Jun 2 6 1984

REGULATION OF CELLULASE ACTIVITY AND SYNTHESIS IN CLOSTRIDIUM THEIRMOCELLUM

ERIC ARTHUR JOHNSON

B.S., University of California, Davis (1976) M.S., University of California, Davis (1978)

Submitted to the Department of Nutrition and Food Science in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

at the

MASSACHUSETTS INSTITUTE OF TECHNOLOGY

November, 1983

c Eric Arthur Johnson 1983

The author hereby grants to M.I.T. permission to reproduce and to distribute copies of this thesis document in whole or in part.

Signature of Author: Department of Nutrition and Food Science, November 21, 1983

Certified by: Thesis Supervisor

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The images contained in this document are of the best quality available. This doctoral thesis has been examined by a Committee of the Department of Nutrition and Food Science as follows:

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Professor A. L. Demain Thesis Advisor

Professor B. Magasanik

Professor A. J. Sinskey _ U -2-

REGULATION OF CELLULASE ACTIVITY AND SYNTHESIS IN CLOSTRIDIUM THERMOCELLUM

ERIC ARTHUR JOHNSON

Submitted to the Department of Nutrition and Food Science on November 21, 1983 in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Applied Microbiology

ABSTRACT

True cellulase activity was demonstrated in cell-free broths from C. thermocellum. Enzyme preparations were highly active on complex and crystalline cellulosic substrates, provided they were supplemented with a sulfhydryl reducing agent and calcium. Under these conditions, low concentrations (0.6 mg/ ml) of cotton, Avicel and filter paper were all extensively solubilized at rates comparable with the cellulase from Trich- oderma reesei but with fifty times less protein in the incubation. Cellobiose was the predominant saccharification product from Avicel.

Cellulase activity was found to be inhibited by cellobiose and inactivated by sulfhydryl reagents and iron chelators. Cello- biose strongly inhibited the C. thermocellum cellulase when the substrate was Avicel but was only mildly inhibitory to the digestion of amorphous cellulose. Cellobiose inhibition was relieved by the addition of 3-glucosidase. Analogues of cellobiose including salicin, lactose, and arbutin mildly inhibited cellulase activity.

The crude dialyzed cellulase was inactivated by incubation in a low concentration (0.2-0.4 mM) of dithiothreitol (DTT). This was caused by oxidation of the low DTT concentration in air to

form H 2 0 2 , which in turn oxidized cellulase sulfhydryl groups. Activity loss was prevented by exclusion of air, or by the addition of catalase, EDTA, or an increased concentration (10 mM) of DTT. Crude cellulase from C. thermocellum was strongly inhibited by sulfhydryl reagents including o-iodosobenzoate (IB), N-ethylmaleimide (NEM), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMB) and copper (Cu). These inhibitions were prevented by 10 mM dithiothreitol. Even in the protective environment of a high DTT concentration, cellulase was inactivated by certain apolar chelating agents including o-phenanthroline and bipyridyl, such inactivation being pre- ventable by the prior incubation of the chelator with a mixture -3-

of Fe ++ and Fe +++ These data suggest that the thermophilic clostridia-l cellulase, unlike the enzyme from aerobic fungi, contains essential sulfhydryl groups and is stimulated by iron. The component of the cellulase susceptible to sulfhydryl inactivation appears to be an enzyme participating in the breakdown of crystalline cellulose (which I assume to be exo-3 (1+4)-glucanase) since the enzyme hydrolyzing amorphous cellulose (which I assume to be endo-3-(1-4)-glucanase) was unaffected by oxidation or thiol reagents.

A minimal chemically defined medium was developed for C. thermocellum. The growth factors required are biotin, pyridoxamine, vitamin B 1 2 , and p-aminobenzoic acid. This medium was used to study the regulation of cellulase formation. It was found that the synthesis of extracellular cellulase is carefully regulated in C. thermocellum. The specific titer (units cellulase activity per g cell) varied more than 100 fold depending on the carbon substrate present in the medium. Cellulase was produced in highest specific titers on cellulose. It was also formed on the cellulose derivatives cellobiose and glucose, and on the sugars fructose and sorbitol, which are not derived from cellulose, indicating that the cellulase system in C. thermocellum is prcduced constitutively. The specific titer of cellulase was increased when the cells were slowed in their formation of cellular energy. This condition was imposed during growth on insoluble cellulose, resulting in limitation of cellobiose, or during adaptation to fructose or sorbitol, which were slowly assim- ilated. Starvation for carbon did not promote cellulase synthesis. Although very high specific titers were detected during the growth lag on fructose and sorbitol, these values declined sharply as the culture gradually adapted, and even- tually reached a specific titer lower than observed on cellobiose. Cells growing on fructose underwent a shift in their pyruvate metabolism and lactic acid production declined to low levels. This resulted in increased oxidative decarboxylation of pyruvate accompanied by an increased ATP yield, and a decline in cellulase synthesis. Cellulase formation was reestablished by inhibitors that shifted pyruvate metabolism towards lactate production or by uncouplers that dissipated the pH gradient across the cell membrane, thereby lowering the energy level in the cells. These results support the conclusion that extracellular cellulase formation is regulated by catabolite repressionin C. thermocellum.

Thesis Supervisor: Dr. Arnold L. Demain Title: Professor of Industrial Microbiology -4-

ACKNOWLEDGMENTS

I would like to thank the many individuals who have gen- erously contributed to this study. I owe a special thanks to

Arnold Demain for his cooperation and support, to Daniel Wang for his leadership, and to Charles Cooney and Anthony Sinskey for their encouragement and guidance. I am grateful to Boris

Magasanik for his interest, suggestions and enthusiasm during this work.

I thank Geoff Halliwell and Irwin Hollander for their collaboration, and Arthur Smith, Frederique Bouchot, Mary

Whitmer, Cristan Orrego, Cindy Allen, Mitsuji Sakajoh, Cindy

Tolman, Sue Groh, Herve Cellard and Beatriz Mendez for participation in various experiments.

I am grateful to W. H. Orme-Johnson, Edmund Lin, and

Elwyn Reese for their valuable suggestions.

I express thanks to Catherine Duong, Gerald Sanchez,

Mary-Louise Piret, Dan Gold, Jon Dordick and Nadine Solomon

for their special contributions, and Ruth Ayers for her expert

assistance.

I acknowledge National Distillers Co., NSF, Eastman Kodak

Co., Department of Energy and Archer Daniels Midland for fin-

ancial support.

Finally, I warmly thank Mary Whitmer for her valuable

help and friendship, and my parents for their everlasting en-

couragement. -5-

TABLE OF CONTENTS

Page

Title Page ...... 1

Abstract ...... 2

Acknowledgments ...... 4

Table of Contents ...... 5

List of Figures ...... 7

List of Tables ...... 10

1. Introduction ...... 12

A. General ...... 12 B. Historical ...... 15 C. Physiological Properties of C. thermocellum ...... 21

1. General Properties ...... 21 2. Carbon Substrate Utilization and Metabolism .. 22 3. Energy Metabolism and Endproduct Formation in C. thermocellum ...... 26

D. Properties of C. thermocellum Cellulase ...... 29 E. Regulation of Cellulase Synthesis in Fungi and in C. thermocellum ...... 38 F. Regulation of Cellulase Synthesis in C. thermocellum ...... 39 G. Selection of Mutants Affected in Extracellular En- zymes ...... 40

2. Experimental Procedures ...... 43

A. Bacteria ...... 43 B. Cultivation of Bacteria ...... 43 C. Determination of C. thermocellum Nutritional Re- quirements ...... 44 D. Source and Preparation of Cellulase ...... 45 E. Measurement of Cellulase Activity ...... 47 F. CM-Cellulase Activity (endo-1,4- -D-glucanase, E. C . 3.2.1.4) ...... 48 G. Units of True Cellulase Activity ---...... 48 H. Analysis of Cellulolytic End Products ...... 50 I. Analysis of Fermentation End Products ...... 50 J. Determination of Phosphate Uptake by Cells ...... 51 K. Assay of ATP Concentration in Cells --...... 51 -6-

Page

L. Assay of Hydrogenase Activity ...... 52 M. Gel Electrophoresis ...... 52 N. Purification of Cellulase ...... 53 0. Chemicals ...... 54

3. Results ...... 55

A. Ca + and Sulfhydryl Reducing Compounds as Require- ments of the Cellulase System of C. thermocellum ...... 55 B. Oxidative Inactivation of C. thermocellum Cellu- lase: Evidence for Essential Sulfhydryls .... 66 C. Effect of Chelating Agents on Cellulase Activity: Evidence that the C. thermocellum Cellulase Requires Iron for Activity ...... 76 D. End Products of Avicel Saccharification ...... 79 E. Inhibition of Cellulase Activity by End Products of Cellulolysis ...... 83 F. Partial Purification of Cellulase ...... 86 G. Construction of a Defined Medium for C. thermocellum ...... 102 H. Control of Cellulase Synthesis in C. thermocellum 103

4. Discussion ...... 135

5. Recommendations for Future Research ...... 145a

6. References ...... 146 -7-

LIST OF FIGURES

Figure No Title Page

1 Biochemical Pathways of Sugar Catabolism in C. thermocellum ...... 23

2 Model for Cellulase Digestion of Insoluble Cel- lulose ...... 32

3 A. First Order Rates of Avicel Solubilization (Measured at O.D. 660 nm) by Varying Concentra- tions of Extracellular Protein. B. Rate of Avicel Solubilization as a Function of Protein Concentration ...... 49

4 Clear Zones Produced by C. thermocellum After 8 Days Growth on Compression Milled Corn Stover (A), Avicel (B), or Amorphous Cellulose (C) ... 56

5 Influence of DTT Concentration on Avicel Hy- drolysis by Clostridium thermocellum Cellulase 58

6 Influence of Ca 2+ on Avicel Hydrolysis by Clos- tridium thermocellum Cellulase ...... 59

7 Inhibition of C. thermocellum Cellulase by EDTA and Its Reversal by Calcium ...... 60

8 Solubilization of Native and Derived Celluloses by Cellulase of Trichoderma reesei QM 9414 and Clostridium thermocellum ...... 62

9 Hydrolysis of Cotton and Avicel under Optimal Conditions by the Cellulases of Trichoderma reesei RUT C-30 (Tr) and Clostridium thermocellum (Ct.) ...... - 64

10 Comparison of C. thermocellum and Trichoderma reesei QM 9414 Cellulase Activities on Phos- phoric Acid-Swollen Avicel and Microcrystalline Avicel. -...... 65

11 Influence of Dithiothreitol Concentration on the Activity of C. thermocellum Cellulase ...... 67

12 Influence of Anaerobic and Aerobic Atmospheres on Inhibition of Cellulase by 0.4 mM DTT or 0.04 mM Hydrogen Peroxide --...... 69 -8-

Figure No Title Page

13 Inhibition of Cellulase by Hydrogen Peroxide or Low DTT in Air ...... 70

14 The Effect of Catalase and Superoxide Dismutase (SOD) on the Inhibition of Cellulase by Low DTT and Air ...... 71

15 Solubilization of 3 g/l Avicel by 7 ig/ml Dial- yzed Extracellular Protein in an Aerobic or Anaerobic (90% N 2:5% CO 2:5% H2) Atmosphere .... 81

16 Product Formation (Cellobiose,0 ; Glucose,O ) During Saccharification of Avicel by C. thermocellum Dialyzed Culture Broth ...... 82

17 Inhibition of Cellulase Activity in Untreated Culture Broths of C. thermocellum and T. reesei by Cellobiose (A) or Glucose (B) ...... 84

18 Inhibition of C. thermocellum Cellulase by Cel- lobiose (o) or Glucose (o) on Phosphoric Acid- Swollen Avicel ...... 85

19 Relief of Cellobiose Inhibition of Cellulase Activity by 3-Glucosidase from Aspergillus phoenicis ...... 88

20 Visible and Ultraviolet Adsorption Spectrum of Dialyzed Cellulase in 20 mrM Tris, pH 8.8 ...... 91

21 DEAE-Sepharose Chromatography of C. thermocellum Extracellular Protein --...... 92

22 Gel Filtration of Dialyzed, Total Extracellular Protein on Ultrogel ACA 22 ...... 94

23 SDS-Polyacrylamide Gel Electrophoretic (SDS- PAGE) Patterns (7% Acrylamide Gel) of C. thermocellum Extracellular Proteins Purified by DEAE- Sepharose and Ultrogel ACA 22 Chromatography .. 95

24 SDS-PAGE of Purified Cellulase Proteins ...... 96

25 Ion-Exchange HPLC Chromatography of Dialyzed, Extracellular Proteins ...... 98

26 Ion-Exchange HPLC Chromatography of Dialyzed, Extracellular Proteins ...... 99 -9-

Figure No Title Page

27 SDS-PAGE of Crude Cellulase from C. thermocellum Grown in Different Carbon Sources and Partially Purified by HPLC ...... 100

28 SDS-PAGE of Fractions Obtained from Preparative HPLC (Lanes A-E) ...... 101

29 Growth and Cellulase Formation in Cellobiose or Fructose Defined Medium ...... 109

30 Cellulase Production (units) as a Function of Dry Cell Weight (mg) During the Lag in Fructose or During Exponential Growth in Cellobiose .... 111

31 Effect of the Addition of Cellobiose on Cellu- lase Formation During the Growth Lag on Fruc- tose ...... 112

32 Growth of C. thermocellum on Cellobiose or Avi- cel ...... 117

33 Transport of Inorganic Phosphate and Formation of ApH by C. thermocellum ...... 120

34 Effect of Serial Transfer on the Specific Titer of Cellulase by C. thermocellum Culture Broths 123

35 Cellulase Synthesis by Cells Adapted to Fruc- tose (Figure _A), Isolated on MJ-Avicel Agar, Picked to Cellobiose Broth for One Transfer, and Reinoculated to Fructose ...... 125 -10-

LIST OF TABLES

Table No. Title Page

1 Isolation of Cellulose Decomposing Microorgan- isms ...... 17

2 Effect of Hydroxyl Radical Scavengers on Oxi- dation of Cellulase by H 20 2 or Low DTT ...... 72

3 Inhibition of Cellulase by Sulfhydryl Reagents and Copper and Prevention by 10 mM DTT ...... 74

4 CM-Cellulase Activity is Not Inhibited by Oxi- dation or Sulfhydryl Reagents ...... 75

5 Effect of Chelating Agents on Cellulase Activ- ity Under Anaerobic, Reduced Conditions ...... 77

6 Reversal of o-Phenanthroline (OP) Inhibition of Cellulase by Prior Chelation of OP with Metals ...... 78

7 Influence of -Glucosidase on Cellulase Hydrol- ysis by C. thermocellum Cellulase ...... 87

8 Inhibition of C. thermocellum Cellulase by Var- ious Carbohydrates ...... 89

9 Vitamin Requirements of C. thermocellum ATCC 27405 ...... 104

10 Composition of CM3, GS and MJ Media ...... 105

11 Formation of Ethanol, Acetic Acid, Lactic Acid, and Avicel Hydrolyzing Activity in MJ and GS-2 Media ...... 106

12 Cellulase Synthesis by Cells Previously Grown in Cellobiose when Transferred as A Small In- oculum (1% v/v) to Different Carbon Sources .. 108

13 Cellulose Synthesis by C. thermocellum Grown on Cellobiose, and Inoculated (1% v/v) to Cel- lobiose or Avicel Media. Cultures were Grown for 60 h ...... 114

14 Growth and Cellulase Formation by Cells Adap- ted to Different Carbon Sources ...... 115 -11-

Table No. Title Page

15 Formation of ATP, End Products and Cellulase During Lags on Fructose and Glucose ...... 118

16 Difference in Product Formation by Cellobiose- or Fructose-Adapted Cells in Cellobiose and Fructose, Respectively ...... 127

17 ATP Levels in C. thermocellum Grown on Soluble Carbon Sources ...... 129

18 Influence of Gas Atmosphere on Growth of Fruc- tose-Adapted Cells ...... 130

19 Derepression of Cellulase Synthesis in Fructose- Adapted Cells by Inhibitors of Pyruvate Decar- boxylation ...... 131

20 Changes in Cellulase Synthesis from Treatments which Affect ATP Accumulation and Formation of ApH ...... 133

21 Effect of Cyclic Guanosine Nucleotides on Cel- lulase Synthesis in Cellobiose Medium ...... --- 134 -12-

1. INTRODUCTION

A. General

The thermophilic anaerobe Clostridium thermocellum is readily isolated from decaying cellulose in a wide variety of habitats including soils, manures, composts, intestinal contents of animals, and marine and fresh water muds (6, 50,

152, 249). Waksman and Skinner (240) suggested that the thermophilic cellulolytic anaerobes, typified by C. thermocellum,

"stand in a group by themselves", and are characterized by their vigorous and rapid fermentation of cellulose. The significance of C. thermocellum in the global degradation of cellulose is not known, but it almost certainly plays a minor role in the recycling of this abundant resource. The primary degradation of cellulose and other plant tissues occurs aerobically through the action of fungi, and is especially prominent in several ascomycetes and imperfect fungi including the genera

Aspergillus, Chaetomium, Fusarium and Trichoderma. The initial aerobic attack rapidly utilizes the amorphous forms of cellulose and leaves as residual material the highly crystalline and lignified forms for digestion by other microorganisms including

C. thermocellum.

A true cellulase system is characterized by its ability to saccharify native cellulose (e.g. cotton fibers) over long -13-

periods of time (84). Cellulase is a complex enzyme system, composed of endo- and exo-3-(1-4)-glucanase enzymes, which act synergistically to degrade insoluble cellulose to cellobiose.

