Cellulases

CHAPTER

2

CELLULASES Afar y Mandel s

U.S. Army Natick Research & Development Laboratories Natick, Massachusetts

I.

INTRODUCTION

In the past few years interest in cellulases has been very strong because of world wide interest in utilizing renewable resources of biomass as a source of chemicals and liquid fuels and the consequent increased availability of financial support for research. One result is a marked increase both in original cellulase papers and review articles. Several recent conferences on biomass conversion and utilization have produced useful symposium proceedings (26,71,196,197), and there have been frequent reviews of cellulase research (41,64,116, 138,140,190,191,220) in the past two years. More specific articles have dealt with cellulase regulation and production (80,181), wood rotters (52), properties and mode of action of the cellulases (130,248,249), hemicellulases (40,176), hydrolysis and saccharification (59,131,137), industrial applications of cellulase including production of liquid fuels (10, 62,63,232), and ethanol production by fermentation to produce alternative liquid fuel (123). The above will introduce the novice to the background and older literature on cellulases. The present review will try to cover more recent advances (1979 , 1980) and the present status of research on cellulolytic enzymes relevant to process development for enzymatic saccharification. Other applications of cellulases and cellulolytic organisms such as waste disposal, improvement of extraction (or digestibility) of plant proteins or other cell contents, or in production of single cell protein are not included.

ANNUAL REPORTS ON FERMENTATION PROCESSES, VOL. 5

35

Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040305-6

36 II.

MARYMANDELS MICROBIAL STRAINS

The most active cellulases are derived from

Trichoderma

reesei, T. viride, T. lignorum (119,153), T. koningii 119), T. pseudokoningii (258) and T. longibrachiatum

(70,91, (119).

There is some confusion about nomenclature in the literature including one report that all T. viride strains have been renamed T. reesei (10). This is not so, and to date T. reesei is properly applied only to QM6a (= ATCC 1361 = IMI 192654 = DMA 769) and its descendants. This strain was isolated from a d e t e r i o r a t e d c a r t r i d g e belt in New Guinea during World War II, was originally identified as a T. viride, and was used for over thirty years in research at Natick. In 1977 it was recognized as a new species belonging to the T. longibrachiatum aggregate, but morphologically distinct from it and named Trichoderma reesei Simmons (204) in honor of Elwyn T. Reese who first recognized its unique ability to secrete very high levels of active cellulase. Three spontaneous variants of this strain exist, QM6c (= IMI 45548), QM6d and QM6e. Numerous mutants have been derived from QM6a; (a) at Natick including enhanced cellulase mutants (QM9123 (= ATCC 24449 = ATCC 28217 = NRRL 3653 = IMI 19265), QM9414 (= ATCC 26921 = IMI 192656 = DSM 769) and MCG77 [= NRRL 11236 (67,68)], cellulaseless mutants QM9136 (= ATCC 26920 = DSM 770), QM9977, QM9978, and QM9979, a white spored mutant QM9170, and a mutant lacking the typical soluble yellow pigment, QM9171; and, (b) at Rutgers including enhanced cellulase mutants NG14, C30, 110, 114, 118, and L5 (145,147,148,149,150). Enhanced cellulase and cellulaseless mutants of T. reesei have also been produced at Cetus Corporation (203), in Portugal (14) and in Finland (156,157). The QM9414 mutant has been widely used in process development research (33,45,48,72,73,92,154) and analysis of cellulase proteins (22,56,57,79,90,105,195,250) because it has been available since 1971. Many of the newer mutants have not been released for general distribution. Trichoderma cultures are available from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, and from the Northern Regional Research Laboratory (NRRL), USDA, ARS, Peoria, Illinois 61600. The Army ceased to maintain the QM culture collection at Natick in 1974, and after several years at the University of Massachusetts, Anherst, it was transferred to the USDA Laboratory in Peoria in 1979. IMI culture numbers refer to the Imperial Mycological Institute at Kew in Great Britain and DSM numbers to the Deutsche Sammlung vor Microorganismen in Gottingen, West Germany.

CELLULASES

37

Several of the Natick and Rutgers T. reesei cellulase mutants have been compared for growth, enzyme production, and properties of the cellulase enzymes (9,23,139). The differences between them and the wild strain appear to be only quantitative; i.e., the mutants produced higher levels of cellulases than the wild strain, but the composition and properties of the enzyme complex were similar for all regardless of the strain or the inducing substrate, and the relative proportions of endo and exo glucanases showed no marked changes (Table 1, Fig. 1). All of the T. reesei strains produce low levels of constitutive cellulases and these are usually increased in the mutants. However, all of the strains investigated by us must be induced for useful levels of cellulase and all are catabolite repressed by glucose and other metabolites. Zonel Zone 2 Zone 3 Zone 4

0

Figure

1.

30

60

MINUTES

90

Separation of Endo- and Exo-$-Glucanases of Trichoderma reesei by HPLC. Bissett, et al. (12). Cultures grown on 6% compressed Milled (FB) Cotton. Zone 1 - $-Glucosidase + Endo-$-Glucanase A Zone 2 - (Shaded) Exo-$-Glucanase A Zone 3 - Endo- ß- G'lueana se B Zone 4 - (Shaded) Exo-ß-Glueana se B

Ball

Ball

Ball

Ball

Lactose Milled Pulp FB Cotton

Milled Pulp FB Cotton

Lactose Milled Pulp FB Cotton

Lactose Milled Pulp FB Cotton

Soluble

3.5 17.8 20.6

1.5 11.9 13.6

9.0 14.8

27. 116. 181.

NT 133.

36. 125. 104.

1.8 7.4 10.7

3.7 9.6 16.2 14.4 21.2

10. 178. 109.

8. 76. 88.

CMC u/ml

Andreotti,

et

Two Roll

NT 31 28

NT 31

44 37 39

NT 37 34

NT NT 34

Mill

NT 69 72

NT 69

56 63 61

NT 63 66

NT NT 66

Endo-$ Exo-$ Glucanase % Total Protein

after

Cellulase

by T. reesei

0.4 5.0 10.0

0.3 0.6 5.0

Filter Paper Cellulase u/ml

production

0.9 8.7 13.6

0.9 0.8 7.4

mg/ml

Protein

on cellulase

NT - Not Tested Cultures grown in fermentors at 28°C with pH controlled not to go below 3.0 Ball Milled Pulp = 200 mesh FB Cotton = Absorbent Cotton processed forl min. (10 mil gap) on Farrel Birmingham Enzyme Units = Micromoles glucose produced per minute in standard assay Endo- and Exo-g Glucanase by HPLC analysis (22,23)

Rutgers C30

Rutgers NG14

MCG77

QM9414

Lactose Milled Pulp FB Cotton

Ball

QM6a

and substrate

Substrate 6%

Effect of strain al., 1981 (9).

Strain

Table 1.

