A Perspective on Cellulase Research - Advances in Chemistry (ACS

1 A Perspective on Cellulase Research R. G .H.SIU

Downloaded by PENNSYLVANIA STATE UNIV on June 8, 2012 | http://pubs.acs.org Publication Date: June 1, 1969 | doi: 10.1021/ba-1969-0095.ch001

4428 Albemarle St., N. W., Washington, D. C. 20016

This symposium on cellulases and their applications represents a most worthwhile contribution. Twenty-five papers are presented from 20 laboratories. These groups represent the best talents in the field. They come from six countries distributed over the northern as well as the southern hemispheres, the eastern as well as the western. The papers are grouped into four sessions. The first emphasizes the nature and components of the cellulase system, including reports on glucotransferase and exoglucanases, the constitutive behavior of cellulases, enzyme-substrate relationships, genic influences, and sites of action. The second session focuses on the question of enzymic accessibility of cellulose, release of cellulase by microorganisms, and association of lignin and cellulose in wood. The third session probes the digestibility and nutritional potential of wood and straw. The fourth session assesses the production of cellulase and commercial applications. Co-chairmen George Hajny and Elwyn T. Reese have organized an excellent conference. The Bridge My personal participation in this symposium was most gratifying. It has been quite a number of years since I last fondled a mass of Trichoderma viride mycelia or looked a cellulase preparation in the eye. As I caught up with the literature I was greatly impressed with the major progress that has been made in this field. The achievements have been remarkable, and all the research workers are worthy of commendation. This excellent progress paves the way for even greater future developments. From my distant view, this symposium appears as a bridge. It is a bridge from the past state of hypotheses and potentiality to the future state of theories and applications. Looking backward, we see the extensive investigations on agronomy and animal husbandry going back over eight decades with such illustrious names as Hoppe-Seyler, Omelianski, and 1 In Cellulases and Their Applications; Hajny, G., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1969.


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CELLULASES A N D THEIR APPLICATIONS

Waksman. W e recognize the great effort of two decades ago on the identification of cellulolytic microorganisms by such eminent taxonomists as White, Weston, and Raper. There is also the report of Selliere about 60 years ago on the first observation of an in vitro cellulase activity, but it was not until the last 20 years, and particularly the past 10, when cellulase attracted the concentrated attention of a group of very capable microbiologists and biochemists employing the most sophisticated scientific instruments. A new generation of investigators has emerged—Reese, Toyama, King, Nisizawa, Selby, Cowling, Erickson, Mandels, Gascoigne, and others. As a result of their efforts, the underbrush has been cleared away, and we are now beginning to see future salients in much clearer focus. The terms cellulolytic enzymes and cellulases now enjoy a workable definition. This has been made possible through the separation of the components of the cellulolytic complex, as well as some clarification of their mechanism of action. New viscometric, isoelectric focusing, gel filtration, and other techniques for the determination of the enzymic components have been devised. Detailed observations on the molecular sites of hydrolytic reactions, the susceptibility of the enzymic reactions to metallic ions, proteolytic enzymes, and other chemicals also provided considerable insight into the nature of cellulase. This summarizes the activities of the past. The papers i n this volume bring us up to date as to the current state of the art. To glimpse the future we must ask: Where do we go from here? What is at the other end of this cellulase research bridge? The other end of the bridge appears to be multiterminal. It leads to a number of fascinating possibilities. Four of these warrant particular attention. T w o are direct extensions of current work; the other two are more speculative i n nature.

Mechanism of Cellulose Action The first and most obvious avenue of progress of course is the continuation of excellent work. More sophisticated biochemical techniques w i l l be developed. This in turn w i l l lead to further clarification of the mechanism of cellulase activity. Particular advances w i l l be made i n our knowledge of the exoglucanases, the initial reactions on the surfaces of cellulosic substrates, and the nature of the polysaccharide-protein complexes formed as intermediary transitory stages. M u c h of this is covered by the first eight chapters in this volume. Hence, further discussion is not warranted.

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

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New Source of Food The second avenue of progress involves the development of new food processing techniques and new sources of food. There is no problem of greater importance to our rapidly expanding human population, expecting to reach the seven billion mark before the year 2000, than i n creased food supply. M u c h research is being conducted to offset the threat of food imbalance. Agriculture is being improved through new varieties and cultivation techniques. Farming the sea is making some headway with exploitation of fish meal. Extraction of proteins from leaves and grasses, followed by precipitation into a form suitable for human consumption is receiving serious consideration. So are o i l seed cake from the protein rich residues of coconut, sesame, peanut, soybean, and cottonseed being proposed for wide use as food supplements. Microorganisms as protein sources are being studied. Even purely synthetic foods have been suggested. It is interesting to note that the general scientific and managerial public has rarely mentioned cellulose in a serious vein as a possible source of foodstuffs. Here is a substance which is the most abundant carbohydrate i n the plant kingdom. It is the most plentiful of all naturally occurring compounds and comprises at least a third of all vegetable matter. Here is this tremendous store of carbohydrates to be exploited— if only it can be converted into a digestible form through practical means. W e know that horse, cow, goat, rabbit, termite, cockroach, fungi, bacteria, and protozoa are able to digest cellulose to varying degrees— that cellulase can increase the digestibility of wood for animals—that cellulase can hydrolyze cellulose to produce concentrations of glucose as high as 30%—that cellulase can be prepared i n fairly large quantities— and finally that a group of very talented and dedicated scientists are working diligently and fruitfully i n the area. In the face of all this, I cannot help but take it as a serious proposition that cellulose w i l l one day constitute a very important source of food for human consumption either directly as a processed form or indirectly as converted into simpler carbohydrates or proteins b y microorganisms and animals. The pressure of the growing population of the future and the millions of hungry people of today place considerable significance upon this potentiality. Incidentally, the actual number of senior capable investigators being supported for their work on cellulase approximates only a score, with another score or so of graduate students or laboratory assistants. Contrast this with the thousands of agronomists, who are doing excellent work no doubt, but nevertheless struggling to expand the arable soils to more than 4 % of the earth's surface. The only conclusion I can draw from this comparison is that an order of magnitude of added support to the