The nature of the processes involved in the anaerobic decomposition of cellulose differs greatly from that found in the presence of oxygen (232, 233, 237, 254). A diversity of microorganisms including protozoa, fungi, actinomycetes, myxo- bacters and aerobic unicellular bacteria thrive on cellulose under aerobic conditions. In the absence of oxygen, cellulose decomposition occurs solely through the activities of bacteria, especially by members of the genus Clostridium. The aerobes are generally versatile, capable of growing on a large number of organic substrates. On the other hand, the clostridia and

soil cytophagas are highly specialized organisms capable of utilizing few if any carbon sources other than cellulose or its

sugar products (50, 213).

The anaerobic fermentation of cellulose is believed (51,

254, 258) to be a cooperative process, carried out by interact-

ing populations of bacteria. The primary hydrolytic decomposers

characteristically produce hydrogen gas and acidic end-products which inhibit further growth unless the end-products are removed

by methanogens and sulfate reducers. The reduced yields of cel-

lular energy in the absence of oxygen requires that cellulose be efficiently digested to support the microbial populations, and

provides a selective pressure for the evolution of an active

cellulase system. -14-

Although the anaerobic bacteria grow very rapidly on crystalline cellulose (136, 242), true cellulase activity (i.e., the ability to completely saccharify native cellulose such as cotton; 82-84), has only been detected in the broths of ascomycetous fungi, especially species of Trichoderma (83, 187, 144).

The anaerobic bacteria are thought to secrete little true cellulase. Eriksson has proposed (55, 56) that oxygen is involved directly in the depolymerization of lignin and cellulose, and in the coupling of their degradations. Furthermore, many rot fungi are known (61, 72, 127) to produce hydrogen peroxide as they enter the stationary phase of growth. H 2 0 2 may in turn react with iron to form oxygen radicals active in the depolymerization of lignin (61, 72) and cellulose (56, 127). Aerobic organisms have developed mechanisms to protect themselves from the reactivity of 02' and have evolved to exploit its high ox- idizing ability. Anaerobes do not have the benefit of being able to use 02 for enzymatic purposes or as an electron accep- tor during growth. Thus, they must use a non-oxidative mechanism for the degradation of plant tissues, e.g. lignin and cellulose, and their inefficient energy metabolism requires that they have efficient degradative enzymes to provide themselves with ample sugar for growth.

The ability of C. thermocellum to grow rapidly on cellulose, and its role in decaying partially attacked cellulose, suggest that it synthesizes an extracellular cellulase system that can efficiently saccharify crystalline cellulose. This -14a-

study was designed to elucidate the biochemical requirements for its activity, and the physiological factors controlling its synthesis. -15-

B. Historical

An understanding of the microbial decomposition of cellulose has developed from investigations on the associative and antagonistic interactions of soil microorganisms, and the biological transformations that they carry out in their natural environment. Winogradsky (251, 235), Omeliansky (173), Waksman

(232, 236, 240) and others recognized that the dead residues of plants are particularly favorable habitats for many microbes.

These soil residues are gradually decomposed by successions of microorganisms (88, 238) first being attacked by sugar fungi

(e.g. zygomycetes), then by ascomycetes and imperfect fungi and finally by bacteria and actinomycetes, which complete the trans- formation into humus (234), which is itself very important for the renewed growth of plants.

The connection between the activities of microorganisms and the decay of cellulose was first established by Omeliansky, the student of Winogradsky. In 1895, he (173) used an enrichment medium with cellulose as the only source of carbon to iso- late a culture of mesophilic anaerobic bacteria that fermented cellulose. Omeliansky found that the mixture of gases produced during fermentation consisted of hydrogen and methane, the two gases being produced by different microorganisms. Heating the dung or mud inoculum destroyed the capacity to produce methane, and the hydrogen-producing bacterium could be obtained free from -16-

the methanogen. It was found to produce terminal endospores, and to cling to cellulose fibers, but he could not maintain it in pure culture.

Omeliansky also studied the rotting of cellulose in manures and composts, which reach temperatures greater than

60*C during decay due to the metabolic activities of microbes

(231). He noticed that the gas composition changed with depth, consisting of methane and hydrogen and completely lacking oxygen at the lower depths of the pile. In 1899, MacFayden and

Blaxall (140) isolated thermophilic bacteria from manure which vigorously fermented cellulose at 65*C.

Following these initial observations, many microbiolo- gists isolated different groups of fungi and bacteria that had the capacity to digest cellulose (Table 1). The existence of various aerobic bacteria capable of degrading was demonstrated in 1904 by Van Iterson and substantiated by Kellerman and his associates (120-122), who with relative ease isolated single colonies that cleared cellulose suspended in agar. The main organism responsible for the decomposition had a gliding motility, and was later identified as Spirochaeta cytophaga (now known as

Sporocytophaga) by Hutchinson and Clayton (110). The ease in purifying the aerobic decomposers, as opposed to the extreme difficulty in isolating the anaerobic cellulose digesters, led

Kellerman to challenge the findings of Omeliansky with respect to the involvement of anaerobic bacteria in cellulose decompo- Table 1

Isolation of Cellulose Decomposing Microorganisms

Generic Name Group (G+C, Molar %) Morphology and Physiology Ecology Cellulolytic Species Comments References

An:erobic Clostridium Rods, usually motile. Spore- Soil,mud, C. thermocellum Vigorous cellulose fer- Omelianski, 1895-1897; cited Bacteria (23-43) former. Mesophilic to ther- gut,ma- C. cellobioparum menters. In Waksman and Skinner, mophilic. nure, 1926. Viljoen et al., 1926. composts Hungate, 1944.

Ruminococcus G+ cocci. Cellobiose pre- rumen R. albus Cellulolytic ruminococci Sijpesteijn, 1949. Bryant et very limited in al., 1958. flungate, 1963. (39. 8-41.4) ferred source, CU 2 and H 2 H. flavefaciens tneir produced. Major non-gas- sugar fermenting eous products are lower abilities. fatty acids.

Bacteroides G- rods. Product mixtures rumen B. succinogenes Cultures show CO 2 up- Hungate, 1950. Bryant and (40-55) of acids including succinic, take in fermentation Doetsch, 1954. formic, lactic, propionic. of cellulose or cello- Non-spore forming. Re- biose. quire p-aminobenzoic and biotin.

Gliding Bac- Sporocytophaga G- rods, motile by gliding. Soil. S. myxococcoides Vigorous aerobic cellu- Hutchinson and Clayton, 1919. teria k Spirochaeta Strict aerobe, respiratory. lose fermenter. Long Stanier, 1940. Dubos, 1928. (aerobic) cytophaga) Only uses cellulose, cello- adaptation to glucose. (36) biose, glucose as C and energy sources. Forms resting stage (microcyst).

Cytophaga Rods, aerobic. Acetate, Soil. C. hutchinsonii Winogradsky, 1929. Fuller (33-42) prdpionate and succinate and Norman, 1943. are end products.

continued... Table 1 (continued)

Generic Name Group (G+C, Molar %) Morphology and Physiology Ecology Cellulolytic Species Comments References

Pseudomonads Pseudomonas Short rods; strict aerobes; Soil, P. fluorescens Probably not strong Winogradsky, 1929. (58-70) Gram-, mesophiles. fresh- var. cellulosa fermenters of cellu- (Cellfailicula) water, Cellvibrio fulvus lose. (Cellvibrio) marine

Actinomrycetes Streyto.ices Aerial mycelium; highly ox-- Soil S. thermoviolaceus Many actinomyc2tes have Waksman, 1919. McBeth and related (69-75) idative: acid generally not a limited capacity to et al., 1913. organisms produced from glucose. attack cellulose.

Cellulomonas Irregular rods; respiratory; Soil. C. flavigena Relationship to 02 is Winogradsky, 1929. (72-73) acid produced from glu- U. uda not clear, most strains Kellerman et al., 1913. cose. Temp. opt. is -3300 give significant yet re- Clark, 1951 C. Coryneform bacteria. duced growth on glucose in absence of oxygen. Thermomono- Facultative aerobe; opt. Fresh T. curvata Henssen, 1957 spora growth 4t-550 C manure, straw, manure compost Nocardia Aerial mycelium; aerobe Soil N. cellulans Metcalf and Brown, 1957 (2) and manure.

continued... Table 1 (continued)

Generic Name Group (G+C. Molar %) Morphology and Physiology Ecology Cellulolytic Species Comments References

Fungi-Ascomy- Fusarium Oxidative metabolism. Pene- Soil. F. solani Although some lower De Bary, 1886. Appel, cetes Trjcioderma trating hyphae. T. viride fungi weakly degrade 1907. Waksman, 1916. C 'haetomium cellulose (e.g. Sapro- Scales, F.M., 1915. Penicillium legnia) most of the Waksman and Skinner, 1926. Verticillium Fungi which secrete Fergus, 1969. Cephalo- potent cellulases are sporium Ascomycetes and Im- perfects. Important In initial, aerobic attack of cellulose. The thermophilic molds show greater cellulolytic ability than thermophilic actinomycetes. -20-

sition. Omeliansky was also challenged by Winogradsky, his professor, who believed cellulose decomposition to be an aerobic process and to occur by an oxidative mechanism (236), e.g. by the formation of uronic acids. The anaerobic, mixed-culture fermentation of cellulose was extremely vigorous, however, and

Pringsheim (186), Kroulik (129), Khouvine (125), and others con- vincingly demonstrated its occurrence. They too, however, failed to purify the bacterium responsible for the degradation.

Khouvine (125) isolated an anaerobe, Bacillus cellulosae dis- solvens, from human feces, but could not obtain single colonies on solid medium. Viljoen et al. (229) in 1926 isolated a thermophilic cellulose digester from manure, which they named Clos- tridium thermocellum, but this organism lost its cellulolytic capacity when subcultured in glucose medium. The same trend continued until the late 1930's, when Pochon (184) suggested that the organisms were pure but were undergoing morphological and physiological changes in a type of life cycle, and Enebo

(51, 53) suggested that life of the anaerobic thermophilic cel-

lulose digesters was dependent on an obligate symbiosis. At last, however, Hungate (102, 104) successfully developed a method for the isolation of pure cultures by meticulously di-

luting mesophilic populations through a series of tubes coated

inside with cellulose mineral salts agar. This technique enabled McBee (151, 152) in 1948 to obtain pure cultures of thermophilic cellulolytic anaerobes, and facilitated the study of their characteristics and physiology in pure culture. -21-

C. Physiological Properties of C. thermocellum

1. General Properties (11, 50, 153, 156, 167, 185, 248)

C. thermocellum occurs morphologically as a Gram- negative rod-shaped bacterium (0.5 x 2.5-5.0 pm), which is generally motile by lateral flagella (153). Cultures appear in liquid media as single cells, in short or long chains, and on solid media as single cells often with swollen terminal spores.

On agar medium, single cells grow into yellow or white lens- shaped colonies and, when growing on insoluble cellulose, produce well-defined clear zones.

In the strains examined, the DNA base composition is approximately 39% G+C (167), low for the clostridia. The temperature optimum for growth is 60-65*C. C. thermocellum does not produce hydrogen sulfide, and nitrate is not reduced to ni- trite. It is not proteolytic. Fermentation products are H 2 '

CO 2 , acetic and lactic acids, and ethyl alcohol. Minor quantities of succinic acid may be formed. Its nutrient requirements are not well defined; it appears to require a number of amino acids and vitamins as growth factors (60). C. thermocellum is probably most closely related to the spore forming, fermentative bacilli (62), and its physiology resembles ruminant cellulose digesters (12, 23-27, 94, 104-107, 208-210) and the fermentative sarcinae (29, 130). -22-

2. Carbon Substrate Utilization and Metabolism

C. thermocellum ferments very few carbon substrates; it trefers cellobiose and cellulose, and it will adapt for growth on glucose (50, 6S , 73, 177), fructose (170), or sorbitol (92). It also grows very slowly on salicin but most strains will not ferment other hexoses, hexitols, organic acids, amino acids, pentoses or polysaccharides (50, 63 , 132). Alexander (178) and McBee (153) reported that C. thermocellum grew on mannitol and xylose, respectively, but these results have not been confirmed.

The biochemical pathways of sugar catabolism and energy generation are summarized in Figure 1. The fermentation of fructose and sorbitol is initiated by phosphoenolpyruvate- dependent phosphorylation and transport (173), and probably occurs by the bacterial sugar phosphotransferase system (PTS;

196). The PTS is very common in saccharolytic anaerobes (100,

195). It may be inducible in C. thermocellum since Patni and

Alexander (173) reported that it was formed only when the cells were grown on fructose or mannitol. In addition, the phospho-

fructokinase responsible for conversion of fructose-l-phosphate

to the common intermediate, fructose-1,6-diphosphate, is induc-

ible in the clostridia (101).

Although PEP was shown to be the phosphoryl donor

in C. thermocellum cell extracts, the direct demonstration of a -23-

Figure 1

Biochemical Pathways of Sugar Catabolism in C. thermocellum

cel lobiose glucose fructose sorbitol I ~2

'II I ~

ATP- PEP- dpendent phospho- t ransf erase

F-6-PF F-i-P

F-1 6-diP

t ATP

NADH

Lactate - - -- PYRUVATE

ferredoxin acetyl-CoA

2 Hi1 AT P *7/ NAD I i K I I V H 2 / Acetate Ethandl -24-

PTS by dissection of the system into its protein components, and

reconstitution with purified constituents from C. thermocellum

or a related organism (e.g. Staphylococcus aureus), has not been

carried out.

Cellobiose is the principal product and glucose a minor product of C. thermocellum cellulase activity. Both of

these substrates are dependent on glycolytically-generated ATP

as the source of energy for their transport (90, 170). Cello-

biose and glucose uptake is inhibited by 1% oxygen and by sodium

arsenate, and partially by DCCD and sodium fluoride (170),

supporting an energy requirement for ATP. Uncouplers (e.g. CCCP

and DNP) do not severly inhibit cellobiose and glucose uptake

(91); it is therefore unlikely that a proton motive force (pmf)

is the direct drive in their transport. The experiments suggest

that ATP may participate directly (15, 197) in the uptake; the

available data does not rule out the direct involvement of

acetyl phosphate, the high-energy precursor of ATP formed after

the pyruvate branchpoint in energy metabolism (see below) and

known to be the direct energy donor in certain other "ATP"-

dependent transport systems (97).

After its energy-dependent uptake, cellobiose is

metabolized by both a cellobiose phosphorylase (4, 5, 7 , 207)

and a -glucosidase (2). The phosphorylase is highly active in

C. thermocellum cell extracts (5, 75), has a Km for cellobiose

approximately 10 times less than the -glucosidase, and is prob- -25-

ably the main enzyme involved in cellobiose cleavage. Cello- biokinase activity has also been detected in C. thermocellum cell extracts (166). A cellodextrin phosphorylase is present in C. thermocellum, which has been purified and characterized as an enzyme distinct from the cellobiose phosphorylase (203).