CELLULASES

39

The degree of catabolite repression or post glucose repression is reduced for some mutants (67,147,148,149,203) and some of the Cetus mutants are reported to produce respectable levels of cellulase constitutively (203) although not equal to the yields of other strains on cellulose. Thus no totally derepressed or constitutive mutant is yet available. Cellulaseless mutants commonly do produce cellobiase indicating that this enzyme is probably under separate genetic controls. Quantitative comparisons between mutants are difficult since both yields and productivities are greatly affected by fermentation conditions and the optimum conditions may not be the same for all strains. Depending on conditions, the best Natick and Rutgers mutants give 3-20 fold the cellulase yield of the wild strain QM6a, but under proper conditions usefully high levels of cellulase can be produced even by QM6a (Table 1) (218). Trichoderma reesei cellulase is commercially available from Worthington, Freehold, New Jersey (QM6a), and from Novo Enzyme Company, Copenhagen, Denmark (QM9414). All T. reesei cellulase preparations contain other carbohydrases; xylanases, mannanases, etc.

40

MARY MANDELS

Trichoderma viride cellulases have also been widely used for process development particularly by Toyama in Japan (228) , Herr in West Germany (97,98) and workers in Russia (165,166) and for analysis of cellulase proteins (77,124,155,201,202, 250). The enzyme complex is similar to that of T. reesei in composition and activity. Improved strains of T. viride have also been developed (Fig. 2 ) . Trichoderma viride cellulase is commercially available from Miles Laboratory, Elkhart, Indiana;

1950 1955 1960 1965 1970 1975 Figure

2.

Enhancement of FPU activity in Japan's Trichoderma viride cellulase preparations. Toyama, et al. (228). Extract of Wheat Bran solid culture. ^Figures show times in min. required for complete disintegration of filter paper by a 1% cellulase solution. FPD units/mg = 30,000 .

CELLULASES

41

Enzyme Development Company, New York, NY; Vanderbilt Company, Norwalk, CT; as Maxazyme from Gist Brocades, Delft, Holland; and from Japanese sources, particularly Meicelase from Meiji Seika Kaisha in Tokyo and Onozuka cellulase from Kinki Yakult in Osaka; T. viride cellulase preparations also contain several other carbohydrases. The advantages of Trichoderma as a source of cellulase are (a) that it produces a complete cellulase with all the components required for total hydrolysis of crystalline cellulose and (b) that very high yields of cellulase protein are attainable (Table 1). There are also disadvantages such as (a) the inability of Trichoderma to metabolize lignin, (b) the low specific activity of the cellulase and (c) low levels of β-glucosidase. Therefore the search for new and possibly better sources of cellulase continues. Work has been reported on cellulases from many other fungi including Aspergillus (119), A. fumigatus (229,230), and A. niger (50,119). foetidus Aspergillus cellulases are usually high in endo-ß-glucanases and β-glucosidases but low in exo-ß-glucosidases so that they are limited in their ability to hydrolyze crystalline cellulose. Aspergillus niger cellulase is commercially available from Miles Laboratories, Elkhart, Indiana. Work has also been reported on cellulases of Botryodiploida theobromae (2 33), Eupenicillium javanicum (222), Fusarium sp (224,231), Geotrichum candidum (119,121), Pellicularia filamentosa (223), Pénicillium janthinellum (175), and Neurospora crassa (46), as well as the thermophiles Humicola insolens (94,95), Sporotrichum thermophile (= Chrysosporiurn thermophile) (30,31,32, 36,86,143), Talaromyces emersonii (65,66,142), Thermoascus aurantiacus (227) and Thielavia terrestris (= Allescheria terrestris) (206,207). Separation and characterization of cellulase enzymes, endo- and exo-ß-glucanases and ß-glucosidases and some hydrolysis of crystalline cellulose have been reported for most of the above, but extensive saccharification under realistic process conditions has not yet been reported. Total hydrolysis of crystalline cellulose by cellulases of, Fusarium solani (250,251) and Pénicillium funiculosum (251) has been demonstrated. Sadana and colleagues have worked with the basidiomycete Sclerotium rolfsii (192,193,198,199) including development of improved mutants and extensive saccharification of pure and complex cellulose materials, including rice straw and bagasse, with the enzyme complex which includes a complete cellulase and high levels of cellobiase and xylanase.

MARYMANDELS

42

Wood rotters have attracted considerable interest (52). White rotters (53) consume all wood components including lignin. Eriksson has written an excellent review (51) of the enzyme mechanisms utilized by Sporotrichum

pulverulent

urn

( = Chrysosporium lignorum (186) = Phanerochaete chrysosporium (210) (perfect stage)) in this process, and he and his colleagues continue their careful and extensive investigation of this organism (8,27,28,29,55,86). The system includes hydrolytic enzymes endo-ß-glucanases, exo-ß-glucanases, ßglucosidases, xylanases; lactonases; as well as oxidative enzymes glucose oxidase, cellobiose oxidase, cellobiose quinone oxido reductase, catalase, laccase, and peroxidase and enzymes involved in degrading ring structures in lignin monomers. This organism shows promise as a means of removing sufficient lignin to save energy in pulping processes (53,54) and for production of single cell protein from lignocellulosic wastes (47) but it has not been suggested as a source of cellulase for saccharification. Cellulaseless mutants of this and several other white rotters have been isolated and studied (53,54). The fungus Irpex lacteus (= Polyporus tulipiferae) produces a complete cellulase including endo- and exo-ß-glucanases which are synergistic with each other, and ß-glucosidases (113,114,115). Irpex lacteus cellulase (Driselase) is commercially available from Kyowa Hakko Kogyo Company, Tokyo, Japan. Brown rotters (101) probably initiate lignin degradation by a non enzymatic system involving H2O2 and Fe + + to cause rapid depolymerization with little weight loss and many show sparse growth on pure cellulose although some strains show clearing on cellulose agar plates. Cell free enzyme preparations are active on carboxymethyl cellulose but inactive on insoluble cellulose (101). Cellulases from the thermophilic actinomycetes including Streptomyces thermodiasticus (37), Thermomonospora curvata (217) and Thermomonospora fusca (37) have also been investigated. These organisms break down cellulose very rapidly during growth, but the cell free enzymes lack ß-glucosidase and show only limited hydrolysis of insoluble cellulose. The cellulase enzymes of Thermomonospora

sp (=

Thermoactinomycete)

have been thoroughly studied at the University of Pennsylvania (61,87,88,89,151,152). This organism produces thermostable endo- and exo-ß-glucanases which are strongly cellobiose inhibited, and a cell bound thermally unstable ß-glucosidase. The enzyme complex, including culture solids, will hydrolyze insoluble cellulose, and can achieve extensive hydrolysis

CELLULASES

43

of phosphoric acid swollen cellulose if periodic additions of cell solids are made to replenish ß-glucosidase. Because of the high temperature (65°) and pH (6.5) optima for this system the fungal ß-glucosidases are unsatisfactory adjuncts. Interest in cellulolytic bacteria continues despite generally low yields of extracellular cellulases. Cell free cellulases have been reported from Ruminococcus albus (254) and a cell bound cellulase preparation from Cellulomonas has been used to saccharify bagasse (35,81). However the greatest attention has been devoted to the thermophilic cellulolytic anaerobes (257) particularly Clostridum thermocellum (1,2,69, 100,107,110,124,126,196,200) and Thermoanaerobium brockii (15,126,127,256) because it is hoped they can be used as the basis of processes for direct conversion of cellulose to ethanol (240) or other solvents. The acetone:butanol:ethanol organism Clostridium acetobutylicum is not cellulolytic, but can produce an extracellular endo-ß-glucanase (3). Zymomonas mobilis is not celluloytic, but is a very efficient ethanol producer (128,185,208). The genetic engineers would like to transfer bacterial cellulase genes to these organisms.

