MINIREVIEW CELLULOSE AND CELLULASES: THOUGHT, FOOD FOR THE MICHAEL

Department

P. COUCHLAN

of Biochemistry,

and

MICHAEL

University

(Rrceined

21 July

College.

FOOD FOR FUTURE? A. FOLAN Galway.

Ireland

1978)

Abstract-Cellulose, a vast and renewable resource and a potential source of food much underutilized. This review deals with the molecule itself. the uses to which and, cellulases, the enzymes that may convert potential to reality.

INTRODUCTION Goti said, “Let the earth produce vegetation: seed-hearing plants, and fruit trees bearing fruit with their seed inside, on the earth”. And so it was. 7he earth produced ljegetation: plants bearing seed in their seceral kinds. God saw that it was good. Evening came and morning came: the third day. God said, “See, I gice you all the seed-bearing plants that are upon the whole earth, and all the trees with seed-bearing fruit; this shall be your food. To all wild bewts, all birds of heacen and all living reptiles on the earth 1 give all the foliage of plants for food”. And so it was. God saw all he had made, and indeed it wa.s t’erq’ good. Evening came and morning came: the sixth day.

These quotations from Genesis tell us, among other things, of the creation of cellulose and that, as this most abundant of organic molecules exemplifies, there has been from the outset an appreciation of the relationship between structure and function. Man was instructed to use cellulose as indeed he has done. However. in a world of increasing population and dwindling resources he may have to rely on it more heavily.

is currently very it might be put

metric tons, is cellulose. Moreover, plant growth provides an additional 4 x 10” metric tons annually. Fortunately, a renewable resource. Structure Cellulose is a linear polymer of D-glucose residues. of which there may be as many as 15,000, joined by b-~-(1-4) linkages. The incidence of hydrogen bonding between OH, of one residue and 0, of the next is very high giving great tensile strength and rigidity to the molecule (Ward & Seib, 1970; Winterburn, 1974). By hydrophobic contacts and by intermolecular hydrogen bonds, chains of cellulose associate to form the microfibrils characteristic of all vegetable fibres. Various proposals regarding the structure and conformation of these components have been reviewed by Cowling (1975). Cowling & Brown (I 969), Rowland (1975) and Cowling & Kirk (1976) have also considered the structure of cellulose in the native state in the context of its hydrolysis. One should note that the cellulose microfibrils in ciao are associated with other molecules such as lignin and hemicelluloses. Thus “pretreatment” may be necessary if hydrolysis is to be effective (see below). Synthesis

1. CELLULOSE Occurrence

Cellulose, the major cell wall constituent of plants accounts for about SO’, of the carbon on earth, The content in plant tissues ranges from 209/O in some grasses to an average of 45% of dry weight in wood and to over 90% in cotton fibre (Stephens & Heichel,

Our knowledge of cellulose formation is based largely on investigations using insoluble enzyme preparations from the bacterium, Acetobacter uplinum, and particulate enzyme preparations from a variety of higher plants (Ward & Seib. 1970; Hassid, 1970). To summarise, one may consider synthesis to take place as follows:

I GTP +

B-D-ghCOpyranOsyl

I p~rapho’phor~‘a~e,

GDP-o-glucose

+ PPi

phosphate 1975). It is also found, but to a much lesser extent, in the animal and mineral kingdoms (Ward & Seib, 1970). Estimates of the total amount in existence vary considerably. Stephens 8~ Heichel (1975), for example,

n(GDP-D-glucose)

+ acceptor

The “activated” glucose monomers are then transferred one at a time to the growing polysaccharide by a transferase enzyme, GDP-D-glucose (l--t4)+~glucan p-D-4-glucosyl transferase.

-+ Acceptor-

(I-4)-b-D-ghKosy)

(cellulose)

n + n(GDP) 1

L

estimate the terrestrial biomass to be 1.8 x IO” metric tons: of this an average of 40”/,. or 7.2 x IO” R.(

IO’2 _A

A cytoplasmic membrane-bound lipid may act as an intermediate carrier of the glucose residues in such 103

104

MICHAEL P. COUGHLAN

synthesis. Thus, glucose is transferred from GDP-glucose to the lipid on the cytoplasmic membrane. The lipid then carries the glucose outside the cell and delivers it to the site of polymerization of cellulose The “empty” lipid then reenters the cell to continue the cycle.