Glucose and glucose-l-phosphate (G-1-P) are generated in the cells by phosphorolysis of cellobiose. G-1-P is converted to fructose-1,6-biphosphate (FDP) by phosphoglucomu- tase, phospho-glucose isomerase, and 6-phosphofructokinase.

Conflicting results have been reported on glucose metabolism.

First of all, several investigators (50, 152) observed that C. thermocellum did not grow on extracellular glucose. Patni and

Alexander (177) and Garcia-Martinez et al. (68) found, however, that growth did occur, but only after a very long lag (> 100 h) in the presence of a high concentration (0.5%) of yeast extract.

Since glucose is generated internally from cellobiose, the delay in growth may be caused by the need for induction of an uptake system (74, 91). This was supported by measurement of the transport of labeled glucose, which was very low in cellobiose- adapted culture of C. thermocellum (74). However, another laboratory has observed uptake of glucose by cellobiose-grown cells of the same C. thermocellum strain (C. Tolman and Dr. M. Roberts, personal communication). Other cellulolytic bacteria, e.g.

Ruminococcus (12) and Sporocytophaga (210, 213) also exhibit extended lags on glucose. In Sporocytophaga, growth occurred -26-

more rapidly on low concentrations of glucose, suggesting that metabolism of glucose may inhibit growth of the culture (210).

Glucose toxicity was also observed in a mesophilic, cellulolytic Clostridium (72). The growth inhibition was not affected by the inoculum density and thus is probably not due to the excretion of a toxic metabolite.

Intracellular glucose is probably phosphorylated by a glucokinase, using ATP as the phosphoryl donor. Gluco- kinase activity was present in cell extracts of C. thermocellum grown in cellobiose or glucose (177, 91), but was low in fructose-grown cell extracts (177). Patni and Alexander (177) suggested that cell extracts contained an inhibitor of glucokinase.

3. Energy Metabolism and Endproduct Formation in C. thermocellum

In anaerobes such as C. thermocellum, large quantities of sugar must be glycolytically catabolized to provide energy for growth (77, 107). ATP for cell growth is obtained by substrate level phosphorylations via 3-phosphoglycerate kinase, pyruvate kinase and acetate kinase. The initial transport and catabolism of sugars by C. thermocellum (see above; Fig. 1) produces the common intermediate, fructose-1,6-diphosphate (F- di-P) which is glycolytically fermented to pyruvate where a branch occurs in the catabolic pathway: (1) reduction to lac- -27-

tic acid by a FDP-activated, NAD-dependent lactate dehydrogenase (132); (2) oxidative decarboxylation to acetyl-CoA, CO 2 1 and reduced ferredoxin by pyruvate:ferredoxin oxidoreductase

(124).

The branch in pyruvate metabolism provides the cells with a mechanism for the regulation of ATP formation and

NADH oxidation. In many anaerobes and lactic acid bacteria a

restriction in the energy supply (e.g. by carbon source limi-

tation in a chemostat) causes drastic shifts in the proportions

of end products formed (28, 45, 70, 78, 95, 182, 220, 221). A

shift toward lactate is often caused by an increased cellular

concentration of F-di-P, signalling energy excess (47, 70),

and activating L(+)-lactic dehydrogenase (70). A switch from

acid to solvent production in Clostridium acetobutylicum is

thought to be associated with decreased rates of growth and

energy metabolism (70a). Such a slowdown can be brought about

by several conditions, including treatment with inhibitors of

energy production or lowering the pH, and appears to be mech-

anistically related to the onset of butnol production. In

the saccharolytic clostridia such as C. thermocellum ATP,

yield is determined by regulating pyruvate decarboxylation,

which occurs by the phosphoroclastic reaction (128, 253).

Acetyl phosphate, carbon dioxide and hydrogen are the products

of this reaction. In Clostridium pasteurianum, phosphoroclas-

tic activity is strongly inhibited by acetyl phosphate (17). -28-

The oxidant acting as cofactor in the decarboxylation of pyruvate is the small iron sulfur protein, ferredoxin

(175). Ferredoxin has a central role in the energy metabolism of C. thermocellum. It functions as a low potential electron carrier from pyruvate or NADH oxidation (110, 175); it serves to release reducing equivalents as hydrogen gas and as a source of biosynthetic reducing power via NADP + :ferredoxin oxidoreductase (118). The oxidation of pyruvate by ferredoxin

(and ATP formation) is tightly linked to the reduction of hydrogen ions (71, 77, 220). Tewes and Thauer (219) have proposed that energy gain in saccharolytic clostridia depends on the availability of oxidized ferredoxin in the cell. Wolfe and O'Kane (253) showed that artificial electron acceptors can substitute for ferredoxin in the phosphoroclastic reaction, including neotetrazolium, and flavins in the presence of oxygen, but not pyridine nucleotides. Artificial oxidants prevent the evolution of hydrogen, but they often stimulate the activity, suggesting that the availability of oxidized ferredoxin may sometimes limit growth in clostridial cells. The biosynthesis of ferredoxin and other iron-sulfur proteins is dependent on an adequate supply of iron in the medium. Re- striction of iron has dramatic effects on the fermentation of carbohydrates in the clostridia. In an iron deficient medium, saccharolytic members of the genus Clostridium produce chiefly lactic acid instead of the usual mixture of hydrogen, CO 2 ' -29-

solvents, and acetic, lactic and butyric acids (71, 123, 176).

This shift in metabolism also occurs in the presence of relatively high concentrations of cyanide (123), which combines with the iron sulfur proteins. Pappenheimer and Shaskan (176) showed that the formation of extracellular lecithinase is highly dependent on the concentration of iron in the medium.

The ferrous iron requirement in the phosphoroclastic reaction is similar to that required by the aldolase of Clostridium perfringens (14). Mueller (160, 161) found that Clostridium tetani lost the ability to ferment glucose in an iron deficient medium, and that this deficiency was reversed by adding small quantities of glutamine (162). These authors also observed that a factor in casein digest, subsequently identified as glutamine, increased the ethanolic fermentation and toxin production by C. tetani (162). In addition, they showed that the gaseous fermentation products had a detrimental effect on toxin production (160). It is clear there is a relationship between fermentation product patterns, iron availability, and toxin production in the saccharolytic clostridia.

D. Properties of C. thermocellum Cellulase (3, 50, 7E, 115, 116, 169)

A true cellulase is an enzyme complex composed of at

least two enzymatic activities (cellobiohydrolase and endo-3- -30-

glucanase) that is capable of completely saccharifying complex cellulosic substrates such as cotton (83). Endo-3-glucanase

is commonly assayed using CMC as the substrate, but there is not a simple assay for cellobiohydrolase in an enzyme mixture.

Many microorganisms produce extracellular fluids which degrade

amorphous or derived forms of cellulose such as phosphoric

acid-swollen cellulose and carboxymethylcellulose, but only

the culture broths of certain fungi (e.g. Trichoderma spp.,

Fusarium spp. and Chaetomium spp.) have been demonstrated to

completely saccharify cotton or filter paper (83, 144 ). Al-

though Clostridium thermocellum and other bacteria grow on

these complex substrates, a potent cell-free cellulase has not

yet been isolated from a prokaryote.

The first report of enzymic decomposition of cellulose

was in 1912, when Pringsheim (186) demonstrated the presence

of a cellulose-destroying enzyme in a culture anaerobically

fermenting cellulose at 551C. He stopped growth by the addi-

tion of iodoform and demonstrated that cellobiose and glucose

accumulate as products. When the temperature was raised to

67*C, cellobiose was the only hydrolytic product, and when

lowered to 20'C, glucose was the predominant product. From

these results, he postulated the presence of two hydrolytic

enzymes, one which converted cellulose to cellobiose and an-

other which hydrolyzed cellobiose to glucose. A classical

study by Reese and Levinson (191) provided further evidence -31-

that cellulase is a multienzyme system. They proposed the "C" concept, C 1 being an enzyme that modifies the crystalline cellulose and renders it susceptible to attack by ordinary hydrolytic enzymes such as C :

Crystalline C1 Reactive C Cellobiose Cellobiose Cellulose > Cellulose - + Glucose Glucose

Non-T c, e Cellulolytic Organisms

True Cellulolytic Organisms

This theory generated a great deal of research and controversy

into the mechanism of cellulose degradation.

Fungal cellulose is not a single enzyme; it is a sys-

tem generally composed of three major enzymic components (84,

187, 246): 1,4- -D-glucan cellobiohydrolase (E.C. 3.2.1.91),

endo--l,4--D-glucanase (E.C. 3.2.1.4) and -glucosidase

(E.C. 3.2.1.21), which acts synergistically to entirely decom-

pose cellulose to glucose. In the currently accepted scheme

(Fig. 2), endo-glucanase randomly cleaves internal glucosidic

bonds within an unbroken glucan chain, creating non-reducing

chain ends which then become the substrates for cellobiohydro-

lase. Cellobiohydrolase erodes away the cellulose crystal by

splitting out cellobiose units; these are then hydrolyzed to

glucose by -glucosidase. -- mu--om-N., -

-32-

Figure 2

Model for Cellulase Digestion of Insoluble Cellulose (Brown, 1982)

A B (EG

UEIG

calobwe

1non-reducn end

C ca D - EG

- ( 2-Glucose

C EG

A schematic representation of cellulase action. (A) The Trichoderma cellulase enzyme system consists of three major enzymes: endoglucanase (EG). cellobiohydrolase (CBH), and 6- glucosidase (O-G). EG binds randomly to the surface of the cellulose microfibril. The catalytic action of EG breaks a glucosyl bond within a glucan chain. (B) EG leaves the microfibril surface. The nick in the glucan chain exposes a reducing and a nonreducing end. (C) CBH can act only on the free nonreducing end of a glucan chain. The catalytic action of CBH cleaves a cellobiose unit from the nonreducing chain end. (D) Cellobiose is released into solution, where it is split into glucose monomers by P-G. CBH moves to the newly created free nonreducing end and continues to cleave cellobiose units from the glucan chain. (E) EG nicks continuous glucan chains, but releases little soluble reducing sugar. CBH is catalytically active only at glucan chain ends. Thus, EG creates sites at which CBH may act. The result is synergistic degradation of cellulose. -33-

The cellulase from Trichoderma has the highly desirable property of being able to act on thick slurries of cellulose. In 1968, Katz and Reese (119) reported the production of 30% glucose from a 50% slurry of heated ball-milled cellulose in 15 days. The reaction mixture contained a high cellulase concentration, and was supplemented with Aspergillus luchiensis cellobiase to alleviate product inhibition. This saccharifying ability has yet to be duplicated by another cellulase system. It should be emphasized, however, that the

Trichoderma cellulase has a very low specific activity on in- s.oluble cellulose, which further decreases with highly crystalline celluloses such as cotton, Avicel, or biomass wastes.

This limitation has hindered its commercial utilization. Cur- rent Trichoderma mutants (RUT-NG-14 and RUT-C30) produce 20 g/l extracellular protein which is predominantly (78.5%) cellulase. The specific activity of the cellulase is only about

0.6 - 0.7 filter paper units/mg protein. To achieve a 40-50% conversion of a 10-30% slurry of cellulose in 24-48 hours requires about 10 filter paper units/g cellulose for a susceptible substrate, and about 20 units/g for a more resistant substrate (7, 146). Thus the requirement is 15-30 g of enzyme protein per kg of substrate, a remarkably high figure. In general, extracellular polysaccharases active on insoluble substrates have a low molecular activity of 102 to 104 bonds cleaved/min/enzyme molecule, compared to 10 to 108 bonds/min/ -34-

enzyme molecule for enzymes acting on small soluble substrates.

A low molecular activity requires that the producing organism make a large quantity of the polysaccharase in order to grow at its maximum rate, and thus the release of fermentable sugar usually limits the growth rate on an insoluble substrate.

By far the majority of studies on cellulases have been done on the aerobic, saprophytic fungi, and it is likely that different biochemical processes are responsible for cellulose degradation by other organisms. Many of the wood-rotting fungi

(e.g. various basidiomycetes) possibly employ an oxidative mechanism for cellulose and lignin decomposition (56, 61, 73-,

127). The biochemical properties of cellulases of anaerobes are not yet known.

A serious problem plaguing the study of bacterial cellulases has been the low cellulase activity detectable in culture filtrates. Cultures of C. thermocellum grow faster than

T. reesei on native cellulose, yet the assay of cellulase activity in the bacterial broth is generally about 100 times less per unit volume of broth (75, 169). It has been suggested that contact between cells and cellulose is necessary in some microorganisms for effective depolymerization, but this does not seem to be the case with C. thermocellum, since large and distinct clearing zones are formed when C. thermocellum is grown on agar media containing cellulose as the insoluble carbon source (116). -35-

C. thermocellum has received some attention for its ability to carry out a limited attack on derived forms of cellulose (3, 169). The presence of CMCase and weak cellulase activity has been demonstrated by Ng and Zeikus (167).

The activity was reported to be "oxygen stable", low in "exo- glucanase", and resistant to inactivation by temperatures up to 700 C. They later found (169) that the cellulase activity was inhibited by Hg +, and that this inhibition was partially relieved by excess dithiothreitol. The cellulase had a pH optimum of about 5.4, much lower than the optimum pH of the organism for growth, which is 7.0-7.5.

Enebo (51-53) showed that the cellulase of C. thermocellulaseum is quite sensitive to inhibition by certain metal iosnluig g+ Cu+ 2+ 3+ 3+ 2+ ions including Ag , Cu , Hg , Fe and Cr , but not to Mn ,

Ca 2+, or Mg 2+. This inhibit-ion could be reversed at low ion concentrations by adding peptone, which probably sequestered the inhibitory metal ions. Ng and Zeikus (169) found that C. 2+ 12+ 2+ thermocellum cellulase is inhibited by Hg , Cu and Zn2+

Mn +, Ca2+ and Mg2+ had no effect. The basis for inhibition of bacterial cellulase by metal ions is not known.

Enebo (53) also demonstrated that thermophilic cellulase activity from C. thermocellulaseum was inhibited by cellobiose, and less strongly by lactose. More recent studies

(76, 205) have suggested that C. thermocellum cellulase is resistant to end-product inhibition, but this discrepancy remains to be resolved. -36-

The composition of the thermophilic cellulase has not yet been satisfactorily determined. It was suggested in 1912

(186), and later in 1954 (53), that the cellulase from C. thermocellum and related thermophilic clostridia consisted of two enzymic components, since different products accumulated in the presence of inhibitors, such as iodoform. The purification of C. thermocellum cellulase has been hindered by low amounts of extracellular protein, binding of enzymes to cellulose, tendency of the proteins to form aggregates, and low recovery of the components, especially exo- -glucanase, during purification. Ait et al. (3) partially purified a protein by preparative polyacrylamide electrophoresis which produced reducing equivalents after 1 h from fibrous cellulose. They recovered 10% of the activity applied to the gel. Petre et al.

(181) were able to separate the cellulase complex into distinct protein fractions by gel chromatography in the presence of urea.

One of these was further purified and characterized as an endo-

-1,4-glucanase. The final purification was about 100 fold and the recovery exceeded 100%; the most highly purified fraction contained 60 percent of the initial CMCase activity. This protein was probably active as a monomer, had a molecular weight of about 56,000 daltons, a pI of 6.2, and an optimum pH of 6.0.

The endo- -glucanase was not affected by EDTA or several mono- valent and divalent ions, was not oxygen sensitive, and was unaffected by several sulfhydryl reagents. Ng and Zeikus (168) -37-

also purified 22-fold an endo- -glucanase from broths of C. thermocellum grown on glucose. From 6.7 g of broth protein, they obtained 21 mg of enzyme, and they attributed this low yield to major losses during ion exchange chromatography. They suggested that this enzyme is a prominent component of the cellulase complex, accounting for over 25% of the extracellular endo- -glucanase activity. The enzyme appears distinct from the endoglucanase purified by Petre et al. (181); it has a molecular weight of 88,000 daltons, contains about 11% carbohydrate, has a pI of 6.7, an optimum pH of 5.2, and is most active at 62*C. The purified protein had a low methionine content and completely lacked cysteine. An endoglucanase from a new thermophilic clostridium has recently been purified (41).