III.

GROWTH AND ENZYME PRODUCTION

T. reesei grows readily in submerged culture on inorganic salts including ammonium as a nitrogen source and any of a wide variety of carbon sources. Other organic growth supplements are not required. Maximum specific growth rates on glucose are 0.12-0.24 g/g per hour under the optimum conditions, pH 4.0 and 35°C. Cell yields are 0.4-0*5 g/g glucose 32-35 g/mmole O2 and 10-15 g/g nitrogen consumed. Maintenance requirements are high, 15-25 mg glucose, 0.5-0.8 mmoles O2/ and 0.5-1.1 mg N per gram biomass per hour at zero specific growth rate in continuous culture (9,189,212). All available strains are inducible by cellulose, lactose, or sophorose and all are repressible by glucose and cellobiose (67,162). The natural inducer of cellulase is not known. The usual assumption is that low constitutive levels of cellulase must react with cellulose to produce a soluble molecule which enters the cell and effects induction. Cellobiose (4-0-ß-D-glucopyranosyl-D-glucose) is a likely candidate because it is a product of cellulase action and since growth

MARYMANDELS

44

on cellobiose will result in cellulase production (181,235). Cellobiose is an inducer of cellulase for Sporotrichum pul-

verulentum

(55), Sporotrichum

thermophile

(30), and Neuro-

spora crassa (46). However cellobiose is not an inducer of cellulase for Trichoderma with washed mycelium (55,213,258). It is effective in shake flask experiments only at high initial concentration (around 1%) and cellulase appears only after the cellobiose is consumed; or in fed batch experiments where cellobiose is fed at a high rate as the limiting nutrient (235). Thus the natural inducer may be a product derived from cellobiose. Sophorose (2-0-ß-D-glucopyranosyl-D-glucose) is a likely candidate since it is a powerful inducer of cellulase, active at 10~5M with washed mycelium of Γ. reesei (55,83,135,213, 214), T. viride (160,161,162), and T. pseudokoningii (258), although it is not an inducer for most other fungi or bacteria. When cellulose was hydrolyzed with T. reesei cellulase a number of reversion disaccharides including sophorose were identified in the syrups (234). Sophorose was also detected in cellobiose solutions exposed to broken cell suspensions of T. reesei grown on glycerol and containing no cellulase activity (234). With washed mycelium, sophorose induces cellulase within 1.5-2.0 hr (83,160,213). Sophorose is taken up by the mycelium and metabolized (135). Most of the cellulase appears after most of the sophorose has been taken up, but enzyme production ceases after depletion of the sophorose or separation of the mycelium from the induction medium (135,213, 258) and several pulses of sophorose are more effective than the same quantity given as a single dose (135). According to most workers, cellulase appears simultaneously in the mycelium and in the medium, and appreciable levels do not accumulate in the mycelium (Fig. 3) (83,135,160,213,235,258) although this is contrary to the findings of Berg and Pettersson for T. reesei (55) that the cellulases are initially cell bound. Sophorose induces both endo- and exo-ß-glucanases (83,213). According to Natick workers the optimum temperature (28°) and

45

CELLULASES

pH (2.8) for cellulase production on cellulose or sophorose are lower than the optima for sugar uptake and growth (9,213). Other workers have used higher pH; 4.0 (135), 5.0 (55,258), or 6.0 (83,160,161), and the Chinese group has used a temperature of 36° for induction work (258). Cellobiase in T. reesei is constitutive and intracellular (135,215,235). The constitutive level is about 0.2-0.3 international cellobiase units/mg of mycelium. Higher levels up to one unit/mg are inducible by methyl-(3-glucoside (215) . In contrast to induction of cellulase by sophorose, there is no

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HOURS

Figure

3.

Location of cellulase and uptake of sophorose during induction. Sternberg and G. Mandels (213), Trichoderma reesei QM6a, washed mycelium, 28°C, pH 2.8, 300 \lg sophorose/ml.

46

MARYMANDELS

lag between inducer uptake and cellobiase induction (Fig. 4) (107,215). In washed mycelium assays extracellular ß-glucosidase is not produced. During growth on cellulose the 3-glucosidase is initially intracellular but later most of it is extracellular. Only about one percent of the extracellular protein is ß-glucosidase, but because of the high specific activity of this enzyme there are usually about 0.20.3 cellobiase units per filter paper cellulase unit in active Trichoderma cellulase preparations. The properties of the intracellular and extracellular enzymes are not the same (Table 2) (78,107,215) but both enzymes hydrolyze all g-glucosides tested, salicin, methyl-ß-glucoside, sophorose,

1

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Uptake of inducer and induction of cellulase and $-glucosidase in Trichoderma reesei QM6a. Sternberg and G. Mandels (215). $-glucosidase induction, pH 3.0, 500 }lgmethyl-$-glucoside/ ml. O O Induced Q-glucosidase (cell bound) £ AMethyl-fi-glucoside (M$G) uptake □ OConstitutive $-glucosidase (No M$G) Cellulase (CMC ase) induction, pH 2.8, 300 \lg sophorose /ml. φ φ Induced cellulase (cell free) A A Sophorose uptake SI M Constitutive cellulase (total) (No sophorose)

47

CELLULASES

Table 2.

Cellobiase

= $-glucosidase

(3.2.1.21.)

Substrates: Cellobiose, other (3 linked glucose dimers Cellodextrins (activity increases as chain length decreases) Aryl $-glucosides (Salicin, p-nitrophenyl $-glucoside) No action on cellulose. Action:

Hydrolysis of $-glucosidic bond (1,2-1,3-1,4-1,6) dimers Retention of &-glucose configuration Transfer of glucose units to other sugars, alcohols.

Product:

Glucose

(use specific

of

procedure).