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area of cellulase investigations is called for if we are to let social relevancy be a major criterion of investment.


There are many ancillary uses of cellulase i n food processing. I need only mention a few examples involving the extraction of essential oils and flavoring materials, tenderizing fruits, clarification of juices, and improvement of filterability of vanilla extracts. Many other examples are given i n this volume. New Chemical Concepts The third avenue of advancement represents sheer speculation on my part. I am not making any recommendations to any one to follow up on my comments, but I would predict that when the proper time comes, some one w i l l do it because he w i l l have no alternative. This person w i l l have accumulated a formidable mass of failure data, which w i l l force him to challenge the current methods of picturing the structure of chemical molecules. H e w i l l find that his observations involving the cellulasecellulose reactions cannot be understood i n the light of the conventional notions. The literature involving enzymic reactions is based on solutions. The cellulase-cellulose system involves an interface between an enzyme in solution and presumably an insoluble substrate surface. The postulation of a C i step i n the cellulase chain of reactions sharpens our focus. It seems to me that as we bear down on the C i activity at that point of the cellulolytic process, we w i l l find ourselves face to face with some very fundamental relationships of such concepts as hydrogen-bonds and crystalline structure as sites of enzymic action. Should this occur, there is no telling as to the greatness of the resulting contributions to the field of chemistry and physics. It may well turn out to be among the most significant of the century.

mux The fourth avenue of research is even more conjectural. Here we see the cellulase-cellulose system as a physico-chemical model of flux. It depicts the transition between the structure and the structureless, between the reserve solids and the mobile solutes. Such transitions exist throughout nature. If our conventional thinking about the reversibility of enzymic reactions in general holds i n the case of the cellulase-cellulose system, it would follow that the system would not only hydrolyze cellulose into glucose but also synthesize cellulose from glucose. This would relate



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the cellulase-cellulose reactions with those of photosynthesis i n a most intimate way. Botanical biochemists have been looking at the starchglucose transformation as the primary surge tank for the glucose of photosynthesis. It seems to me that the cellulose-glucose transformation should be given equal attention. A host of research questions immediately come to the surface: what compound in the cellulose-glucose series of reactions is the primary reserve for metabolic purposes? Is it one of the oligosaccharides or cellobiose, rather than cellulose? What are the crossover compounds from the cellulose-glucose chain to the other metabolic cycles in plants? C a n lignin be considered the plant's regulator i n protecting the dissolution of the celulosic structure against further metabolic transformation? Is it possible to find lignin-free celluloses, which readily undergo the cellulose-glucose reactions in both directions? A n d so on. A l l of these are fascinating research speculations. But I would like to single out one of them for a few extra words. It concerns the dimensions of structure. A t some point i n the glucose-to-cellulose transition, structure appears i n the elementary form of crystalline cellulose, fibrils, and the like. Are there enzymes which guide the elaboration of physical structure i n contrast to the formation of chemical compounds? O r must we seek a new kind of responsible agents? If we extend the implications of our current ambiguous definition of the C i step of cellulase, we must conclude that either enzymes are responsible for governing the physical structure of cells and organisms through the whole gamut of "Van-derWaalase," "hydrogen-bondase," "left-handed-twistase," "crystallinase," and so on, or that the C i step represents a new class of non-enzymic physico-chemical entity, which works i n conjunction with enzymes i n living tissues, or that the current notions of the role and nature of enzymes must be drastically extended. In any case, the deeper study of the cellulase-cellulose system puts us face to face with a most important facet of a most challenging problem of science and philosophy—i.e., putting form into the formless. The Wild Flower I would like to emphasize again the social importance of the current research on cellulases and their applications. It is an interesting observation that this field of cellulase research began to gain support because of attempts to prevent the destruction of textile materials. Yet it is now showing its greatest potential contributions in the direction of augmenting the world's food supply and i n opening up new salients i n fundamental thinking of physics, chemistry, and philosophy. As I gaze intently into


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the mysteries of the cellulase-cellulose complex, I cannot help but hear the beautiful words of W i l l i a m Blake's most fitting stanza: To see the world in a grain of sand, And a heaven in a wild flower, Hold infinity in the palm of your hand, And eternity in an hour.


RECEIVED October 14, 1 9 6 8 .


A Perspective on Cellulase Research - Advances in Chemistry (ACS

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