The enzyme is large, having a molecular weight of 91,000 to

99,000.

Recently, workers at the Pasteur Institute (39) have managed to clone into Escherichia coli two endoglucanases from

C. thermocellum. The genes for the two enzymes were examined by nucleic acid hydridization, and were found not to be homolo- gous, and to be separated on the C. thermocellum chromosome.

Other workers have also cloned and expressed endoglucanases

from cellulolytic organisms including Cellulomonas fimi (248) and Thermomonospora sp. (37). These genetic studies will

facilitate detailed characterization of endoglucanase activ-

ities but no one has yet been able to clone cellobiohydrolase -38-

or an enzyme responsible for true cellulolytic activity in

these bacteria.

E. Regulation of Cellulase Synthesis in Fungi and in C. thermocellum (50, 67, 68,144, 187)

The nutritional and environmental factors influencing

cellulase formation have been little studied in bacteria, but

have been investigated in the fungi, especially in Trichoderma

(144). Trichoderma is a saprophytic, aerobic fungal genus,

generally imperfect, but of ascomycetous origin. Members of

this genus secrete a true cellulase.

The rapid metabolism of cellobiose, glucose or other

soluble carbon sources drastically decreases cellulase synthe-

sis in cellulolytic fungi and bacteria (171). The decrease in

cellulase synthesis during rapid growth on the products of

cellulolysis indicates this regulation is due to carbon catab-

olite repression (144, 171, 187).

The cellulase in T. reesei is reported to be an induc-

ible enzyme system, which is formed in highest quantities when

the fungus is grown on cellulose. Since cellulose is insoluble,

limiting quantities of solubilized products must trigger induc-

tion. Cellulase in Trichoderma and many other cellulolytic

organisms (144, 187) has been reported to be induced by a product derived from cellulose, originally thought to be cellobiose -39-

(148). Mandels et al. (147) later showed that sophorose (2-0-

-D-glucopyranosyl-D-glucose), probably formed from cellobiose as a transglycosylation product (138), is a much more potent inducer. In 1979 (214), it was confirmed that sophorose stim- ulates CMCase synthesis in T. reesei. Synthesis of CMCase stopped when the mycelium was removed from the inducer.

The slow metabolism of sophorose may be responsible for its effectiveness as an inducer of CMCase. The uptake of sophorose by mycelia was quite slow (1/5 to 1/10 the rate of other sugars) (214) and the disaccharide was catabolized to

CO 2 and H 2 0 (138). Induction was maximal at the slowest re- spiration rates and was influenced by varying the pH or carbon source. These data suggest that it may be the slow metabolism of sophorose, and not its combination with a repressor protein, which is responsible for its stimulation of cellulase synthesis. Cellulase synthesis in Myrothecium verrucaria is reported to be regulated solely by carbon catabolite repression (84).

F. Regulation of Cellulase Synthesis in C. thermocellum (50, 68

An understanding of the control of cellulase synthesis in bacteria has been hindered by the difficulty in obtaining true cellulase activity from the culture broths. In C. ther- -40-

mocellulaseum, very closely related to C. thermocellum (53,

152), broths with high activity on derived cellulose were obtained when insoluble cellulose was the carbon source; the addition of cellobiose or glucose to C. thermocellulaseum cellulose fermentations markedly lowered the amount of cellulose fermented (53). Cellobiose was the more inhibitory of the two sugars, presumably because of its 3-fold higher rate of metabolism. This suggested that cellulase synthesis by

C. thermocellulaseum is sensitive to catabolite repression.

In contrast to these earlier findings with C. thermocellulaseum, which apparently differs from C. thermocellum only in its ability to ferment maltose and xylose, catabolite repression has not been reported to influence cellulase syn-

thesis in C. thermocellum. CMCase appears to be produced con-

stitutively (3, 68:, 76, 87, 152), and nutritional and environmental conditions that improve cell growth (e.g. prevention of a pH drop) generally also result in improved CMCase volumetric activity.

G. Selection of Mutants Affected in Extracellular Enzymes

An initial objective of this study was to develop se-

lection methods for the isolation of C. thermocellum mutants

affected in extracellular enzyme formation or activity. There

have been many techniques and strategies developed for the se- -41-

lection of microbial mutants that produce increased quantities of an intracellular enzyme or metabolite (46). Environmental conditions can be devised so that an individual (B) producing more of an intracellular enzyme or product than the parent (A) will be better fit to multiply in the environment and will increase at a growth rate p A(1+S) = yB where P is the specific growth rate and S is the selection coefficient. The selection coefficient is therefore defined as S = (yB~4A A. When S is greater than 0, the fitness (f) of B is greater than A, and positive selection occurs for B, i.e., pB >A* When S is 0, the environment is neutral with respect to the trait under con- sideration and no selection occurs, and when S is negative, selection occurs against the B strain. Several techniques are known for increasing the fitness of a strain over-producing an intracellular enzyme. For example, Horiuchi et al. (100) were able to select for hyperproducers of -galactosidase by limiting the supply of lactose in continuous culture. The development of selective conditions for extracellular enzymes is not as straightforward. In a mass culture, it would not be advan- tageous for an individual cell to overproduce an extracellular enzyme, which could be exploited by other members of the popu-

lation. Furthermore, such a trait would be unstable in a homogeneous environment such as a fermentor because the occurrence of a non-producing mutant would result in faster growth than the overproducer. The process of extracellular enzyme -42-

evolution is interesting because it requires that selection in a population operates on a trait which is potentially harmful to an individual possessing the trait. It is a difficult problem to devise environmental conditions that would enable extracellular enzyme producers to outcompete non-producers and increase in proportion in a mass population. A direct approach is to study the regulation of the enzyme activity and synthesis in the producing organism. An understanding of its control and functions in the individual cells might explain its capacity to evolve in a microbial population (228). -43-

2. EXPERIMENTAL PROCEDURES

A. Bacteria

The ATCC 27405 strain of Clostridium thermocellum was used throughout this study. It was periodically plated in a

Coy anaerobic (90% N 2 :5% CO2:5% H 2 ) glove box on defined medium containing Avicel as the carbon source. A cellulolytic colony was recovered with a toothpick, grown in cellobiose broth and reisolated on an Avicel plate. This was grown in cellobiose minimal broth and stored at 4*C. The clostridial cultures could be maintained by lyophilization or at -80*C in

50% V/V glycerol.

B. Cultivation of Bacteria

The development of a minimal, defined medium (MJ) is described in the Results section of this thesis, and was used for growth experiments unless otherwise noted. It was prepared by mixing the basal salts (1.5 g KH 2PO 4, 2.9 g K 2 HPO4 , 2.1 g urea, 1.0 g cysteine hydrochloride, 10.0 g morpholinopropane sulfonic acid, 1.0 mg resazurin and 3.0 g of sodium citrate in 850 ml H 20). This was then boiled to remove 02 and transferred to an anaerobic chamber with an atmosphere of 90%

N 2, 5% CO2 and 5% H 2. The medium was dispensed and capped in -44-

Hungate pressure tubes (Bellco) inside the chamber, and ster-

ilized for 15 min at 121 0 C. A 1OX trace salts solution (10.0

g/l MgCl 2 .6H 20, 1.5 g/l CaCl 2 .2H 2 0 and 12.5 mg/l FeSO 4.7H 20) was autoclaved separately and combined with a filter-steril-

ized 1000 X vitamin solution (20 mg/l biotin, 200 mg/l pyri-

doxamine hydrochloride, 40 mg/l p-aminobenzoic acid and 20 mg/

1 vitamin B 1 2), which was then added by syringe. The carbon

sources were then added at 5 g/l, except where noted. All

cultivations were done in Hungate tubes at 60*C and growth was

measured by optical density in a Turner model 330 spectropho-

tometer. One mg dry cell weight per ml corresponds to 1.8 op-

tical density units at 660 nm. Sampling of tubes during ex-

periments was usually done in the anaerobic glove box to pre-

vent leakage of air and changes in the physiology of C. ther-

mocellum. Growth on cellulose (Avicel PH105) was determined

by microscopic counts in a Petroff-Hausser chamber. Viable

counts were obtained by plating on GS-2 medium (114) in the

anaerobic chamber.

C. Determination of C. thermocellum Nutritional Require- ments

To determine amino acid and vitamin requirements, an

initial inoculum of C. thermocellum ATCC 27405 was grown in

GS-2 medium for 24 h at 60 0 C. It was then added at 2 ml/liter

to GS-2 medium lacking yeast extract. Cells were cultured for -45-

24 h, the carryover of growth factors allowing growth to occur in this first transfer. The value of such a starvation step in developing inocula is well documented (42). These cells were used to inoculate 10 ml of GS-2 medium lacking yeast extract, but containing various combinations of growth factors

(second transfer). The third transfer was made to identical media, and growth (absorbance at 660 nm) was measured after

40 h of incubation.

D. Source and Preparation of Cellulase

Initially, a crude cellulase preparation was obtained from B. Faison who used C. thermocellum ATCC 27405 grown in a

12-liter Microferm fermentor (New Brunswick Scientific Co.) on CM-4 cellobiose medium ( 92 ) at 60*C and 60 rpm for 68 h.

In her work, the broth was chilled to 4*C and then centrifuged at 18,000 X g for 20 min to remove cells. The supernatant fluid was treated with solid (NH 4 )2so 4 at 80% saturation and stored overnight at 4*C. The precipitate was harvested by centrifugation, dissolved in 50 mM sodium citrate buffer (pH

5.7), reprecipitated by the addition of 4 volumes of saturated

(NH4 )2 so 4 , and again stored overnight before dissolution in citrate buffer. This preparation was desalted by passage through

Biogel P-2 (Bio-Rad Laboratories, Richmond, CA), diluted to 1 mg of protein per ml, and stored frozen (B. Faison, S.M. The- sis, M.I.T., Cambridge, MA, 1981). -46-

A second batch of enzyme was prepared similarly but with C. thermocellum grown in a 100-liter fermentor contain-

ing 0.5% Solka-Floc SW-40 (Brown Co., Berlin, NH) as carbon source. The fermentor was stirred slowly at 50 rpm and gassed with N 2 for the first 19 h of the 60 h fermentation. At 60 h, cysteine hydrochloride was added to a final concentration of

0.1% and the cells were removed in a Sharples centrifuge

(type M47-16Y, Sharples Corp., Philadelphia, PA). This extra-

cellular preparation has retained its cellulolytic activity

for at least a month at 4*C, and for at least 2 years at -20*C.

The broth [- 200 pg protein ml by the Coomassie blue method

(19)] was freeze-dried for 48-72 h in shallow enamel pans in a

Virtis freeze drier, reconstituted in water and dialyzed at

room temperature against four changes of 20 mM Na succinate

buffer (pH 5.8) for 6 h. The clear yellow-green preparation

obtained (- 312 pg protein ml~ ) was stored at -20*C, and when required was thawed and used without further purification.

Dried powders from T. reesei were prepared at the Na-

tick Laboratories (T. reesei QM 9414 cellulase, 0.61 mg of

protein per ml, 0.32 filter paper units [FPU] per mg. and T.

reesei RUT C-30, 0.53 FPU/mg). These were prepared in solu-

tion at 5 FPU/ml and 9 FPU/ml, respectively, which is equiva-

lent to their broth concentrations. -47-

E. Measurement of Cellulase Activity

A simple procedure was developed in this study for routine measurement of cellulase activity. Activity was de-

termined by decrease in turbidity (660 nm) of an Avicel suspension (Type PH 105, 20 jiM particles, FMC Corp., Marcus Hook,

PA) (see Results). Three mg samples of Avicel were suspended

in Hungate tubes in 3 ml of 0.1 M sodium succinate buffer (pH

5.8), 0.5 ml of 0.1 M DTT, 0.5 ml of 0.1 M CaCl 2 .2H 2 0 plus

various volumes of enzyme and water to 5 ml. This concentra-

tion of cellulose (0.6 mg/ml) was used so that cellulolysis would proceed to completion (81-84) within a relatively short

time with minimum interference from products.

In collaboration with M. Sakajoh and G. Halliwell,

cellulase activity was also -measured on Avicel and nonpowdered

celluloses by loss in dry weight. Three milligrams of filter

paper (Whatman No. 1 filter paper circles) or nonabsorbent

cotten (SP cotton coil, C-9355-3, American Scientific Products,

Bedford, MA) were incubated with the Clostridium cellulase

under the same conditions as with Avicel. Residual cellulose

was determined colorimetrically with K2 20 7 (116).

When inhibitors or stimulants were added to the cellu-

lase reaction mixture, they were first dissolved in water or

ethanol. Stock metal ion solutions were prepared at 1 M in

dilute HCl. The chelating agents o-phenanthroline and 2,2'-

dipyridyl were dissolved at 1 M in ethanol. -48-

F. CM-Cellulase Activity (endo-1,4-3-D-glucanase, E.C. 3.2.1.4)

CM-cellulase activity was determined by reduction in viscosity of a 0.25% (w/v) carboxymethyl cellulose (CMC) (Type

7H4, Hercules cellulose gum, lot 48594) solution in 25 mM Na succinate (pH 5.8). The activity is expressed as the recipro- cal of time (min) required for 5 ml to flow from an Ostwald viscometer at 60*C, and is corrected for the flow of buffer.

The activity was linear with protein concentration up to at least 15 pg protein per 5 ml assay. Units of CM-cellulase activity are given as [l/min (assay) - Ymin (H20)] X 10. The standard deviation for assays done on different days was 0.8.

G. Units of True Cellulase Activity

The C. thermocellum cellulase activity can be measured by loss in weight of non-absorbent cotton, micro-crystalline

Avicel, or filter paper (see Results and (116)). The loss in dry weight correlates with the decrease in optical density at

660 nm of an Avicel suspension. The principal sugar product

formed is cellobiose (see Results). Under conditions of a low

Avicel concentration (0.6 g/l), the decrease in optical density

is first order and correlates with protein concentration (Fig-

ure 3) up to approximately 5 pg/ml of extracellular protein.

In a Turner model 330 spectrophotometer, the degradation by -49-

Figure 3

A. First Order Rates of Avicel Solubilization (Measured at O.D. 660 nm) by Varying Concentrations of Extra- cellular Protein.

B. Rate of Avicel Solubilization as a Function of Pro- tein Concentration.

L - p 0 Oug N 1.6

3.1

-N 3 62

-8 1UNIT x - 125 2 0o -4 go 18,1

I I a I 0 40 80 0 20 40 hours ug protein -50-

this concentration of cellulase (5 iig/ml) corresponds to an

O.D. decrease of 0.25 in 24 h at 601C. For the purpose of this thesis, one cellulase unit is defined as the amount of cellulase which gives a first order rate that completely degrades 1 mg of Avicel in 24 h. This degradation corresponds to a first order rate content of 0.02 h 1. This assay avoids the trouble- some measurement of reducing sugars in the presence of DTT, and measures the complete saccharification of microcrystalline cellulose.

H. Analysis of Cellulolytic End Products

Products of saccharification were determined by HPLC on a column of HP X-87-H (Bio-Rad) at 60*C with a flow rate of 0.5 ml/min. The solvent was H2SO4 in glass distilled H 20, pH 2.1-2.2.

I. Analysis of Fermentation End Products

Ethanol and acetic acid were measured by gas-liquid chromatography on Chromosorb 101 columns with samples acidi- fied with 1.5 M HCl and n-propanol as an internal standard.

Lactic acid was measured enzymatically (Sigma Chemical Co.,

Bulletin No. 826 UV). -51-

J. Determination of Phosphate Uptake by Cells

Cells were grown in CM-4 medium, centrifuged at 44C, and washed and resuspended in NMR buffer (5 mM KH 2 PO 4, 5 mM

Na 2 HPO 4 , 100 mM PIPES, 50 mM MOPS and 85 mM NaCl). Cells were kept cold and anaerobic in 3 ml aliquots in NMR tubes. When assayed for phosphate uptake and membrane energization, 0.1 ml of 20% (w/v) carbon source was added to cells previously warmed to 60*C. They were immediately inserted into the Bruker magnet, and 3 1 P-NMR was employed to monitor intracellular and extracellular inorganic phosphate.