Properties: Soluble glycoprotein enzyme, can be immobilized Inhibited by glucose, gluconolactone, nojirimyein. Intracellular enzyme (107,215) T. reesei MW 98,000 opt. pH 6.5 Thermal Stability 40° 1 hr 95% loss, 60° 1 hr. 100% loss Km cellobiose 0.9-3.3 mM, sophorose, 1.4-6.7 mM. Extracellular enzyme (19,78) T. viride MW 47,000-76,000 (T. reesei 34,000) opt. pH 4.0-5.0 Thermal stability 40°-60° 1 hr no loss Km cellobiose 1.5-2.6 mM. Activity: International units = \l moles glucose > 100 cellobiase units per mg protein Can be measured in crude preparations.

per

minute

Remarks: When aryl $-glucosides are used as substrates, activity can be measured by following the release of the aglucone such as saligenin, p-nitrophenol, etc. Cellobiose is the substrate of choice for cellulase work since aryl (3glucosidases exist which do not hydrolyze cellobiose. References: Aspergillus phoenicis (4,7,24), Botryodiploida theobromae (223), Candida cacaoi (237), Clostridiumthermocellum (1), C. acetobutylicum (3), Lenzites trabea (99), Pénicillium janthinellum (175), P. funiculosum (251), Pyricularia oryzae (102), Schizophyllum commune (43), Sclerotium rolfsii (193), Sporotrichum thermophile (32, 143), S. pulverulentum (42,210), Talaromyces emersonii (142), Thermoascus aurantiacus (227), Thermomonospora sp (89), Trichoderma reesei (79,83,107,215), and T. viride (19,78,244).

48

MARYMANDELS

cellobiose, gentiobiose, etc. The uptake of sophorose or cellobiose by washed mycelium is inhibited by nojirimycin (214) thus suggesting that ß-glucosidase is involved in sophorose uptake and is located near the surface of the T. reesei cell. Several workers have found that sophorose also induces aryl ß-glucosidase (as measured on p-nitrophenyl-ß-glucoside) (83), T. viride (160), and T. pseudokoningii in T. reesei (258), but the Natick workers found that sophorose did not induce cellobiase and in fact at 10" 7 M repressed induction (by methyl-ß-glucoside) of cellobiase (Fig. 5) (214,215). So with increasing concentration sophorose is a repressor of ß-glucoside production, an inducer of cellulase, degraded by ß-glucosidase (Fig. 5) (107) and finally a repressor of cellulase production (135). It tempts one to speculate on some interesting regulatory mechanisms. — i

100

80 X c

60 S c o

>

Φ

40

a Φ

Φ >

20 Φ

100 Sophorose (/xM) 5.

Effect of sophorose concentration on its role in Enzyme production by Trichoderma reesei QM6a. Sternberg and G. Mandels (214). Qy •O Repression of $-glucosidase induction (methyl-$-glucoside inducer) Δ-~--Α induction of cellulase fry nj Sophorose as a substrate for $-glucosidase, Km 1.4 mmolar

Figure

CELLULASES

49

Yields of cellulase are lower when Trichoderma is grown on lactose than when it is grown on cellulose (9,139). However, the use of a soluble substrate has many advantages over cellulose in studying quantitative physiology. Ryu, et al. , have evaluated some of the factors involved in cellulase biosynthesis by separating growth and enzyme production in a two stage continuous culture of strain MCG77 growing on lactose. The first stage was optimized for growth with pH 4.5, temperature 32° and a high dilution rate. The second stage was optimized for enzyme production with pH 3.5, temperature 25°, and a low dilution rate. Uptake rates, yield, and maintenance factors for carbon, oxygen, and nitrogen were estimated. A maximum specific enzyme productivity of about 5 filter paper cellulase units/g biomass per hour was attained in the second stage when the specific growth rate was equal to or slightly less than zero and a maximum productivity of about 50 units per liter per hour when the dilution rate was 0.028 (188). Allen and Mortensen attained 11 filter paper cellulase units/ml in a carbon limited fed batch fermentation with T. reesei strain C30 growing on enzymatic hydrolysis sugars from cellulose. The syrup contained predominately glucose, cellobiose, and xylose plus minor components. Induction was apparently due to a reversion compound in the syrup since glucose or cellobiose alone, or an artificial syrup containing glucose, cellobiose, and xylose in equivalent concentration did not induce cellulase (6). The highest yields, 2% extracellular protein, and produc tivitie^, 150 mg enzyme protein per liter per hr. of cellulase are still attained in batch cultures on cellulose (5,9,137). Enzyme yields are proportional to initial cellulose concentration (212) with maximum yields of about 0.3 g of extracellular cellulase protein per g of cellulose consumed (9,23). Cellulose concentrations greater than 6% are difficult to stir and create problems with oxygen transfer. Temperature and pH profiling frequently result in increased yields and productivities (5,154). Indirect means of estimating biomass during growth on insoluble cellulose by rates of oxygen consumption and CO2 production have been developed (92). A mathematical model of enzyme hydrolysis and fermentation of cellulose has been attempted (168). Good yields of cellulase have been attained in continuous culture on cellulose with cell recycle and in fed batch fermentations, with cellulose as the limiting substrate, for QM6a (218) and QM9414 (73). Binder and Ghose observed that cellulose is strongly adsorbed on the mycelium of strain QM9414, and if this is prevented by placing cells

50

MARYMANDELS

and cellulose in separate mesh bags, cellulase was not produced, even if dilute enzyme was added to the system (21). Vohra, et al., reported that lignin and certain lignin components repress cellulase production in strain QM9414 (238), but this could not be duplicated at Natick (unpublished data).

IV. ENZYME PROPERTIES, ANALYSIS AND ASSAY Cellulase is not a single enzyme, bat a unique system in which several enzymes act together to carry out the hydrolysis of cellulose which no one of them, acting alone, can achieve. Many papers continue to appear on separation and characterization of the cellulase enzymes of various microorganisms. An excellent review article on this subject is available (130). New separation procedures on Concanavalin A (77,251) or cross linked cellulose (244) or by high pressure liquid chromatography, HPLC, (22,23) and techniques for recognizing individual enzymes by immunological procedures (56,163,195) have been developed. Suggestions that some of the multiplicity of components is due to proteolytic action continue to appear (76, 79,81,83,153,155,167,247,253). However, many components have been separated and well characterized as individual enzymes with different modes of action. Most of the cellulolytic fungi appear to have similar cellulase systems containing one to several g-glucosidases (Table 2), endo-ß-glucanases (Table 3) and exo-ß-glucanases (Table 4) that act synergistically to hydrolyze insoluble cellulose (Table 5). Cellobiase (Table 2) is not a cellulase and may be produced by non cellulolytic organisms. It enhances cellulase action (Fig. 6, Fig. 7) by removal of inhibitory cellobiose (24,169,219,236,237). The level of 3-glucosidase is low in Trichoderma preparations for practical saccharification due to glucose inhibition. Supplemental 3-glucosidase from another source such as Aspergillus phoenicis may be added to enhance the rate and extent of cellulose hydrolysis. Cellulase preparations from Pénicillium, Sclerotium, and Thermomonospora have higher 3-glucosidase levels. Endo-ßl,4-glueanases (Table 3) are common in filtrates from cellulolytic fungi and bacteria. Most of these preparations lack exo-3-glucanase and therefore have only limited action on crystalline cellulose. Most organisms that produce endo-3-glucanase also produce cellobiase, but the enzymes appear to be under separate controls so that either can be

51

CELLULASES

Table

3.