Ceilulose is. as might be expected, the principal source of energy in ruminant nutrition due to its fermentation in the rumenoreticulum by cellulolytic microflora. It is also found in building materials, textiles and paper. Moreover. various derivatives of cellulose are used in the manufacture of such diverse products as explosives. lacquers, adhesives, inks, plastics, film. rayon and in the sizing of leather and paper. Much of crop and forest wastes are degraded in sirrr by cellulase-producing microorganisms thereby contributing signjficantly to soil fertility. Such facts would have to be borne in mind when considering prospective additional uses for cellulose.

In considering prospective uses for cellulose one must think. initially at any rate. of using “waste” materials. Fortunately, in this context. the amount of cellulosic waste generated each year runs into many hundreds of millions of tons. Wood pulp and paper mill wastes are the most attractive sources of substrate because of localized concentration and because the material has been subjected to “pretreatment” (Stone. 1976; Spano. 1976). However forest. agricultural. industrial and municipal wastes, animal manures and residues from fruit and vegetable processing should all be considered seriously (Inman. 1975; Bassham, 1975; Diaz. 1975; Stephens & Heichel, 1975; Stone. 1976: Sloneker. 1976: Cooper. 1976). Stephens & Heichel (1975) has discussed the energetics of producing agricultural cellulose while Humphrey (1975). Inman (1975) and Dunlap (1975) have considered the economics of utilizing this substrate as a chemical or energy resource.

and

MKHAFI. A.

FOLAS

2. C’ELLLILASES

lmportuncc Cellulases are essential to the growth and ripening of fruit. By catalyzing the decay of forest and agricultural residues they help to maintain the carbon cycle and soil fertility. Moreover, because of the celiulases produced by symbiotic Inicroorganisms. livestock can utilize cellulose as a foodstuff. The damage done by cellulolytic organisms to wood, wood-products and textiles each year is enormous. in the L1.S. in 1924 the cost was $800 million white by 1929. 25 million gal of preservative were being used to protect timber alone (Gascoigne & Gascoigne, 1960). Much of the impetus for the current interest in cellulases came from military anxiety at the rate at which clothing and equipment decayed in the jungles of the South Pacific (Augustine, 1976). Moreover, the most powerful cellulolytic organism known, Trichoriurmcl I%?&, was first isolated from a rotted cartridge belt found in New Guinea (Augustine. 1976). However, man has the ability to turn adverse situations to his own advantage. Consequently many groups of investigators seek to increase our understanding of these enzymes.

These enzymes arc produced by bacteria. fungi, actinomycetes, protozoa. insects and molluscs. Indeed the list of cellulases (from diverse sources) whose properties had been examined was already extensive by 1960, occupying sixteen pages of the book by Gascoigne & Gascoigne. The U.S. Army Natick Development Ccntre has over 14,000 cellulolytic microorganisms in its mycology arsenal (Augustjne, 1976). Although cellulases are widespread. the fungal enzymes offer the best possibilities for commercial exploitation (Mandels & Weber. 1969). These organisms grow readily on simple media into which they secrete the enzymes in question. thereby facilitating isolation. However, while many fungat species readily degrade amorphous regions of cellulose or its soluble derivatives few produce high levels of enzyme capable of extensively hydrolyzing the crystalline substrate in vitro (see Mandels & Weber. 1969: Mandels. 1975 and references therein).

.4citl t’s eri-_>,rnir i~~~r~~l~sjs

Thr twzym~ “c~mplt~r”

Much of the prospective uses of cellulase will its degradation by cellulase enzymes. reqttire Although hydrolysis of this substrate by acid may be more complete, that catalyzed by cellutase is the more efficient; one molecule of enzyme at 50 C doing the work of 10” molecules of acid under similar conditions (Reese. 1956). Since 1956 “better” cellulases have been discovered and this with pretreatment of the substrate allows of greater degradation, The enzymic process also scores in that it specifically hydrolyzes p-( 1-4) linkages whereas acid attacks all types of glycosidic linkage and thus all types of polysaccharides. Moreover. at the temperatures and acid concentrations needed to effect hydrolysis of the less accessible regions of the substrate decomposition of glucose can ensue (Goldstein. 1976), products toxic to subsequent fermentation may be formed and expensive acid resistant hardware is required.