K. Assay of ATP Concentration in Cells

The procedures of Cole et al. (35) were used for sample preparation and Holm-Hansen and Karl (96) for the luciferase assay. Two milliliter samples of growing cells were removed with syringe, and rapidly added into 0.5 ml of ice-cold perchloric acid. After 10 min on ice, the extract was vortexed and neutralized with 1 M KOH; the precipitate was removed by centrifugation and the extract kept at -20*C until the ATP determination. This was done exactly by the published procedure

(96) and the error between identical standard samples was less than 3%. A Packard scintillation counter with one channel at

1385 V and the coincidence control switched out was used for -52-

the measurement. Counting was done for 0.1 min, and 2-20 mM

ATP standards were run at the same time.

L. Assay of Hydrogenase Activity

Hydrogenase was assayed in centrifuged broths at 50*

C in a glove box atmosphere (88% N 2 :5% CO 2 :7% H 2 ) or in a N 2 control. To 3 ml of 50 mM Tris-HCl, pH 8.5 containing 1.5 mM methyl viologen was added 0.1 ml to 0.5 ml broth, and the increase in O.D. at 578 nm was recorded.

M. Gel Electrophoresis

Polyacrylamide gel electrophoresis (SDS-PAGE; 131) was used to qualitatively characterize C. thermocellum's extracel-

lular proteins. Slab-gels containing 3 percent (stacking gel) or 5 percent acrylamide were prepared from a stock solution of

30 percent by weight of acrylamide and 0.8 percent by weight of N,N'-bis-methylene acrylamide, according to the following recipe (per gel):

Running Stacking Gel (5%) Gel

H 20 17.1 ml 6.3 ml 1.5 M Tris-HCl, pH 8.8 7.5 " - 0.5 M Tris-HCl, pH 6.8 - 2.5 " 10% SDS 0.3 " 0.3 " Bis-Acrylamide Mixture 5.0 " 1.0 10% Ammonium Persulfate 0.1 " 0.1 -53-

This solution was degassed, mixed with 10-20 il of

TEMED, and immediately poured using a syringe. The running gel was overlayed with isobutanol, and blotted dry with a Kimwipe before pouring the stack. The electrode buffer (pH 8.3) contained 0.025 M Tris, 0.192 M glycine and 0.1 percent SDS.

Samples (80 il containing 5-50 jg protein) were mixed with 20 pl of 5-fold sample buffer (1.25 M Tris, pH 6.8 (10 ml); 25%

SDS (10 ml); glycerol (25 ml); 0.75% bromophenol blue (1 ml); water (4 ml) ), and immersed in a boiling water bath for

5 min. High molecular weight protein standards (30,000 -

200,000; Sigma MW-SDS-200) were always electrophoresed as markers. The standards were carbonic anhydrase, 29,000 MW; egg albumin, 45,000 MW; bovine albumin, 66,000 MW; phosphorylase B, 97,400 MW; E. coli -galactosidase, 116,000 MW; and myosin, 205,000 MW. Electrophoresis was done at 20 mA/ gel loading, and 40 mA/gel running, until the bromophenol dye reached the bottom of the gel (about 3-5 h). The proteins were stained for 1-2 h in Coomassie blue (filtered solution of

1.25 g Coomassie blue Brilliant R, 250 ml methanol, 250 ml water and 45 ml glacial acetic acid) and destained in 10% methanol/7% acetic acid.

N. Purification of Cellulase

The extracellular cellulase from C. thermocellum was partially purified by ion exchange chromatography and gel fil- -54-

tration. Ion exchange chromatography was performed with a

DEAE-sepharose open column (- 2.5 x 12 cm) or by HPLC (cour- tesy of Cindy Allen at Waters Associates) using a vinyl ion- exchange resin. The cellulase and ion-exchange resins were equilibrated with 20 mM Tris-HCl, pH 8.8, and after adsorption, the cellulase was eluted in a KCl gradient to 0.5 M salt

(see Results). Gel filtration was done with an Ultrogel ACA

22 column (- 1.8 x 85 cm), equilibrated with 50 mM Tris-HCl, pH 7.1 containing 100 mM NaCl or with a Fractogel Column equilibrated with the same buffer containing 300 mrM NaCl.

Standards (Sigma Chemical Co.) for molecular weight calibra- tion in gel filtration were hemocyanin, 3.1 x 106 MW; thyro- globulin, 669,000 MW; ferritin, 440,000 MW; catalase, 232,000

MW; and aldolase, 156,000.

0. Chemicals

Bovine blood superoxide dismutase, sulfhydryl compounds, reducing agents, cellobiose and metal chelators were from Sigma. Hydrogen peroxide was from Mallinckrodt. Asper- gillus niger catalase was from U.S. Biochemical Corp. Glucose was from Anachemia and fructose and sodium citrate were from

Baker. Cellodextrins were provided by Herve Cellard. Cello- triose was a gift from M. Ladisch. All other chemicals were of analytical quality. -55-

3. RESULTS

A. Ca 2 + and Sulfhydryl Reducing Compounds as Requirements of the Cellulase System of C. thermocellum

A major impediment to the use of bacterial cellulases has been their reportedly weak activity compared to the fungal enzymes. In this first section of my thesis, I describe experiments which resulted in improvement of C. thermocellum cellulase activity.

C. thermocellum plated on agar medium containing insoluble cellulose gave rise to colonies surrounded by clear zones in 5 to 7 days, suggesting the presence of an extracellular cellulase (Fig. 4). However, extracellular broth from the organism, prepared on cellobiose medium, showed only weak cellulolytic activity (0.2 to 0.4 mg reducing sugars ml broth h- ) in the filter paper assay (.145), confirming earlier findings

(76, 169). Since C. thermocellum is an anaerobe, it seemed plausible that reducing conditions may be necessary for cellulase activity. However, reducing agents interfere drastically with assays measuring liberation of reducing sugars, and it was therefore necessary to use a method that did not depend on the detection of reducing equivalents. I decided to develop a turbidimetric assay with powdered Avicel as the substrate. A slight but significant decrease in turbidity was observed when

succinate replaced citrate as the buffer and a dramatic in- -56-

Figure 4

Clear Zones Produced by C. thermocellum After 8 Days Growth on Compression Milled Corn Stover (A), Avicel (B), or Amor- phous Cellulose (C)

0 0 0 0~

0 *0A -57-

crease in activity occurred when a sulfhydryl reducing compound was added (Figure 5).

DTT had little effect on the initial rate of Avicel hydrolysis but greatly increased the ultimate extent of breakdown

(Fig. 5). In the presence of 5 mM DTT, Avicel was completely

solubilized; in the absence of DTT, solubilization stopped after 24 h. Other effective reducing agents included cysteine,

sodium dithionite, glutathione and mercaPtoethanol. DTT (5 mM)

had no effect on the enzymatic solubilization of phosphoric acid-swollen Avicel or trinitrophenylcarboxymethyl-cellulose

(205).

The cellulase activity of the C. thermocellum enzyme

preparation was stimulated by Ca2+ (Fig. 6). EDTA (10 mM) in-

hibited completely the cellulolysis of Avicel by the Biogel-

treated preparation even in the presence of DTT (Figure 6).

Addition of MgCl 2 (7 mM) improved cellulolysis slightly whereas

7 mM CaCl 2 was far more effective and enabled the enzyme prep-

aration to achieve complete solubilization of the substrate. A

dialyzed preparation of C. thermocellum culture broth was

strongly inhibited by EDTA (Figure 7); this inhibition was re-

versed by calcium confirming the necessity of this ion.

In the presence of calcium and DTT, the first order rate

of Avicel solubilization is proportional to broth protein added

(Fig. 3, Materials and Methods), but only at protein concentra-

tions less than - 6 vig/ml. This proportionality can be extended -58-

Figure 5

Influence of DTT Concentration on Avicel Hydrolysis by Clostrid- ium thermocellum Cellulase. Fifty Micrograms of Biogel-Treated Extracellular Protein were Incubated with 3 mg Avicel and The Absorbance (660 nm) of the Suspension was Determined with Time. Succinate Buffer with No Ca2 + was Used.

0.4

No DTT

- 0.3- E C

S 0.2

2mM DTT

S0.1.-

5mM DTT

0 40 80 120 160 Hours of Hydrolysis -59-

Figure 6

Influence of Ca 2+ on Avicel Hydrolysis by Clostridium thermocellum Cellulase. Reaction Conditions were the Same as in Fig. 2, but the Incubation Mixtures Con- tained 5 mM DTT and Either 20 mM EDTA, 7 mM CaCl 2, or 7 mM MgCl 2.

0.3 EDTA

E o 0.2 0

CaCC2

0 20 40 60 so 100 Hours of Hydrolysis -60-

Figure 7

Inhibition of C. thermocellum Cellulase by EDTA and Its Reversal by Calcium. Fifty Lil of Dialyzed Enzyme were Used.

+2,5 or 10 mM 0.4 EDTA

E C 0 (0 0.3

C 0 +10 mM EDTA (n Z-2H 2 0

(n. 0.2

=3 No EDTA, No Ca 2 + 0.1 +5 mM EDTA 10 mM Ca 2 + +2 mM EDTA 0 10 mM Ca 2 + +10 mM Ca 2 + I I 0 20 40 60 Hours -61-

to higher protein concentrations by increasing the substrate conc. Therefore, the rate of hydrolysis of insoluble Avicel appears to be limited by the availability of susceptible sites for attack.

The following experiments were done (in collaboration with Drs. M. Sakajoh and G. Halliwell) to determine whether

C. thermocellum cellulase in the presence of Ca2+ and DTT is capable of extensively solubilizing complex celluloses including cotton and filter paper and whether it could do it as fast as the cellulase from Trichoderma reesei. Equal broth volumes

(1 ml) of the C. thermocellum (0.2 mg protein ml 1) and T. reesei QM 9414 (9.5 mg protein ml~ ) cellulase were examined quantitatively for their ability to solubilize cotton, filter paper, and Avicel as measured by loss in weight. The crude

Trichoderma cellulase acted rapidly on filter paper, less so on Avicel and slowest on cotton (Figure 8), whereas the Clos- tridium enzyme showed the reverse pattern, cotton being saccha- rified most rapidly and faster than the Trichoderma cellulase.

Filter paper, however, showed greater resistance to hydrolysis by the clostridial cellulase, particularly in the early stages.

Of note was the Clostridium enzyme's ability to achieve essen- tially complete hydrolysis of all three forms of cellulose in the manner expected of a true cellulase.

The previous experiment comparing T. reesei and C. thermocellum cellulases (Figure 8) employed a temperature of 37*C -62-

Figure 8

Solubilization of Native and Derived Celluloses by Cellu- lase of Trichoderma reesei QM 9414 and Clostridium thermocellum. One Milliliter of Culture Broth (Fresh or Re- constituted) was Incubated at 370 C and pH 4.8 Acetate Buffer (Trichoderma) or 60*C and pH 5.5 Succinate Buffer (Clostridium) with Cotton, Filter Paper or Avicel. The Clostridial Enzyme Incubation Mixture Contained 7 mM CaCl 2 and 10 mM DTT.

100

~80. \T. reesei

------C. thermocallum

4 FILTER PAPER \ A AVICEL o 60- \COTTON

0 4 0-

20-

Q.)

0 21 42 63 84 96 Hours of Hydrolysis -63-

for Trichoderma, this temperature being optimal for the cellobiohydrolase (C1 ) component of T. koningii over long periods

(85). However, in the short-term (1 h) assay employed by Man- dels et al. (145), 50*C is used, being optimal for the CM- cellulase component in the culture filtrate. Therefore, it was of interest to compare the cellulase of T. reesei at 50*C with the clostridial cellulase (at 601C). For this experiment, 1 ml of the best available T. reesei preparation [RUT C-30;

Mandels (144)] was used at a culture broth strength (9 FPU ml ).

The results measured turbidimetrically on Avicel (Figure 9a) and as loss in weight of Avicel and cotton (Figure 9b) confirm those

found in Figure 8. In addition, the Trichoderma enzyme showed

slightly improved activity on both substrates at 50*C compared with 37*C, rates that were equalled or exceeded by the Clostrid-

ium cellulase.

The activities of the Clostridium and Trichoderma cel-

lulases were also compared on a specific protein basis (Figure

10). Interestingly, the Clostridium cellulase showed a higher

specific activity on Avicel, but a much lower activity on phosphoric acid-swollen cellulose. The solubilization of Avicel requires exo- and endo-glucanase activities; digestion of swollen

cellulose requires mainly endo-glucanase. These data together with the preference of the bacterial cellulase for highly crys-

talline cotton suggests the presence of a prominent exo-glucan-

ase component in the C. thermocellum complex. -64-

Figure 9

Hydrolysis of Cotton and Avicel under Optimal Conditions by the Cellulases of Trichoderma reesei RUT C-30 (Tr) and Clostridium thermocellum (Ct). The Trichoderma Enzyme Incubation was Done in pH 4.8 Acetate Buffer and the Clostridium Enzyme Incubation in pH 5.8 Acetate Buffer. (A) Turbidimetric Measurement of Avicel Hydrolysis. (B) Colorimetric Determination of Resid- ual Cellulose using Avicel or Cotton. For each Organism, 1 ml of Culture Broth (Fresh or Reconstituted)was Used.

100 -100 A B

Cotton SO Avicel 0 0

0 0 0 60 - W Tr 37C 0 %4- 0 (- Tr 37*C - \ \ s0*C .0., U) 40 Ct 604C

U) Tr 2 20 AVICEL 3rC ;20 \Ct 60*C Tr

Ct r6 609C 0 21 42 0 21 42 Hours of Hydrolysis -65-

Figure 10

Comparison of C. thermocellum and Trichoderma reesei QM 9414 Cellulase Activities on Phosphoric Acid-Swollen Avicel and Microcrystalline Avicel. Fifty *ig of Crude Broth Protein were Used in the Incubations.

' ~ *4 E Avicel Swollen Avicel c

I C .3 .3

- CT C) .2 ,21

C .1 II

CT TA

I I I -J. i 0 L- I I - Id 0 40 80 120 2 4 6 22 Hours Hours -66-

B. Oxidative Inactivation of C. thermocellum Cellulase: Evidence for Essential Sulfhydryls

The stimulatory effect of DTT suggested that sulfhydryl reduction may be important for Clostridium cellulase activity.

When dialyzed culture broth was treated with various concentrations of DTT, it was observed that stimulation occurred at high DTT (10 mM) and surprisingly that inhibition occurred at a lower concentration (0.1 - 0.5 mM) (Figure 11). Activity loss in low DTT was completely prevented or reversed (within 24 h) by addition of 10 mM DTT. Some other enzymes (e.g. rhodanese and glyceraldehyde-3-p-dehydrogenase) show similar behavior with

DTT or other thiols (40, 226), and it has been postulated that

inhibition at a low concentration of thiol is due to the formation of hydrogen peroxide, which inactivates the enzyme:

H 2 C SH H 2 C S

H-C-OH H-C-OH + 02 metal ions +HO HO-C-H 2 HO-C-H

H 2 C SH H 2 C S

The hydrogen peroxide could oxidize cellulase directly or could be converted to hydroxyl radical, which is a very po-

tent oxidant: -67-

Figure 11

Influence of Dithiothreitol Concentration on the Activity of C. thermocellum Cellulase. Activity is Expressed as the Change in O.D.(660 nm) of an Avicel Suspension after 24 Hours.

I I I

--. 2 ,2

.2 .4 r1 5 10 15 20

dithiothreitol c onC c.(mm) -68-

3 Fe 2+ + H 202 -- OH a + OH + Fe +

destructive

To test this, I treated C. thermocellum cellulase with 0.4 mM

DTT under aerobic and anaerobic conditions (Figure 12). Under anaerobic conditions, H 2 0 2 should not be formed, and inactivation should not occur. There was no inhibition of cellulase activity by 0.4 mM DTT in the absence of air (Figure 12); in the presence of air, cellulase was strongly inhibited. Fur- ther, cellulase was inhibited by 0.04 mM H 2 0 2 under aerobic or anaerobic conditions (Figure 12). The inhibition by H 2 0 2 was prevented when 10 mM DTT was added to the reaction mixture

(Figure 13). Inactivation of cellulase by low DTT was partially prevented by adding catalase (10 U/ml) from Aspergillus niger and removing the azide from the incubation, which is inhibitory to catalase (Figure 14). Cellulase was not protected by bovine

superoxide dismutase (10 U/ml), by copper EDTA which is known

to dismutate superoxide ion, or by hydroxyl radical scavengers

including mannitol or sodium formate at 20 mM (Table 2). By

far the best protectant from oxidation was millimolar quanti-

ties of DTT.