Endo-$l,4-glucanase ase) (3.2.1.4)

= carboxymethyl

cellulase

(CMC-

Substrates:

CMC, other soluble cellulose derivatives (low degree of substitution) "Walseth" cellulose - amorphous cellulose, dissolved, reprecipitated, never dried Cellodextrins (activity increases as chain lengh increases) Almost no activity on cellobiose Almost no activity on crystalline cellulose by purified enzyme.

Action:

Random hydrolysis of $-1,4-glucosidic gous to a amylase Retention of ß glucose configuration.

Products: Glucose, Cellodextrins

bonds,

analo-

cellobiose (transient).

Properties: Soluble enzyme, natural substrate insoluble Molecular weight 11,000-65,000 glycoproteins Optimum pH 4.8, temp. 50° (Trichoderma) Inhibited by methylcellulose, cellobiose Thermostable. Activity

:

International units = μ moles reducing sugar (as glucose per minute (CMC 50T) in crude preparations -20 units per mg protein Viscosity decrease - arbitrary units Can be measured on CMC in crude preparations.

Remarks: Purified endo-$-glueanases give a rapid fall in visto production of sugar from Walseth cosity of CMC relative cellulose. Represents about 30% of the extracellular protrin in T. reesei cultures (22,23,83). References: Clostridium thermocellum (200), Irpex lacteus (114,115) , Pénicillium funiculosum (251), Ruminococcus albus (254), Talaromyces emersonii (142), Thermoascus aurantiacus (227), Trichoderma reesei (79,83,90,201,202), T. viride (77,124,244).

52

MARYMANDELS

Table 4.

Exo-$l,4-glucanase (3.2.1.91)

- cellobiohydrolase

= CBH

Substrates: "Walseth"cellulose (acid swollen, reprecipitated) Avicel = microcrystalline cellulose Cellodextrins (activity increases as chain length increases) Almost no action on cotton by purified enzyme Limited action on CMC No action on cellobiose. Action:

Hydrolysis of $1,4-glucosidic bonds removing dimer (cellobiose) from the non-reducing end of the chain, analogous to ß amylase Inversion of ß glucosidic configuration.

Product:

Cellobiose,

α anomer

Properties

: Soluble enzyme, insoluble substrate, strongly sorbed by cellulose Molecular weight 50,000-65,000 glycoproteins Optimum pH 4.8, temp. 50°C (Trichoderma) Strongly inhibited by methylcellulose, cellobiose Less stable than endo-$-glucanases.

Activity: Cannot be directly because of synergism.

measured

in crude

ad-

preparations

Remarks: Exo-$-glucanases which remove monomer (glucose) from the non-reducing end of the cellulose chain are also known, but have been less intensively studied than the cellobiohydrolase of Trichoderma. Represents about 70% of the extracellular cellulase protein of T. reesei (22, 23,83). References: Irpex lacteus (111), Pénicillium funiculosum (251), Talaromyces emersonii (142), Thermoascus aurantiacus (227), Trichoderma koningii (91), T. reesei (56,57, 79,105), T. viride (19,84,85,244) .

53

CELLULASES

Table 5.

Cellulase = filter ase, etc.

paper cellulase

Substrates: Filter paper Cellulose powder Avieel Cotton Various wastes as received

or after

system,

avicel-

pretreatment

Action: Swelling Disintegration, fragmentation Hydrolysis of insoluble cellulose Products: Glucose, cellobiose Other sugars such as xylose are usually present in due to hydrolysis of other polysaccharides by other zymes . insoluble Properties : Soluble enzyme mixture, Optimum pH 4.8, temp 50° (Trichoderma)

digests en-

substrate

Activity:

International units = y moles reducing sugar as glucose per minute Specific activity depends on nature, concentration, and pretreatment of the cellulose substrate and on assay procedure and extent of hydrolysis but is normally less than one unit per mg protein.

Remarks: Initial rates of accessible cellulose is to zero if the cellulase values should be based sion (1 - 4%, 0.5 - 2.0

hydrolysis are rapid as the most hydrolyzed, but fall off rapidly is incomplete. Therefore unit on equal and significant convermg reducing sugar).

54

MARYMANDELS

100

ß GLUC0SIDASE ADDED—.

/

GO OC 60

BW200

o o 40h

o cc

20

J

L 1.0

5.0

10.0

20.0

FP CELLULASE UNITS/GRAM INITIAL CELLULOSE

Figure

O •

6.

Effect of enzyme:substrate ratio and added $-glucosidase on percent conversion of milled cellulose pulp (BW200) (139). Hydrolysis at pH 4.8, 50° shaken, for 48 hours. 5%-15% substrate concentration. 0.125-1.0 filter paper cellulase U/ml. O BW200 (Milled Pulp) · BW200 + $-glucosidase added to equal 1 unit/FP unit.

55

CELLULASES

100 r

CO

ß GLUCOSIDASE ADDED

60

40

>

AVICEL

20 h

1.0

5.0

10.0

20.0

FP CELLULASE UNITS/GRAM INITIAL CELLULOSE

Figure

7. Effect of enzyme : substrate ratio and added $-glucosidase on percent conversion of microcrystalline cellulose (Avicel). Hydrolysis at pH 4.8, 50° shaken, for 48 hours. 5%-15% substrate concentration. 0.125-1.0 FP cellulase U/ml. Δ Δ Avicel A ~ " Ά Avicel + $-glucosidase added to equal 1 unit/FP unit.

56

MARYMANDELS

produced in the absence of the other by use of appropriate fermentation conditions or mutants. Endo-ß-glucanase preparations usually contain 3-5 or more enzyme components which differ in physical properties such as molecular weight and isoelectric point and may also differ slightly in their mode of action on cellulose. Exo-ßl,4-glucanases (Table 4) are rare having been identified only from cellulases of a few organisms. Cellulase preparations containing exo-ß-glucanases but lacking endo-ßglucanases have not been reported. Some, but not all, endoand exo-ß-glucanases act synergistically to hydrolyze crystalline cellulose. Synergism occurs between enzymes from different fungi provided the fungal source produces a complete cellulase complex capable of extensive hydrolysis of crystalline cellulose (Table 6) (250). The cellulase of Cellvibrio gilvus does not hydrolyze crystalline cellulose even though it contains both a cellobiohydrolase and an endoß-glucanase (216). When a crude enzyme preparation shows extensive hydrolysis of crystalline cellulose, it must contain both endo- and exo-glucanases. When hydrolysis conditions or added substances affect action on crystalline cellulose (Avicel, cotton) but have little effect on action on CMC it can be inferred that the effect is on the exo-ß-glucanase. Recently synergism between the two cellobiohydrolases of T. reesei has been reported (57). The use of Ci as a synonym for exo-ßl,4-glucanase is misleading. The confusion arises because Ci was originally postulated to be the "first cellulase" which produced amorphous cellulose as a substrate for the Cx enzymes which are the endo-ßl,4-glucanases. Since cellulases which hydrolyze crystalline cellulose were postulated to contain Ci and since such cellulases were always found to contain exo-ßl,4-glucanases , it was easy to assume they were one and the same. Now it seems more likely that action by an endo-glucanase to produce free chain ends as substrates for the exo enzymes is the initial step. Prior action to decrystallize cellulose, a proposed mode of action of Ci, has not been demonstrated although it has been shown that when Trichoderma cellulase acts on cotton in the presence of the cellulase inhibitor methylcellulose, there is an increase in alkali soluble cellulose. Reese now believes that Ci may be a very random endo-ß-glucanase (177). This enzyme has never been isolated and cannot be measured in any meaningful way.

