the sense that there is little hydrolytic action going on, I consider it purely as a peptization effect." Leatherwood: "In other words you consider it inert?" Selby: "Yes, a breaking of secondary bonds that are holding these particles together. This was the idea." T . K. Ghose: "I wish to refer to your data showing percent cellulase activity retained against heating period of the enzyme i n hours. You know that the p H of such a system is likely to exercise considerable influence on the rate and nature of the loss taking place. I was wondering if you had done any study on this question. What was the p H of the system you worked with?" Selby: "As to p H , we haven't done a full p H temperature optimum scan on this. A n d as you say, it does vary; the optimum p H stability w i l l vary as you modify the temperature. W e determine optimum p H for C i as about 5 0 and we have worked at about p H 4 . 5 . In fact, this is not really complete until you have said, 'Let's change the p H at 6 0 or 6 5 and see what happens/ "


4.

SELBY

C

t

51


M. Mandels: "I would like to ask what you think your A factor from Myrothecium is now? (Since in your present work you equate wet strength loss with C ) You d i d have that factor separated, as I remember it, from C ? " K. Selby: "Yes, we d i d separate the factor that was responsible for tensile loss, but we never saw any synergistic effect. This needs reexamining. But really it isn't such a good solubilizing system. I am now trying to say to anybody who has a good solubilizing enzyme, 'Let's have a look at it and see if we can get a C i out of it/ " R. G . H . Siu: "In the past, my colleagues and I have indicated that systems of the C i - C type are probably not limited to cellulase, but that they may exist wherever there is a need for the biological degradation of highly oriented polymeric materials, such as wool, walls of living cells, chitin, etc. I wish to call your attention to reactions of markedly different types where similar systems of proteins are involved (Table I ) . The second column here represents the C] ( = A ) and C ( = B ) story. T w o proteins, each relatively inactive, together they are highly active. The nature of the activity by one of the components is known; that of the other protein ( C i ) u n k n o w n . x


x

x

x

Table I.

Synergistic Action of Proteins Activity

Protein A Protein B Protein A and B

r

IIa

lib*

nr

6 0 260

1 5 102

3.0 6.0 96.0

1.5 0 100

b

(2), (b) Wood (4). = Lactose synthetase. Brew, Vanaman, and Hill (1). ° = Assoc. constants. (Antibody—Antigen) Tanford (3). = Enzymatic hydrolysis of cotton (T. viride Ci and C ) (a) Selby and Maitland

0

b

x

"In the third column is another enzyme system, lactose synthetase, an enzyme synthesizing lactose in mammary glands. Here again, two proteins are required. One ( A ) has known synthetic ability, it can synthesize 2V-acetyl lactosamine. The other ( B ) is a-lactalbumin, with no known catalytic effect in itself. Alone each is inactive; together they synthesize milk sugar. Finally, in the first column, are components A and B, derived from larger antibody molecules. The binding constants of each component with antigen is small. O n re-association of A and B, the binding constant is much higher, approaching that of the undegraded antibody. " W e hope that elucidation of the action of such systems may lead to an understanding of C i . "


52

CELLULASES A N D THEIR

Literature

APPLICATIONS

Cited


(1) Brew, K., Vanaman, T. C., Hill, R. L., Proc. Natl. Acad. Sci. 59, 491 (1968). (2) Selby, K., Maitland, C . C., Biochem. J. 104, 716 (1967). (3) Tanford, C., Accounts Chem. Res. 1, 161 (1968). (4) Wood, T. M . , Biochem. J. 109, 217 (1968).


Cellulases and Their Applications - American Chemical Society

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