The stimulatory effect of DTT on cellulase activity and

the reversible inhibition by H 2 0 2 suggests that there is essen-

tial thiols necessary for activity. Dialyzed cellulase was

treated with various sulfhydryl reagents which differ in their -69-

Figure 12

Influence of Anaerobic and Aerobic Atmospheres on Inhibition of Cellulase by 0.4 mM OTT or 0.04 m.M Hydrogen Peroxide. Al- so Shown is-the Effect of 10 mM OTT. Anaerobic Atmosphere was Obtained by Sealing the Hungate Tubes in a Coy Anaerobic Chamber (90% N 2 :5% CO 2 :5% H 2).

C Aerobic Anaerobic

0.4 E C 0 0.04 mM H20 2

C 0.3- 0.04 mM H 0 0 0.4 mM DT T 2 2 .) C

co 0.2-

C!)

0.1- 10 mM DT T 0.4 or 10 mM DTT

0 40 80 120 0 40 80 120 Hours -70-

Figure 13

inhibition of Cellulase by Hydrogen Peroxide or Low DTT in Air. Open Symbols Represent the Addition of 10 mM DTT after 1 h.

A A

0.4 0.4 mM H 202 E c 0 (0

0.3 0.04 mM H T

(n C

0.2

0.0 0 4 mM H2 0 2 ,. DTT t 0.4 nM 10 mM H20 2 0 .4 mM OTT 0.1 {or d OC

0 20 40 60 Time (Hours) -71-

Figure 14

The Effect of Catalase and Superoxide Dismutase (SOD) on the Inhibition of Cellulase by Low DTT and Air.

0.4-

E C 0 0 (D 0.3

() C- 0.2. 0.4 mM DTT

C- 0.4 mM DTT +SOD a>

0.1

0.4 mM DTT d + Catalase 10 mM DTT 0 10 20 30 40 50 60 70 Time (hours) -72-

Table 2

Effect of Hydroxyl Radical Scavengers on Oxidation of Cellulase by H 20 2 or Low DTT.

%activity

DTT(mM) H 20 2 (mM) Scavenger aerobic anaerobic

1 10 0 none 100 104 2 0.1 0 none 65 100 3 0. 1 0 sodium formate, 20mM 78 100 4 0.1 0 mannitol, 20mM 45 100 5 0. 1 0 ethanol, 300mM 46 101 6 0 0.05 none 59 47 7 0 0.05 sodium formate, 20mM 46 63 8 0 0.05 mannitol, 20mM 56 56 9 0 0.05 ethanol , 300mM 50 47 10 0.1 0.05 none 63 99 11 0.1 0.05 sodium formate, 20mM 70 99 12 0.1 0.05 mannitol, 20mM 89 98 13 0.1 0.05 ethanol, 300mM 54 94 14 0.1 0 DTT, 10mM 100 104 15 0 0.05 DTT, 10mM 96 102 16 0.1 0.05 DTT, 10mM 97 102

100% activity corresponded to the complete degradation of 3mg Avicel by 15.6ug extracellular protein in 65h. -73-

mode of action including N-ethylmaleimide (NEM; addition to double bond), 5,5'-dithiobis-(nitrobenzoic acid) (DTNB; oxidation); o-iodosobenzoate (IB; oxidation); p-chloromercuribenzoic acid (p-CMB; mercaptide formation), and iodoacetate (IA; alkylation). Inhibition occurred in the presence of all these reagents (Table 3) and protection or reversal by DTT varied with the reagents. Complete protection by DTT was observed in the case of DTNB or TB and only partial restoration of activity was observed with NEM, pCMB or IA. The C. thermocellum cellulase was strongly inhibited by 5 iM copper, known to catalyze air oxidation of sulfhydryls. Low concentrations of metal chelators protected the cellulase from air oxidation

(data not shown).

The cellulase of T. reesei is known to be comprised of endo-3-1,4-glucanase and exo--1,4-glucanase components. It was of interest to determine if the endo-glucanase activity in the C. thermocellum cellulase is susceptible to oxidation.

Endo- -glucanase can be measured by depolymerization of CMC, but there is no direct way of measuring exo-3-1,4-glucanase activity in a crude extracellular preparation. I observed that endo-S-glucanase activity in C. thermocellum cellulase was not inhibited by low concentrations of DTT or by H 20 2 (Table 4).

The CMCase activity was also not affected by p-CMB, DTNB, or

NEM, although these are strong inhibitors of true cellulase activity. These results suggest that the exo-f-1,4-glucanase activity is the component sensitive to oxidation. 74-

Table 3

Inhibition of Cellulase 1 by Sulfhydryl Reagents and Copper and Prevention by 10 mM DTT

Addition of % 2 Reagent Added 10 mM DTT Activity

None 71 + 100

1 mM H 2 0 2 29 + 77

2 mM o-iodosobenzoate 5 + 78

0.2 mM 5,5'-dithiobis-2-nitrobenzoic 15 acid (DTNB) + 92

1 mM N-ethylmaleimide (NEM) 35 + 62

1 mM iodoacetic acid (IA) 50 + 70

0.02 mM p-chloromercuribenzoic acid 2 (~pCMB) + 27

5 i'M CuSOg'5H 2O 11 + 98

1 15.6 Lg of dialyzed, extracellular protein was used. Incubations were aerobic + 10 mM DTT.

2 Decrease in turbidity after 48 h. 100% activity corresponded to the complete depolymerization of 3 mg Avicel in 60 h. -75-

Table 4

CM-Cellulase Activity is Not Inhibited by Oxidation or Sulfhydryl Reagents

Treatment Activity(min' )i

None 10

DTT, 10 mM 12

H 2 0 2 , 0.5 mM 11

DTNB, 0.2 mM 10

p-CMB, 20 M 10

7.5 pg of dialyzed enzyme protein was used. No activity was observed in the absence of enzyme. -76-

C. Effect of Chelating Agents on Cellulase Activity: Evi- dence that the C. thermocellum Cellulase Requires Iron for Activity

The experiments described above indicate that inactivation of cellulase in low DTT takes place via the formation of hydrogen peroxide, which then oxidizes essential thiols. Un- expectedly, the apolar chelators o-phenanthroline and 2,2'- dipyridyl (but not the polar chelators EDTA or 8-hydroxyquinoline) were found to strongly inhibit DTT-protected cellulase in an anaerobic atmosphere (Table 5). o-Phenanthroline inhibition did not occur if the chelator was first combined with a mixture of ferrous and ferric iron (Table 6), and was partially prevented if the chelator was mixed with zinc and ferric ions. How- ever, inhibition still occurred if the chelator was first combined with ferrous iron alone. o-Phenanthroline binds zinc and ferrous ions quite strongly, but ferric ions only weakly (Bragg,

1974). It is likely that zinc or ferrous iron tied-up the inhibitory chelator, and enabled ferric ion to stimulate the cellulase activity. It is also possible that the combination of ferric and ferrous ions provided an oxidation potential in which the enzyme was active. Characterization of the role of iron in purified cellulase and redox titrations should dis- tinguish between these hypotheses.

The possibility of iron being a component of cellulase was investigated in collaboration with Drs. Sue Groh and W. H. -77-

Table 5

Effect of Chelating Agents on Cellulase Activity Under Anaerobic , Reduced Conditions

Chelator Conc. (mM) % Activity 2

None - 100

o-phenanthroline 1 77 4 71 10 45 20 21

8-hydroxyquinoline 1 100 2.5 99

EDTA 1 100

2,2'-dipyridvl 5 83 10 78 20 54

i Experimental conditions identical to Table 3 except that air atmosphere was replaced by 90% N2:5% CO2:5% H2. DTT concentration was 10 mM.

2 100% activity corresponded to the complete depolymerization of 3 mg Avicel in 65 h. -78-

Table 6

Reversal of o-Phenanthroline (OP) Inhibition of Cellulase by Prior Chelation1 of OP with Metals

Addition % Activity

None 100

20 mM OP 29

+ 6 mM Fe2+ 34

+ 6 mM Zn2+ 34

3 + 0.5 mM Fe 29

+ 6 mM Fe 2 + + 0.5 mM Fe 3 + 100

+ 6 mM Zn2+ + 0.5 mM Fe3 + 69

Metals (as dissolved as chloride salts in HCl) were combined with OP before addition to enzyme incubation. Ex- perimental conditions identical to Table 5. Inactive were manganese, cobalt, copper, molybdenum, magnesium and nickel. -79-

Orme-Johnson of MIT's Chemistry Department. Chemical analysis of dialyzed, crude cellulase (312 pg protein per ml) showed the presence of 0.12 mM iron. Electron paramagnetic spectroscopy

(epr) revealed that there was a high concentration of high spin

ferric iron associated with the proteins. The iron signal increased in intensity in a preparation of cellulase partially purified by ion-exchange chromatography. The epr analysis

showed that the iron, however, is bound non-specifically, i.e.,

it is not present in heme or an iron-sulfur cluster, and there-

fore the only definitive way to show its involvement in cellu-

lolytic activity is by complete purification of the cellulase

and detection of iron. The presence of ferric iron in extracellular hydrolytic enzymes is very unusual, having only been

reported in the periplasmic acid phosphatase from the yeast,

Saccharomyces rouxii (10).

D. End Products of Avicel Saccharification

In the previous results of this study, I showed that the

cellulase from C. thermocellum has a specific activity on crys-

talline cellulose 50 to 70 times higher than reconstituted

Trichoderma reesei (146). This high activity is observed when

a low concentration of purified, crystalline cellulose (e.g.

cotton or Avicel) is used as substrate and aerobic incubation

is carried out in the presence of a sulfhydryl reducing agent

and calcium. However, when the Avicel concentration was in- -80-

creased to 3 g/l '(5-fold increase), cellulolysis in air stopped after one-quarter of the cellulose was digested (Figure 15), probably due to oxidation of the cellulase. In an atmosphere of 90% N2 : 5% H 2 : 5% CO 2 (anaerobic) the reaction went to completion.

Achieving complete saccharification anaerobically of a reasonable cellulose concentration (3 g/1) provided the means to determine sugar products formed during the digestion of microcrystalline Avicel. Previously, Gordon (75) showed that cellobiose was the principal product from the digestion of phosphoric acid-swollen cellulose, which is non-crystalline.

Small quantities of cellotriose and glucose were also formed.

Similarly, I found cellobiose to be the major product of Avicel saccharification at all stages of digestion (Figure 16). Cel- lobiose was assayed by HPLC; this major peak cochromatographed with a cellobiose standard, and was converted entirely to glucose when treated with -glucosidase from Aspergillus phoenicii

(data not shown). I also detected smaller concentrations of glucose, which increased in the later stages of saccharification, especially at the higher substrate level. No cellotriose was detected. -81-

Figure 15

Solubilization of 3 g/1 Avicel by 7 ig/ml Dialyzed Extracellular Protein in an Aerobic or Anaerobic (90% N2:5% C02:5% H2) Atmosphere. DTT and CaCl 2 were 10 mM.

E C 1.2- 0 Aerobic

C 0

c.0.8

- 0.4 0 Anaerobic

41 0 40 80

Hours -82-

Figure 16

Product Formation (Cellobiose, O ; Glucose, 0 ) During Saccharif- ication of Avicel by C. thermocellum Dialyzed Culture Broth. The Reactions on 3 g/l or 0.6 g/l Avicel were Conducted in Hun- gate Tubes in an Anaerobic Atmosphere. Saccharification was Followed by Turbidity (o) and the Products were Determined by HPLC on a Column of HPX-87-H (Bio-Rad). DTT and CaCl 2 were 10 MM.

3 g/l Avical A 1.2 3

1'0

E 0.8 Cellobios e42

0 (D)0 0.6

Glucose 0 0.4 0 C: 0.2 0 0 Q.)

0 0 20 40 60 0 0.4w 0.6 g/l Avical a -- 0.3 0.6 0 Cellobiose <0 00

0.1 0.2 Glucose 0 20 30 Hours of Hydrolysis -83-

E. Inhibition of Cellulase Activity by End Products of Cellulolysis

The next series of experiments describes the inhibition of C. thermocellum cellulase by the end-products, cellobiose and glucose. When microcrystalline cellulose (Avicel) and untreated culture broth were incubated in the standard assay (10 mM DTT and Ca 2+, aerobic, see Materials and Methods) it was found that

C. thermocellum cellulase is strongly inhibited by cellobiose and is much less susceptible to inhibition by glucose (Figure

17). Approximately 50% inhibition occurred at 2.5 mg/ml cellobiose and the cellulase was completely inhibited at 20 mg/ml. A maximum of 20% inhibition was observed with 60 mg/ml glucose.

The C. thermocellum cellulase was not inhibited strongly when the substrate was non-crystalline; a maximum of 50% inhibition by cellobiose was observed on phosphoric acid-swollen cellulose

(Figure 18) and no inhibition occurred when TNP-CMC was the substrate (data not shown).

Inhibition of the Clostridium and Trichoderma cellulases were compared (Figure 17). The fungal cellulase was less inhibited by cellobiose than the bacterial cellulase at equal broth volumes. The T. reesei cellulase was much more strongly inhibited by glucose; this may be due to the presence of -glucosidase in the fungal broth, which is sensitive to inhibition by glucose. a-glucosidase has not been found to any significant level in the Clostridium culture broths. -84-

Figure 17

Inhibition of Cellulase Activity in Untreated Culture Broths of C. thermocellum and T. reesei by Cellobiose (A) or Glucose (B). Various Volumes of Culture Broth were Used. Avicel was the Substrate and the Duration of the Experiment was 20 h. Maximal (100%) Activity for C. thermocellum Corresponds to the Degradation of 0.73 mg (0.1 ml Containing 20 ,.g Protein), 0.93 mg (0.2 ml; 40 ig protein) and 1.7 mg (0.5 ml, 100 ,ig Protein). For T. reesei, 100% Corresponds to 1.7 mg (0.5 ml Containing 6 mg Protein). A. Cellobiose 100

80 Tr (0.5ml)

Cl (0.SmI) 40

Ct (0.I2mI 2d~

C (0.1"t)

4-rn I 0 A I B. Glucose C Ct (0.5m) 0

0~ Ct (0.(m)

40 T r (o0 SmO)

20I

foJ 0 S 10 15 20 40 60 Sugar (mq/ml) -85-

Figure 18

Inhibition of C. thermocellum Cellulase by Cellobiose (e) or Glucose (o) on Phosphoric Acid-Swollen Avicel. Activity was Measured by Decrease in Turbidity of a Swollen Cellulose Sus- pension after 3 h Incubation at 601C. Fifty 4g of Crude De- salted Enzyme was used in this Experiment.

Avicel Swollen Avicel 100 100

o Glucose 80 80

60- 60

Cellob iose 40 40

20. 20

Cel lobiose 0 I I it 0 0 10 20 30 40 50 60 0 10 20 30 40 50

Sug ar ( m g/ m I) Sugar (mg/ml) -86-

As shown above, cellobiose but not glucose is an inhib-

itor of C. thermocellum cellulase activity. To determine whe-

ther -glucosidase would benefit the activity of clostridial

cellulase by destroying cellobiose, a -glucosidase preparation was incubated (10 units/ml) with the bacterial cellulase at 45*C

(Table 7). The addition of S-glucosidase promoted a 16% in-

crease in cellulose hydrolysis in the absence of added cello-

biose and increased the activity five-fold in the presence of

cellobiose. Similar results were obtained with a heat-stable

a-glucosidase from Aspergillus phoenicis (Figure 19). Glucose

did not affect activity in the presence or absence of added B-

glucosidase.

A number of sugars other than cellobiose and glucose

were tested to see whether they inhibited C. thermocellum cellu-

lase (Table 8). Certain glucosides and galactosides, e.g. sali-

cin, lactose and arbutin were fairly inhibitory, but none was

as inhibitory as cellobiose.