57

CELLULASES Table

6.

Synergistic effects of cellulase activity shown by combination of C\ and Cx from various fungal sources (after Wood and McCrae (250)).

Source of Ci Component Exo-ß-Glucanase Trichoderma

koningii

II

Fusarium II

II

solani II

Pénicillium

funiculosum

Source of Cx Component Endo-$-Glucanase T.

koningii

Solubilization Cotton (%) Ci

Cx

alone

alone

Nil

Nil

of Cl

&Cx 54

F. solani

II

II

79

F. solani

II

II

59

T. koningii

II

II

51

P. funiculosum

II

" It

"

45

units

of

II

II

2\ koningii

II

II

II

F. solani

II

All assays contained the same number of Cx (CMCase) activity and 200 \\g of C\ protein.

72 51

A difficult question frequently raised is "which component is limiting in practical saccharification?". All active cellulase preparations which have been analyzed contain both endoand exo-ß-glucanases. As purified components they show limited action on crystalline cellulose (Table 6). When isolated components from Trichoderma koningii were recombined, the original proportions were optimum for maximum solubilization of cotton, and increasing either component beyond this up to two fold did not increase hydrolysis (247). The stimulatory effect of cellobiase on hydrolysis of pulp or Avicel appears to reach a maximum at about 1.5 cellobiase units per filter paper cellulase unit but this would represent only about 1% of cellobiase on a protein basis in the mixture with the balance about 30% endo- and 70% exo-ß-glucanase protein (83). When optimum proportions are achieved, increasing the total cellulase will increase rate and extent of hydrolysis at least up to 25 filter paper cellulase units per gram of substrate (Fig. 6, Fig. 7). Thus any component may be limiting, but as it is increased other components will in turn become limiting. Once an active enzyme is available, the best means of increasing hydrolysis is to add more of the complete enzyme. At present, the critical limiting factor for all available cellulases is low specific activity.

58

MARYMANDELS

Stability of the cellulases of several organisms under use conditions has been investigated. In general the (3-glucosidases and endo-ß-glucanases are more stable to heat, pH extremes, and chemical inhibitors than are the exo-ß-glucanases (39,180). Inactivation of T. reesei cellulases under shaken conditions occurs because of shear stress (183) apparantly at the air-liquid interface (118). This can be alleviated by increasing enzyme concentration (182) or by addition of fluorocarbon surfactants or high molecular weight polyethylene glycols in low concentration (178),179). These act by reducing the surface excess of protein. It is most desirable that cellulase assays should be standardized, but this is difficult because cellulose is not one substrate and cellulase is not one enzyme. It seems unlikely that any single assay procedure for enzyme mixtures acting on native cellulosics can satisfy biochemists trying to understand synergism, microbiologists evaluating strains, and chemical engineers involved in process development. The use of antibiotic test discs as an easier substitute for the filter paper assay has been proposed (146). Dyed Avicel is a useful substrate for some applications such as ctutomated assays (132,158). New methodologies for recognizing active cellulase producers on agar plates continue to be sought (38, 239). In the final development of a process, enzymes must be evaluated under realistic hydrolysis conditions (139). The quantity of cellulase in a cellulase preparation can usually be estimated from the protein content as about 0.7 filter paper cellulase units per mg of protein.

V.

SACCHARIFICATION SUBSTRATE: PROPERTIES AND PRETREATMENT

The critical bottlenecks for an enzymatic process to convert cellulosic substrates to fermentable sugars and liquid fuels are (a) availability of suitable substrate, (b) pretreatment, and (c) the high enzyme requirement (49,164,211, 246). Substrate availability (171,246) is outside the scope of this review. The ideal substrate would be available in large quantities the year round, easy to collect, high in cellulose, and would require little or no pretreatment. Low cost is essential since at 40% conversion it will require about 20 kg of substrate to produce one gallon of ethanol (138,211,246). At present municipal wastes, agricultural

59

CELLULASES

residues, paper mill wastes, and in the long run, biomass such as poplar from energy plantations are the most promising substrates. Many of these will give a 40%-50% yield of fermentable sugars from enzymatic hydrolysis provided they have received a suitable pretreatment (211). The most important properties of cellulose affecting its susceptibility to enzymatic hydrolysis are crystallinity (194 ,221) because in crystalline cellulose hydrogen bonds as well as glucosidic bonds must be broken before soluble products are formed, and accessibility of the cellulose to enzymes in solution as determined by such factors as particle size, available surface, affinity for cellulase (adsorption) and admixture with impurities such as lignin (Fig. 8) (12,33, 58,59,60,62). Therefore the objectives of pretreatment are to reduce crystallinity and to increase available surface by various chemical and physical means. For enzymatic hydrolysis the aim is maximum destruction of fiber structure and interaction between cellulose molecules (12,103,134,136). This can be accomplished very well by dissolving cellulose in cadoxen, CMCS (Fe, tartrate, OH) solvent, cuprammonium, or strong acid 60 50

^ * .

30

3 O

»

20

O M

!>

10

s •s UJ

3

3

10

20

30 40 5060 1 04

O-WSSA/^OOO-Crl) ·

Figure

8.

Correlation of the extent of hydrolysis after 8 hours, XQ, as a function of specific surface area (SSA) and crystallinity index (CrI) of cellulose after various pretreatment s. Fan, et al. (60).

MARYMANDELS

60

(60,103,125,134) and then reprecipitating and washing. However, because of economic and pollution control constraints, the solvent must be recovered (4-5 grams of solvent are required to dissolve one gram of cellulose) and this would be a formidable operation. Furthermore, it is much easier to dissolve pure cellulose than to extract the cellulose in a lignocellulose material. Extraction in dilute acid with some removal of hemicellulose (16,108,109,122,172) or swelling in dilute alkali (60,82,103,226) are more economical but the product will require washing or pH adjustment before exposure to enzymes. Partial removal of lignin by white rot fungi (44,54, 186,245) is a slow process and would entail some loss or degradation of the residual carbohydrate. Steam explosion (240° C, 40-50 atm. pressure, 20-80 sec.) by the Iotech process (112,134) is economical ($25-30/ton) and very effective for hardwoods and agricultural residues, less effective for softwoods and municipal wastes (225). Ball or attritor milling (144) and compression milling (225) are effective for all substrates tested and provide a product of high bulk density permitting use of 20%-30% slurries in the saccharification reactor. Kelsey and Shafizadeh (117) have made an interesting finding that wet milling during saccharification enhances cellulase action. Other pretreatments proposed include high energy irradiation (11) and even conversion to a soluble cellulose derivative (255). The most effective and economical pretreatment will differ from one substrate to another.