Almost 30yr ago Reese ef uf. (1950) explained latter observations as follows: crystalline cellulose

-

C,

amorphous cellulose

-

the

C, soluble products

The hypothetical non-hydrolytic C, fraction was thought to cause disaggregation of cellulose chains in the native material thereby allowing of hydrolysis of the products by the C, enzymes. The latter enzymes are produced by all cellulolytic organisms, Ci and C, by relatively few. C, and C, components have since been isolated from certain fungal culture filtrates (King & Vessal. 1969; Leatherwood, 1969; Mandels & Weber. 1969: Selby, 1969). Neither component alone causes significant degradation of crystalline cellulose. However. together they effect extensive hydrolysis of such substrates. Moreover, cross-synergism

Cellulose and cellulascs

is observed between the C, component of one fungal species and the C, component of another (e.g. Wood. 1975). More recently highly purified C, material has been isolated and its identity with the hydrolytic enzyme, cxo-p-1, 4-glucanase, demonstrated (Nisizawa ef ul.. 1972; Pettersson rr al.. 1972; Wood, 1972; Berghem & Pettersson, 1973; Shikata & Nisizawa, 1975; Wood, 1975). While these findings have not met with universal acceptance (see e.g. Reese, 1976), the Ci-C, hypothesis has of necessity been refined (e.g. Wood & hlccrae, 1978). Thus hydrolysis of native cellulose is thought to result from synergistic action of endoand exo-glucanases which form multienzyme complexes on the surface of the substrate molecule. One may envisage the endo enzymes opening glycosidic linkages at random thereby providing “ends” to be attacked by the exo enzymes. In the absence of the exo-glucanases the cleaved glycosidic links would rapidly reform due to the highly-ordered nature of the substrate. In the absence of the endo-glucanases, “ends” for exo attack would not be available. T/w component

enzymes

105

Assay procedures used include measurement of loss in weight of insoluble substrate, decrease in tensile strength of libres, change in turbidity of cellulose solutions, release of reducing end-groups and of glucose from soluble and insoluble material, and decrease in viscosity of solutions of cellulose derivatives (Eriksson, 1969; Mandels & Weber, 1969). The latter is a particularly sensitive test for endoglucanase action since a few random breaks will cause a marked alteration in chain length. Comparison of the rate at which reducing groups (or glucose) are released with that of decrease in viscosity can be used to distinguish between endo- and exogfucanases and to examine the action patterns of various endoglucanases (Wood & McCrae, 1978; Shoemaker & Brown, 197Xa). Mandels rr nf. (1976) have discussed the complexity of measuring cellulase action and of the confusion that has arisen because of the various “units” of activity used. They recommend adoption of the “filter unit value, it paper assay” and the corresponding being simple, reproducible and predictive of practical saccharifying ability. Huang (1975) has considered the kinetics of cellulose-cellulase systems. These are also complicated by the multiplicity of enzymes involved. the structural polymorphism of the substrate and the fact that the reaction products, cellobiose and glucose. are potent inhibitors of enzyme action.