F. Partial Purification of Cellulase

Partial purification of cellulase was done to determine

the proteins involved in cellulolysis and to provide a means by

which changes in activity observed during growth on various car-

bon sources (see later) could be attributed to activation of

the cellulase or to changes in the biosynthesis of cellulase

proteins in the broth. Table 7

Influence of O-Glucosidase on Cellulase Hydrolysis by C. thermocellum Cellulasel

Additions Activity (mg Avicel Variation 0-glucosidase Cellobiose Glucose Cellulase hydrolyzed in 24 h) (10 units/ml) (10 mg/ml) (10 mg/ml)

1 - + 1.16

2 + - + 0.24 I 3 + - + 1.35 00 4 + + - + 1.29

5 + + + + 1.29

6 + + 4 1.29

7 + + ~ 1.16

8 + 0

1 Reactions were done at 4500 in succinate buffer with Avicel as substrate as described in Methods. -88-

Figure 19

Relief of Cellobiose Inhibition of Cellulase Activity by S- Glucosidase from Aspergillus phoenicis. Fifty uil of Dial- yzed Culture Broth were Used.

0.4<

-..+ Cellobiose (10 mg/ml) C 0 0 0.3 + Cellobiose +,-Glucosidase + Glucose (10 mg/ml) 0

C > 0.2-

U) +Cellobiose +jS-Glucosidase (10 Units/mI)

0.1

No +,-Glucosidase or oAdto +-Glucosidase +Glucose

0 20 60 100 Hours -89-

Table 8

INHIBITION OF C. THERMOCELLUM CELLULASE BY VARIOUS CARBOHYDRATES'

Concentration Activity Inhibitor (mg/ml) (%) none 100 glucose 40 76 2-deoxyglucose 40 67 glucose- 1-phosphate 3 100 methyl- -D - glucoside 40 33 gentiobiose 40 42 maltose 40 62 trehalose 40 83 sucrose 40 100 cellobiose 10 10 20 0 40 0 lactose 10 67 20 50 40 17 60 12 arbutin 10 67 20 51 40 18 60 12 salicin 10 45 20 36 40 17 xylan 3 100 laminarin 3 100

1 Assays were done with Avicel as substrate. Maximum (100%) activity corresponds to 1.21 mg Avicel solubilization in 24 h. -90-

The source of cellulase was the cell-free fluid from a culture grown in a 100 liter fermentor on Solka-floc (see Mate- rials and Methods). It has retained approximately 80% of its activity during 2 years of storage at -200C. The proteins were concentrated by freeze-drying, reconstituted in water, and dialyzed for 4 h at room temperature against 4-5 changes of 20 mM

Tris-HCl, pH 8.8. It was found that large losses of activity

(> 80%) occurred during ammonium sulfate precipitation, and this method was therefore not used for concentration.

The dialyzed, crude extracellular preparation had a greenish-brown color, and had a slight absorbance in the visible range (Figure 20). In addition to the usual protein absorbance at 280 nm, the dialyzed-concentrate had a very high absorbance at 260 nm. It is not known whether this is due to binding of nucleotides or other UV-absorbing materials by the extracellular proteins.

The cellulase was initially purified by column chromatography on DEAE-sepharose (Figure 21); the cellulase bound tightly to the ion-exchanger, and most of the contaminating proteins were eluted before the cellulase in a KCl gradient.

The cellulase eluted with part of the main protein peak; therefore it was probably contaminated with much protein; in addition, large losses of activity (- 80-95%) took place during ion exchange chromatography. The active fractions were then concentrated and applied to a gel filtration column (Ultrogel -91-

Figure 20

Visible and Ultraviolet Adsorption Spectrum of Dialyzed Cellu- lase in 20 mM Tris, pH 8.8

- -V:

7- f------HTET~z.z~z I

- -

17,~ 4- t C0 - }- -

- S- ~ -- ~ -

, - 4---- -

0 PQ w 0n 0 02 WAVELENGTH -92-

Figure 21

DEAE-Sepharose Chromatography of C. thermocellum Extracellular Protein. The Protein was Dialyzed Against 20 mM Tris-HCl, pH 8.8, and Applied to the Column Equilibrated with the Same Buf- fer. A KCl Gradient (0-0.5 M) was Run from Fractions 8 to 40. Five ml Fractions were Collected and Analyzed for Protein and Cellulase Activity.

180-

Cellulase -3

0.5M KCI

Protein

100 - A / 2

xE A-1= 100 -,

20- - 20 a0.

0 10 20 30 40 fraction -93-

ACA 22). The cellulase chromatographed as a large complex, eluting in the void volume (MW > 1.5 x 10 6) (Figure 22). Active fractions (18-22) were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 7% acrylamide gel (Figure 23); three major bands of large molecular weight were observed (MW

218 K, 112 K and 85 K). In addition, several minor bands were observed. A major contaminating protein in the DEAE-treated protein was removed by gel filtration (lane C).

A second preparation was purified as described above.

Again, a large loss (92%) of activity occurred during chromatography on DEAE-sepharose, whereas the recovery from gel filtration exceeded 100%. The purified preparation was electrophoresed by SDS-PAGE (using a 5% acrylamide gel) and compared to the crude (Figure 24); it appears that cellulase comprises 2 major and 1 minor bands of - 100 K, 78 K, and 220 K, respectively. It is difficult to test whether these proteins are components of a pure enzyme, since it forms a large complex that barely migrates into a native polyacrylamide gel (3% - 12% gradient concentration of acrylamide) and chromatographs in the void volume of Ultrogel ACA 22, whose exclusion limit is > 1.5 6 x 106. Thus, it is necessary to find a gel filtration medium with a large exclusion, e.g. 2-5 x 10 6, or to use ultracentrifu-

gation to test for purity. It is interesting that other anaerobic bacteria are also known to produce extremely large cellulo-

lytic complexes (183, 25 ). This characteristic is different 300

,ftI a A I IuIlase c c 0 0 T 10 200 0 L Ui) c Protein

4i) 0 C,)

\00 7 - 5 4- C

0 9 w I L -L 10 20 30 40 50 Fraction

Figure 22

Gel Filtration of Dialyzed, Total Extracellular Protein on Ultrogel ACA 22. The Protein (2 ml, - 15 I,/ml, - 0.5 mg protein/ml) was Ap- plied to Ultrogel Previously Equilibrated with 50 mM Tris-HCl, pH 7.1, 100 mM NaCl, and 2.7 nil Fractions were Collected. -95-

Figure 23

SDS-Polyacrylamide Gel Electrophoretic (SDS-PAGE) Patterns (7% Acrylamide Gel) of C. thermocellum Extracellular Proteins Pur- ified by DEAE-Sepharose and Ultrogel ACA 22 Chromatography. Lane A, Molecular Weight Standards (Sigma Kit MW-SDS-200): My- osin, 205,000 Daltons; -Galactosidase, 116; Phosphorylase B, 97,400 D; Bovine Albumin, 66,000 D; Egg Albumin, 45,000 D; Carbonic Anhydrase,29,000 Daltons. Proteins were Electropho- resed at 20( mA to Load a 3% Stacking Gel, then at 40 mA per Running Gel, Removed and Stained in Coomassie Blue. Lane B, Cellulase Activity Fraction (80 pl) after DEAE-Sepharose and Ultrogel Treatment; Lane C, Non-Cellulolytic Fraction (80 'l) from Same Treatment Recovered from Ultrogel; Lane D, Same as B except 20 pl were Applied. - 95a-

a b C d

-218 K 205Km-

~ L

116Kbmo - M-112 K 97 K-

8 85K

66 K- ie. ~

45K-

I- A

q.s.ummd -96-

Figure 24

SDS-PAGE of Purified Cellulase Proteins. Lane A, Standards (see Legend to Fig. 23); Lane B, Crude, Dialyzed Extracellu- lar Proteins; Lane C, Purified Cellulase. Conditions were the Same as in Fig. 23 except a 5% Acrylamide Gel was Used. a b c

4f .

.IIL -97-

from the cellulases of aerobic fungi, which readily separate in

solution into exo- and endo-glucanases.

The large losses of activity (~ 90%) that occur during

ion-exchange chromatography were eliminated by using HPLC with an ion-exchange vinyl resin column. Using HPLC, 70-80% of the

cellulase activity was routinely recovered. This procedure was very rapid (30-60 min), and resolved the extracellular protein mixture into many more peaks than had been obtained on an open

column (Figure 25, compare to Figure 21). The versatility of

the machine allowed the programming of non-linear gradients to achieve optimal separation (Figure 26). Two separations by HPLC were compared. In the first, 3 ml of crude cellulase was

applied, and a linear gradient was run to 0.5 M, during which most of the contaminating proteins were separated. Then, the

salt concentration was stepped to 1 M, eluting the remainder of

the cellulase. However, the cellulase preparation obtained was

contaminated with many non-cellulolytic proteins (Figure 27,

lane a). We decided (seeFigure 26) to run a non-linear gra-

dient to 0.3 M salt to get rid of contaminating proteins, then

to step to 0.7 M KCl to elute the cellulase, and finally to step

to 1 M to eliminate further contaminants. This technique was

applied on a preparative scale: 40 ml of crude (- 20 mg pro-

tein, 600 units cellulase) were applied, and chromatographed as

described above. The cellulase recovery was 75%, and the prep-

aration obtained had 3 major bands (Figure 28). It is likely -98-

Figure 25

Ion-Exchange HPLC Chromatography of Dialyzed, Extracellular Proteins. Three ml of Dialyzed Broth was Applied to the Ion- Exchange Column, the Column was Washed with 20 m.M Tris-HCl, pH 8.8, a Linear KC1 Gradient to 0.5 M KC1 was Applied, and Finally the Salt Concentration was Stepped to 1 M to Elute the Remaining Proteins. The Recovery of Cellulase was 79%.

------r I O.D. 280 nm Cellulasel 4

2 K C I

f\ ,

0 20 40 Time (min) -99-

Figure 26 Ion-Exchange HPLC Chromatography of Dialyzed, Extracellular Proteins. Same Procedure as Fig. 25 except 2 ml Crude Cel- lulase was Applied, and a Non-Linear Gradient was Applied as Described in the Text. Recovery was 74%.

-- CIM PI I I 60 jI ri 0.D. 280 nm....I~ h II 401 0.5 ci, CeIuIa% se Co I~~tii liii II I ci) ,4vAsj () 20F- I \ // I 01 0 -I -J II 0 30 60 Time (min) -100-

Figure 27

SDS-PAGE of Crude Cellulase from C. thermocellum Grown in Dif- ferent Carbon Sources and Partially Purified by HPLC (see Fig. 25). Lane A, Active Cellulose Fraction from HPLC; Lane B, Broth from Cells Grown in MI-cb Medium; Lane C, Broth from MJ- Avicel Medium; Land D, Broth from MJ-Fructose. Eighty pl of Sample were Electrophoresed. For Examination of Broths, Three ml Samples were Dialyzed, Lyophilized, and Redissolved in 0.5 ml 50 mM Tris-HCl, pH 7.1. Arrows at Left of Figure Point to Probable Cellulase Proteins Based on Previous Characterization. Lane E Contains Molecular Weight Standards.

a b C d e

!PV

* . - -101-

Figure 28

SDS-PAGE of Fractions Obtained from Preparative HPLC (Lanes A- E) (see Text). Also Electrophoresed are Crude Broths; Lane F, MJ-Fructose; Lane G, MJ-Avicel; Lane H, MJ-Cellobiose. Molec- ular Weight Standards were Run in Lane I. Arrows at Left Point to Probable Cellulase Proteins.

a b C d e f g h r

218-

-205

-116 112-- -102-

that these are components of the C. thermocellum cellulase. The final purification of cellulase from C. thermocellum depends on the utilization of a technique after HPLC which can separate the cellulase based on its size.

G. Construction of a Defined Medium for C. thermocellum (113)

To study the regulation of cellulase synthesis by C. thermocellum it is desirable to use a minimal, chemically defined growth medium. Unfortunately, only a complicated defined medium has been described previously (60), which probably contains several unnecessary nutrients. The next few experiments describe the development of a minimal medium.

Early studies by Madia and Demain (personal communication) noted requirements for biotin and vitamin B 6 ; the latter was supplied by pyridoxine. However, this medium failed to sus-

tain growth through repeated transfers. Pyridoxal or pyridoxamine was next shown to support higher growth rates than pyridoxine, but this was not sufficient for serial subculture.

Superiority of pyridoxamine and pyridoxal over pyridoxine has

been observed with other bacteria (42). It was next noted that methionine addition to biotin and pyridoxamine showed a posi-

tive effect on growth, but again it was not sufficient for serial

subculture. Since auxotrophic requirements for vitamin B 1 2 or -103-

p-aminobenzoic acid are sometimes compensated for by methionine

(43), these vitamins were added to biotin plus pyridoxamine.

Not only was good growth obtained (Table 9), but also the culture remained vigorous through at least 10 subcultures.

The chemically defined medium (MJ medium) is described in Table 10. It is identical to the complex medium GS-2, with the exception that 6 g of yeast extract per liter is replaced by 2 mg of pyridoxamine hydrochloride, 0.4 mg of p-aminobenzoic acid, 0.2 mg of biotin, and 0.2 mg of vitamin B 1 2 per liter.

This minimal medium supports good growth, product formation, and cellulase synthesis by C. thermocellum (Table 11), and also supports the growth of other C. thermocellum strains including

LQ8 (170), NCIB (2), and the C-9 ethanol-tolerant mutant developed by Herrero and Gomez (92). However, the ethanol-tolerant, lactate non-producing strain S7-19, developed by Avgerinos (un- published), was found to require supplementary addition of tryp- tophan for growth (data not shown).

It can be seen from Table 10 that MJ medium contains sodium citrate (3 g/l) to prevent precipitation of salts. This addition, however, may cause a decrease in cellulase titer (K.

Shimada and A. L. Demain, personal communication).

H. Control of Cellulase Synthesis in C. thermocellum

Previous studies (3, 50, 68, 87) on the regulation of synthesis of C. thermocellum cellulase have concluded that car- Table 9

Vitamin Requirements of C. thermocellum ATCC 274051

Additives to GS-2 Medium Without Yeast Extract (mg/liter)

Biotin Pyridoxamine p-Aminobenzoic B12 Pyridoxine Pyridoxal Growth at 40 h (0.2) (2) Acid (0.4) (0.1) (2) (2) (absorbance at 660 nn)

+ + + + - - 0.70

H + - + + - + 0.75 Q

- + + + - - 0.10

+ - + + - - 0.01

+ + - + - - 0.02

+ + + - - - 0.02

+ - + + + - 0.05

1 GS-2 medium supported growth to an absorbance of 0.87. -105-

Table 10

Composition of CM3, GS and MJ Media

Ingredients CM3 GS' MJ

KH 2 PO 4 1.5 g 1.5 g 1.5 g

K 2HPO 4 2.9 g 2.9 g 2.9 g

(NH4 )2 SO 4 1.3 g - - Urea - 2.1 g 2.1 g

MgCl2 . 6H2 0 1.0 g 1.0 g 1.0 g

CaC 2 .2H2 0 150 mg 150 mg 150 mg

FeSO 4. 6H 2 0 1.25 mg 1.25 mg 1.25 mg Cysteine-hydrochloride 1.0 g 1.0 g 1.0 g Resazurin 2.0 mg 2.0 mg 2.0 mg Cellobiose 10.0 g 10.0 g 10.0 g Morpholinopropane sul- - 10.0 g 10.0 g fonic acid Yeast extract 2.0 g 6.0 g - Pyridoxamine hydrochloride - - 2.0 mg- Biotin - - 0.2 mg p-Aminobenzoic acid - - 0.4 mg

Vitamin B1 2 - - 0.2 mg

Sodium citrate.2H 2 0 - - 3.0 g

Final volume 1 liter 1 liter 1 liter pH 7.0 7.4 7.4

GS-2 is identical to GS except it contains 3 g/l sodium citrate. -106-

Table 11 FORMATION OF ETHANOL, ACETIC ACID, LACTIC ACID, AND AVICEL HYDROLYZING ACTIVITY IN MJ AND GS-2 MEDIA'

Dry 2 Acetic Lactic cell wt. 1 Ethanol Acid Acid Blue Medium (g/liter) (h ) (g/liter) (g/liter) (g/liter) Avicelase

MJ 0.43 0.29 1.15 0.45 0.90 0.10

GS-2 0.47 0.42 1.20 0.52 0.73 0.25

Cultures were grown for 24 h at 600C.