VI.

HYDROLYSIS, PROCESS DEVELOPMENT

A good review on hydrolysis kinetics is available (131). Saccharification with T. reesei cellulase is generally carried out at pH 4.8, 50°C, in a stirred reactor. Even after pretreatment, a great deal of enzyme is required to saccharify cellulose if a high percent conversion is desired, because the amount of sugar produced is a function of the logarithm of the ratio of enzyme to substrate (Fig. 6, Fig. 7). To achieve a 40%-50% conversion of a 10%-30% slurry in 24-48 hours requires about 10 filter paper cellulase units per gram substrate for a susceptible substrate (Fig. 6) and about 20 filter paper units per gram for a more resistant substrate (Fig. 7). Since the specific activity of cellulase on filter paper is only about 0.6-0.7 units/mg protein the requirement

61

CELLULASES

is for 15-30 g of enzyme protein/kg substrate. One enzyme unit should produce 0.18 mg (one ymole) of reducing sugar per minute, but this is true only under the assay conditions of short time and low percent conversion. As hydrolysis proceeds the rate declines so that the enzyme efficiency (ratio of achieved conversion to that predicted by the enzyme unit value) is only 10%-20% when conversion is high (Fig. 9) (5,13, 138,139,153). The enzyme protein requirement for cellulose hydrolysis is about 100 times that for starch hydrolysis.

10

Ü

CO 120 CD z 100

o ^^ Q LU OC

au 60 40 20 0

2

4

6

8

10

12

14

16

18

20

FP CELLULASE U/g INITIAL CELLULOSE Figure

9.

Effect of enzyme ficiency (139). Fig. 6, Fig. 7. cellulase unit in "glucose."

substrate ratio on cellulase efConditions and symbols same as The theoretical yield from one 48 hours is 0.18 x 60 x 48 mg

62

MARYMANDELS

Why do cellulases have such low specific activities and efficiencies? The reasons seem to be related to the insoluble and recalcitrant (to hydrolysis) nature of cellulose and the necessity for synergistic action by several enzymes. As percent conversion increases, hydrolysis rate slows due to increasing resistance of the residual substrate, product inhibition (45,141), and enzyme inactivation (104,180). Cellulose strongly adsorbs cellulase (33,45,129) but not cellobiase (72). Separated cellulase enzymes are inactive (Table 6) so the enzymes must be adsorbed in close proximity, perhaps as a complex, for activity (173,250). The endo- and exo-a-1,4 glucanases can carry out their action on starch sequentially. This high cellulase requirement and the cost of producing the cellulase is the major economic barrier that must be lowered before enzymatic saccharification of cellulose becomes economically feasible. To date the search for new strains and mutants of existing strains of microorganisms has not turned up a cellulase with a significantly higher specific activity than Trichoderma cellulase. Pretreatments do reduce the crystallinity of cellulose and increase enzyme accessibility, but the above enzyme requirements are for pretreated substrates. The use of additives, surfactants to increase enzyme stability (178,179,180), or enzymes to remove hemicelluloses and improve accessibility of the cellulose to the cellulases (72,205) is favorable. Cellulases acting on insoluble cellulose are strongly inhibited by cellobiose (18,91,141). Cellobiose is less inhibitory and may even be an activator when carboxymethyl cellulose is the substrate (120). ß-glucosidases are strongly inhibited by glucose (24,78,83,98). Therefore the addition of supplemental ß-glucosidase, an enzyme which does not act on cellulose, alleviates product inhibition and can reduce the cellulase requirement for any desired conversion by 50% (Fig. 6, Fig. 7) (43,169). Fermentations to produce Aspergillus ß-glucosidase in high yield have been developed (4,7). Because of the high specific activity of this enzyme, it is much less expensive, per unit, to produce than is cellulase. Since the substrate is soluble ß-glucosidase can be immobilized. This increases its stability to pH and temperature extremes and permits reuse (24,133,219,236). An alternative way to alleviate product inhibition is to remove the hydrolysis products by use of membrane reactors (96,98,111), or by simultaneous saccharification and yeast fermentation (SSF) to ethanol (170). Ethanol is also inhibitory to the cellulase system but less so than glucose or cellobiose (169).

CELLULASES

63

Another means of using cellulase more effectively would be to recover and reuse the enzyme from the hydrolysis reactor. Cellulase immobilization in radiation polymerized microspheres has been reported (252) but the interactions between an immobilized enzyme and an insoluble substrate must be severely limited. Enzymes remaining in the syrups after hydrolysis can be tannin or solvent precipitated or recovered by adsorption on fresh substrate (25,33,34) or by ultrafiltration (228). Stabilization of enzymes becomes more critical when recovery is contemplated. Eventually it may be possible to elute enzymes from hydrolysis residues if means are found to reduce the affinity of the enzyme for its substrate. This might even increase specific activity by permitting greater mobility of enzyme components. Castanon and Wilke (34) found that the addition of 0.1% Tween 80 to the hydrolysis reactor resulted in a larger fraction of enzyme remaining in solution throughout hydrolysis, increased enzyme efficiency, and improved enzyme recovery from syrups. The effect of the Tween 80 is to reduce the surface inactivation which occurs during rapid agitation of the reaction mixture (178,179). Production of ethanol from hydrolysis syrups (74,75) and various means of lowering the cost of ethanol production (93) are being investigated. Most hydrolysis syrups contain high levels of xylose that are not fermentable by the usual strains of Saccharomyces yeasts used for ethanol production. Rosenberg has prepared a review of fermentation of pentose sugars by microorganisms (187). Another approach is to use the enzyme glucose isomerase (= xylose isomerase) to convert xylose to xylulose (D-threo-pentulose) which is fermentable by Saccharomyces yeasts (76,241,242,243). Normally the equilibrium for this reaction is at only 16% xylulose, but the addition of borate at 0.02 M shifts the equilibrium to 60%-65% xylulose (209). In the United States process development has been high technology and the economic projections are based on very large plants producing typically about 25 x 106 gallons of ethanol per year. Toyama, et al. (228), have proposed a simple small scale technology suitable for a farmer, or small cooperative, saccharifying their own agricultural residues such as rice straw or bagasse which have been chopped and delignified by a simple procedure such as boiling in 1% NaOH. Enzyme would be produced commercially, but by a solid state fermentation growing Trichoderma viride in trays of moistened bran or other cellulosic material for several days. The resulting "Koji" can be dried and used as is, or the enzyme can

MARYMANDELS

64

be extracted in water or buffer. The delignified substrate is acidified with 0.5% citric acid and packed in "Sho Chu" jars (Fig. 10). The low pH and exclusion of oxygen by the narrow necked jars and lack of stirring retard undesired contaminations. This process can yield concentrated sugar solutions (Table 7) to use as a substitute for cane or beet juice or for production of food yeast or ethanol. The enzyme can be recovered from the sugar solution for reuse by precipitation with 0.5% tannic acid (MW 1700). The enzyme tannic acid complex is centrifuged out and regenerated with a 1% polyethylene glycol (MW 4000) solution which solubilizes the enzyme. The insoluble tannic acid polyethylene glycol complex can be removed by centrifugation (228).