By the combined actions of the component enzymes. insoluble cellulose is hydrolyzed to soluble glucose. (ri The 1, 4-fi-D-glucan 4-glucanohydrolases (endoI. 4+r+glucanases; EC 3.2.1.4) preferentially hydroFroductia~ of ceiiuiases lyze internal glycosidic linkages of cellulosic subThe laboratory scale production of cellulase. an instrates. Shoemaker & Brown (1978a,b) and Wood AL ducible system, has been reviewed by Mandels & McCrae (1978) have isolated and characterized 4 difWeber (1969) and by Sternberg (1976). Cellobiose, ferent endoglucanases from culture filtrates of T. produced by the action of the small amount of “conriride and T. koninyii. respectively. These are distinct stitutive” enzyme, is thought to be the physiological enzymes, rather than isoenzymes, differing in their inducer. At high concentrations, however, cellobiose molecular weights, amino acid and carbohydrate actually represses cellulase synthesis as indeed does composition and specific activities with various subglucose. Consequently, for best yields of enzyme one .StKltCX uses celluloses that are not too readily hydrolyzed. (ii) The 1.4fi-D-glucan cellobiohydrolases (exoIn Japan, these enzymes are produced in good yield cellobiohydrolases; EC 3.2.1.91) preferentially cleave by a koji process. However, subsequent isolation is cellobiose units from the non-reducing ends of cellumore complicated than that for submerged cultures. lost chains. Gum & Brown (1977) have purified and The latter process is also the more economical (Reese. characterized 4 electrophoretically distinct exoglu1976). canases from culture filtrates of T. ciride. Unlike the For much of the investigative work on cellulase endoglucanases, these would appear to be “differenproduction, relatively pure sources of cellulose have tially glycosylated” forms of the same polypeptide. been used in growth media. However, with an eye (Iii) /&glucosidase (cellobiase; EC 3.2.1.21) cleaves to practical application production of the enzyme on cellobiose units produced by the endo- and exoglusuch sources as bagasse (Srinivasan & Han, 1969) canases to give glucose, waste paper (Mandels et al.. 1974) and straw (Peitersen. 1975) has been demonstrated. For commercial Purification and assa) processes large amounts of enzyme would be All of the usual protein purification procedures required. With this in mind Nystrom & Allen (1976) have been applied to the concentration and isolation investigated the mechanics and economics of pilot of cellulases (Eriksson, 1969). Refinement of technique plant scale production. continues. Recently. Halliwell & Griffin (1978) separated the indjvidual components of the cellulase system of T. ~oningji by exploiting differences in tenacity of adsorption to the insoluble substrate. Much of the practical applications of these enzymes A variety of celluloses, cellulose derivatives and will depend on the availability of large quantities of cello-oligosaccharides have been used for the reactive cellulosic materials. Accordingly, the extent measurement of enzyme activity (Eriksson, 1969; of saccharification of a large variety of possible subMandels & Weber, 1969). Carboxymethyl-cellulose is strates has been investigated as have the details of usually used in determining endoglucanase activity the saccharification processes involved (Srinivasan & whereas hydrolysis of cotton, the most resistant subHan, 1969; Ghose & Kostick, 1969; Toyama & strate, is the best indicator of the complete cellulase Ogawa, 1972, 1975: Mandels et al., 1974; Toyama. system. 1976; Andren et al., 1976a,b).

106

MI(XAI.I. P. C'OIIGHLAN and

The susceptibility of such substrate to enzymatic hydrolysis depends on particle size (surface area), degree of crystallinity and extent of lignification. Various physical and chemical pretreatments. including ball-milling or swelling in alkali. can increase, in some cases markedly. the extent of degradation (Millett ut ul., 1975. 1976; Nystrom. 1975). However, such procedures may add considerably to overall operational costs. With regard to cost analyses of enzymatic saccharification processes. the figures quoted by Brandt (1975). Wilke & Mitra (1975) and Wilke t’r ul. (1976) make interesting reading. Toyama (1976) points out the fact that while sugar may be produced from cellulosic wastes using expensive commercial cellulasc preparations. a more economical approach would be to grow cellulase-producing organisms on such wastes and achieve the same result. Elimination of the fibre fraction of urban wastes by enzymic means may be considered economically viable pt’r \c. In such cases the resultant production of glucose is a welcome bonus. The technological details of producing and utilizing cellulases in commerical processes have been documented (Pathak & Chose, 1973: Wilke & Mitra. 1975; Nystrom & Andren, 1976).

MICHAEL A. FOLAU

Table I. Current and potential

Food pro~.~.\in

Cellulose and cellulases: food for thought, food for the future?

MINIREVIEW CELLULOSE AND CELLULASES: THOUGHT, FOOD FOR THE MICHAEL Department P. COUCHLAN of Biochemistry, and MICHAEL University (Rrceined 21...
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