Figure

10.

Simplified resources

saccharification with cellulase.

process of Toyama, et al.

cellulosic (228).

65

CELLULASES Table

7.

Thick sugar solution attainable with parations using cellulosic wastes.

Substrate

Rice

Enzyme

Cone.

Incubation Sugar (%)

pre-

96 hr Decom. (%)

3

21.89

65.7

1

15.92

47.8

3

21.06

63.2

1

14.90

44.7

Straw

Bagasse

Powdered substrates were delignified by boiling for 3 hr. Substrate cone. 25%, pH 5.0, 45°C. lase CEP-233. Toyama, et al. (228). VII.

cellulase

with 1% NaOH Enzyme: Meice-

ECONOMICS, OUTLOOK

The practical objective of most of the work reviewed here is utilization of biomass as a renewable resource, particularly for the production of ethanol as a liquid fuel. A number of economic analyses of the enzyme process have been made (48,49,106,164,211,246) and it would appear that the process is not yet economically viable without a very cheap substrate and some subsidy. Nevertheless researchers in the field remain optimistic because the long range outlook is for increasing scarcity and high cost of petroleum, and because the starch to alcohol process has very serious drawbacks including increased food prices, reduced grain exports, increased soil erosion, and an unfavorable energy balance. If in the future less alcohol is produced from grain so that our increased distillery capacity is underutilized then the pressure for a cellulose conversion process will be very strong. The study of microbial cellulases continues to attract an increasing group of enthusiastic investigators because of the intellectual challenge of understanding the interactions of a multiple enzyme system with its complex insoluble substrate, and the practical challenge of reducing the enzyme requirement so that an abundant waste can be economically converted to a more convenient and usable product. The critical research areas are (a) enzyme production including the search for new strains, improvement of existing strains, greater understanding of controls over enzyme production and secretion; the role

66

MARYMANDELS

of proteases in enzyme secretion, stability, and multiplicity, and further optimization of fermentation to produce enzymes and (b) hydrolysis including a search for better pretreatments that are efficient and economical, enzyme kinetics and the nature of synergism, and enzyme stabilization and desorption from hydrolysis residues to permit greater enzyme recovery and reuse. Such research will be interesting to carry out and may have valuable commercial and social implications.

ACKNOWLEDGMENTS

I am grateful to the many colleagues who have provided me with publications and data, often in advance of formal publication, to Elwyn T. Reese, Gabriel R. Mandels, and C. Patrick Dunne for assistance in the preparation of this manuscript and to the Department of Defense and Department of Energy for financial support.

REFERENCES

1. Ait, N., Creuzet, N., and Cattanés, N.

Biophys.

Res.

Commun. 90, 537.

2. Ait, N., Creuzet, N., and Forget, P.

Microbiol.

113, 399.

3. Allcock, E. R., and Woods, D. R.

Environ.

Microbiol.

41, 539.

Biochem.

(1979). (1979).

(1981).

J.

Appl.

Gen. and

4. Allen, A. L., and Andreotii, R. E. (In press). Proceedings International Symposium on Wood and Pulping Chemistry, Stockholm, Sweden. June 1981. 5. Allen, A. L., and Blodgett, C. R. (In press, 1981). Proceedings AIChE Symposium, Boston, MA, August 1979. 6. Allen, A. L., and Mortensen, R. E. (submitted 1981).

Biotechnol.

Bioeng.

7. Allen, A., and Sternberg, D. (1980). in "Biotechnology in Energy Production and Conservation" (R. D. Scott, e d . ) ,

Biotechnol.

Bioeng.

Symp. No. 10, p. 189.

8. Ander, P., Hatakka, A., and Eriksson, K. E.

Microbiol.

125, 189.

(1980).

Arch.

9. Andreotti, R., Medeiros, J., Roche, C., and Mandels, M. (In press, 1981). Proceedings Second Bioconversion Symposium, IIT Delhi, March 1980.

CELLULASES

67

10. Aunstrup, K. , (1978). In "Annual Reports on Fermentation Processes" (D. Perlman and G. Tsao, eds.) Vol. 2, p. 125. 11. Beardmore, D. H. , Fan, L. T., and Lee, Y. H. (1980). Biotechnology Letters 2, 435. 12. Beardmore, D. H., Lee, Y. H., and Fan, L. T. (1979). Proc. Ann. Biochem. Eng. Symp. 9th, 66. 13. Beja da Costa, M. (1980). Cienc. Biol. (Coimbra) 5, 59. 14. Beja da Costa, M., and Van Uden, N. (1980). Biotechnol. Bioeng. 22, 2429. 15. Ben-Bassat, A., Lamed, R., and Zeikus, J. G. (1981). J. Bacteriol. 146, 192. 16. Ben-Ghedalia, D., and Miron, J. (1981). Biotechnol. Bioeng. 23, 823. 17. Berg, B., and Pettersson, G. (1977). J. Appl. Bacteriology 42, 65. 18. Berghem, L. E. R., and Pettersson, L. G. (1973). Eur. J. Biochem. 37, 21. 19. Berghem, L. E. R., and Pettersson, L. G. (1974). Eur. J. Biochem. 46, 295. 20. Berghem, L. E. R., and Pettersson, L. G. (1975). Eur. J. Biochem. 53, 55. 21. Binder, A., and Ghose, T. K. (1978). Biotechnol. Bioeng. 20, 1187. 22. Bissett, F. H. (1979). J. Chromatog. 178, 515. 23. Bissett, F. H., Andreotti, R. E., and Mandels, M. (In press, 1981). Proceedings Second Bioconversion Symposium, IIT Delhi, March 1980. 24. Bissett, F., and Sternberg, D. (1978). Appl. Environ. Microbiol. 35, 750. 25. Blotkamp, P. J., and Emert, G. H. (2 Sept. 1980). U. S. Patent 4,220,721 (Chem. Abstr. 93:236977). 26. Brown, R. D., Jr., and Jurasek, L. (eds.). (1979). Adv. in Chem. Ser. 181. American Chem. Soc., Washington, DC, p. 399. 27. Buswell, J. A., Ander, P., Pettersson, B., and Eriksson, 103, 98. K. E. (1979). FEBS Letters 28. Buswell, J. A., and Eriksson, K. E. (1979). FEBS Letters 104, 258. 29. Buswell, J. A., Hamp, S., and Eriksson, K. E. (1979). FEBS Letters 108, 229. 30. Canevascini, G., Coudray, M. R., Rey, J. P., Southgate, R. J. G., and Meier, H. (1979). J. Gen. Microbiol. 110, 291. 31. Canevascini, G., Fuchin, M., and Trachsel, S. (Submitted 1981). 32. Canevascini, G., and Meyer, H. P. (1979). Exp. Mycol. 3, 203.

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MARYMANDELS

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