2 Specific growth rate.

3 Expressed as change in optical density (595 nm) after 24 h by 0. 1 ml culture broth. This degree of activity is equivalent to that shown by a lyophilized broth filtrate of Trichoderma reesei QM 9414 when used at 0.1 mg of protein per ml of assay mixture. -107-

boxymethylcellulase (CMCase) is a constitutive enzyme that is produced in relatively constant specific titers irrespective of the conditions of growth. With the finding of true cellulase activity in culture fluids from C. thermocellum and the construction of a defined medium, it became possible to rein- vestigate the regulation of cellulase. It is shown in this section of the progress report that true cellulase formation is strongly influenced by the metabolism of the carbon substrate.

The ability to grow on cellulose is a stable character in C. thermocellum populations. When C. thermocellum was ser- ially transferred to cellobiose medium, and then plated on cellulose agar in a glove box under anaerobic conditions, the entire population grew and formed clearing zones. Single colonies were picked and grown in cellobiose broth as inoculum, and then transferred to fructose, sorbitol or glucose (Table

12). C. thermocellum initially grew very poorly on these carbon sources from a cellobiose inoculum. The poor growth in fructose and sorbitol (but not the poor growth in glucose) was accompanied by a 9-fold increase in the specific titer of cellulase. When broths from cellobiose and fructose cultures were mixed, no inhibition of cellulase activity was observed, suggesting that the difference in activities was due to different amounts of cellulase proteins.

The kinetics of growth and cellulase formation were examined in fructose and cellobiose (Figure 29). Growth was rapid -108-

Table 12

Cellulase Synthesis by Cells Previously Grown in Cellobiose when Transferred as A Small Inoculum (1% v/v) to Different Carbon Sources

axim um DCW1 Maximum Cellulase Medium lag (h) (mg/ml) (units/ml) (units/mg DCW)

No C Source 0 0.08 0.16 2.0 (starved)

Cellobiose 0 0.48 3.6 7.5

Glucose 120 0.26 2.7 10.7

Fructose 110 0.13 5.9 45.4

Sorbitol 84 0.17 6.4 37.6

1 Cultures were harvested after reaching stationary phase. -109-

Figure 29

Growth and Cellulase Formation in Cellobiose or Fructose De- fined Medium. Carbon Sources were 10 g/l. The Inoculum (1% v/v) was a Culture Growing Exponentially in Cellobiose. 4 4

cellobiose

2- -2

fructose X

044

2- 2

0 40 8 0 hours -110- in cellobiose and cellulase volumetric titer paralleled growth.

Cells inoculated to fructose from a cellobiose inoculum had a

60-70 h growth lag, during which cellulase readily accumulated in the medium. The accumulation of cellulase as a function of increase in dry cell weight was fifty times higher during adaptation to fructose than during growth on cellobiose (Figure 30).

When the cells began to grow on fructose, the differential rate of cellulase synthesis (159) dropped to 12, still 5-6 times higher than on cellobiose (Figure 30).

The addition of cellobiose to a culture in the lag phase on fructose (Figure 31) caused a rapid burst of growth and ces- sation of cellulase synthesis. The extent of growth and decrease in cellulase synthesis depended on the dose of cellobiose. When the added cellobiose was exhausted, the growth lag on fructose was reestablished and formation of cellulase again occurred at its high rate. Therefore, the addition of a desired carbon source to cells slowly growing on fructose promoted rapid growth and a sharp decline in cellulase synthesis.

The response to cellobiose/fructose is analogous to 3- galactosidase synthesis during diauxic growth of methyl-3-D- thiogalactoside- induced Escherichia coli on glucose plus lactose (54, 194). The glucose is initially metabolized at a rapid rate and little -galactosidase is made. After exhaustion of glucose, -galactosidase is made at a high differential rate during the lag on lactose, which drops slightly when growth be- gins. Under appropriate conditions of growth, not only glucose, -111-

Figure 30

Cellulase Production (units) as a Function of Dry Cell Weight (mg) During the Lag in Fructose or During Exponential Growth in Cellobiose

41-

fru

C', .4.' 0

G) C',

C., 2 F-

0 0.2 0.4 dcw -112-

Figure 31

Effect of the Addition of Cellobiose on Cellulase Formation During the Growth Lag on Fructose 1Or

C.29/1

no additions 6H gi

CE 19/1

2 g/ I

I I I I I 0 0.1 0.2 0.3 0.4 dcw(mg/ml) -113-

but all readily utilizable carbon sources, act as repressors of 3-galactosidase. In an E. coli strain constitutive for 3- galactosidase synthesis, the enzyme titer is inversely corre- lated with the doubling time of the culture (149) and with the quantity of "high energy" phosphate in the cells (165,

142); the poorer the carbon source, the higher is the enzyme activity.

The derepression of cellulase formation during adaptation to fructose, suggests that rapid catabolism of cellobiose

(the principal product of cellulolysis and C. thermocellum's preferred energy source) leads to catabolite repression of cellulase synthesis. This concept was supported by limitation of catabolism by growth on insoluble crystalline cellulose. Under this condition, growth is limited by the supply of soluble carbon source (cellobiose) (136, 243). I observed a major increase in cellulase titer with cellobiose limitation (Table 13). Fur- thermore, when cellobiose was limited by fed-batch addition, the cellulase titer increased. In this experiment (Table 13), surprisingly little variation in protein titer was observed, suggesting the limiting cellulase activity may be a minor component of the extracellular proteins.

The derepression of cellulase formation in fructose was not limited to prior growth in cellobiose; cells previously adapted to glucose and inoculated to fructose also substantially

increased their specific titer of cellulase (Table 14). This high specific titer also dropped when growth was established, Table 13

Cellulase Synthesis by C. thermocellum Grown on Cellobiose, and Inoculated (1% v/v) to Cellobiose or Avicel Media. Cultures were Grown for 60 h.

Maximum Maximum Extracellular Carbon Source DCW Cellulase Protein (final conc. 2 g/1) (mg/m] (units/ml) (units/mg DCW) (pg/m1)

Cellobiose (cb) 0.33 1.4 4.3 62.5 0.22 6.4 29.2 65.0 Avicel (Av) 1-h H cb + Av 0.24 5.6 23.2 70.0

cb1 0.19 2.9 15.3 38.0

Av1 0.14 5.1 36.4 65.0

1 Added in three doses of 0.5 g/l after limitation of growth at each stage. Table 14

Growth and Cellulase Formation by Cells Adapted to Different Carbon Sources

1 maxium Adapted 2 -1 DCW Cellulase Cellulase Inoculum Medium lag (h) p (h ) (ng/hl) (units/ml) (units/mg DCW)

3 Cellobiose Sorbitol 110 nd 0.12 6.5 47 Fructose 110 nd 0.13 6.0 46 Cellobiose 0 0.35 0.38 3.2 8.4 Glucose 125 nd 0.26 2.0 7.9 no C source 0- 0.08 0.12 1.5

Glucose Sorbitol 70 nd 0.14 7.3 53 Hn Fructose 30 nd 0.11 5.1 47 I Cellobiose 0 0.21 0.44 3.3 7.6 Glucose 30 0.18 0.41 1.6 4.0 no C source 0 - 0.04 0 0

Fructose Sorbitol 5 0.20 0.31 0.6 2.0 Fructose 7 0.30 0.43 0.5 1.1 Cellobiose 3 0.45 0.56 0.5 0.9 Glucose 12 0.14 0.47 0.1 0.2

Sorbitol Sorbitol 15 0.20 0.31 0.79 2.5 Fructose 10 0.25 0.39 0.86 2.2 Cellobiose 0 0.25 0.40 0.94 2.3 Glucose 15 0.25 0.38 0.26 0.7 no C source 0 - 0.05 0 0

1 2 Inocula developed for at least 8 serial transfers. Carbon sources were 0.5% (w/v). 3 Exponential growrth did not occur. -116-

indicating that the catabolism of the sugars was responsible for the repression.

It is clear that the formation of extracellular cellulase in C. thermocellum is regulated by the carbon source and growth rate of the cells. Cellobiose is not a unique repressor; the rapid metabolism of other substrates also controls cellulase synthesis. Limiting the supply of cellobiose by growth on Avicel substantially increases the formation of extracellular cellulase. These data suggest that the rate of catabolism and energy production by the cells controls the levels of cellulase.

To test this, I measured ATP levels in cells grown in carbon- source excess (cellobiose) or under carbon-source limitation

(Avicel) (Figure 32). It was observed in cellobiose that ATP concentration reached a very high level during rapid growth, but plummeted when the culture entered stationary phase. In contrast, ATP levels remained low during growth on Avicel, and declined slightly when growth stopped. As previously observed, the cellulase titer was higher in the Avicel medium.

Carbon substrate limitation also occurred during adaptations to fructose, sorbitol or glucose, from a cellobiose inoculum, but only in fructose and sorbitol was an increased specific titer of cellulase observed. Adaptations to fructose and glucose were studied in more detail (Table 15). During the lUO h adaptation to fructose the culture was able to glycolytically ferment the sugar to low quantities of end products and to form relatively low amounts of ATP. A high differential rate of W

-117-

Figure 32

Growth of C. thermocellum on Cellobiose or Avicel

S a I m U *1 ~1 -J 'U CELLOBIOSE CELLS AVICEL U -ATP CELLS

CELLU LASE OA

CELLULASE

ATP

O 40 80 0 40 80 HOURS Table 15

Formation of ATP, End Products and Cellulase During Lags on Fructose and Glucose

Tine Cells Cellulase ATP Acetate Ethanol Lactate C Source (h) (mg/ml) (U/ml) (U/mg) (nruoles/mg) (mg/ml) (rng/ml) (mg/ml) fructose 23 0.06 0.2 3.3 0.57 0.08 0.05 0.03 " 57 0.06 0.45 7.5 0.72 0.11 0.12 0.02 " 100 0.083 1.6 20.0 0.88 0.15 0.17 0.02 I H glucose 23 0.05 0.1 2.0 0.50 0.19 0.03 0.03 03 " 57 0.06 0.17 2.8 0.10 0.13 0.06 0.05 " 100 0.06 0.27 4.5 0.02 0.11 0.08 0.04 no C~source 100 0.05 0.0 0 0.10 0.0 0.0 0.0 cb control 60 0.38 1.1 2.9 n.d. 0.27 0.54 1.97 2 fru control 60 0.40 1.0 2.5 n.d. 0.32 0.96 0.28

1 Fructose inoculum; other inoculated from cellobiose inoculum. 2 Not determined in this experiment, see Table 17. -119-

cellulase synthesis was detected during this slow glycolytic metabolism. In contrast, cells exposed to glucose declined

drastically in ATP levels, and did not accumulate fermentation products, indicating they were blocked in glucose uptake or in

glycolysis. The extremely low ATP concentrations and absence

of glycolytic metabolism in cells exposed to glucose or in the

absence of a carbon source precluded the synthesis of cellu-

lase proteins.

Hernandez suggested (91) that the extended lag (> 100 h)

on glucose from a cellobiose inoculum is caused by the need for

induction of a transport system. However, the lack of uptake

is probably not solely responsible for the lag. Experiments in

Dr. M. Roberts' laboratory (C. Tolman, personal communication)

have demonstrated by 1 3C-NMR that labeled glucose is transported during short incubation periods (< 1 h). Similarly, I observed

that cellobiose-grown cells, washed and resuspended in buffer,

are able to transport glucose, as. indicated by their ability to

form a pH gradient across the membrane (Figure 33). The proton

gradient was rapidly formed in cellobiose or glucose, but not

in fructose or in the absence of a carbon source (data not

shown). The proton gradient was measured by the uptake of in-

organic phosphate (Figure 33), which depends on ApH for its

transport (197), formed by ATP hydrolysis by the membrane ATPase

(197). Thus, cells exposed to glucose deplete their ATP levels

by the ATPase and possibly by glucokinase (177), and cannot re-

generate the energy due to a block in glycolysis, as reflected -120-

Figure 33

Transport of Inorganic Phosphate and Formation of ApH by C. thermocellum

The cells were grown in cellobiose medium and washed and resuspended in NMR buffer (see Materials and Methods). They were dosed with glucose, cellobiose or fructose and the rate of Pi uptake determined by 3 1P-NMR. The numbers indicate minutes of incubation in the presence of sugar. The tall peak is extracellular Pi; intracellular Pi de- velops as peak immediately to the left. Refer to ApH rapidly formed in cellobiose. -121-

glucose cel lobiose fructose

12 1 15

5 5 ~vI~Nv~p 30 10 8

tJV\PV\45 ~AAPPA (~PJ12

Ar 60 -122-

by the absence of end product formation (Table 15). The luciferase assay confirmed that ATP concentration drops to a drastically low level in glucose (Table 15), much lower than detected in the absence of a carbon substrate (Table 15). These results suggest that exposure to glucose causes a depletion of ATP and a paralysis of metabolism. The depletion of ATP might be caused solely by the membrane ATPase, which in Clostridium pasteurianum has the interesting property of being activated by phosphoenolpyruvate and fructose 1,6-bisphosphate (Morris, personal communication). Depletion might also occur by a kinase/phosphatase cycle (86) or by the formation of a reserve polysaccharide such as glycogen (106).

Although the specific titer of cellulase was high in cells adapting to fructose or sorbitol (Table 12), continued cycles of growth on these substrates led to a decrease in cellulase production, until specific titers were obtained which were consistently less than cultures maintained in cellobiose or glucose (Figure 34). It was observed that cells only gradually adapted to fructose. Initially the lag phase was 1OU h, decreas- ing to 30 h on the second transfer, than to 14 hours, and finally to 3 to 4 h; growth reached a constant final cell den-

sity of about 0.4 g dcw/l. This adaptation and rapidity of

fructose utilization occurred concomitantly with the drop in cellulase titer.

Fermentation broths were analyzed by SDS-polyacrylamide gel electrophoresis to determine whether the quantities of cel- -123-

fructose

E sorbitOl

20- 7S

cellobiose

glucose

01 6 24 serial transfer Figure 34

Effect of Serial Transfer on the Specific Titer of Cellulase by C. thermocellum Culture Broths. Cultures were Grown in Cello- biose and then Inoculated to the Various Carbon Sources. Each Transfer Corresponds to Five Mass Doublings. -124-

lulase proteins varied after growth in fructose, cellobiose or

Avicel (Figure 27, page 100). Identical volumes of broth were electrophoresed. Growth was approximately the same on each of the carbon sources and the cellulase activities were: Avicel,

4.0 U/ml; cellobiose, 1.2 U/ml and fructose, 1.0 U/ml. The results show that growth in Avicel yields higher concentration of cellulase components, and confirms that cellulase synthesis is regulated in C. thermocellum.

The drop in cellulase titer on serial transfer in fructose was not caused by the selection of cellulase non-producing mutants, as shown by plating experiments. In the fructose adapted culture, equal numbers of cells grew on cellulose or fructose agar and the cell recovery was more than 70%. Furthermore, when individual cells from the Avicel agar plates were isolated, growth in cellobiose, and transferred back to fructose, they underwent another cycle of increased cellulase synthesis followed in later transfers by a very low specific titer (Figure 35).

These experiments demonstrate that a genetic change had not occurred in the cells.

I considered the possibility that cycles of growth in

fructose or sorbitol depleted the cells of an inducer which was made during growth on the cellulose derivatives (cellobiose or glucose) but not on fructose or sorbitol. Cellobiose or cellodextrins would be plausible inducers of cellulase, especially

since they are initial products of cellulolysis (75), and are known to be made in C. thermocellum extracts from glucose and -125-

10 F.

i U

S U) C

U) U

WE U 20 5i. 0

I I I I I i =A- 20 .40 20 40 mass doublings

Figure 35

Cellulase Synthesis by Cells Adapted to Fructose (Figure 12), Isolated on MJ-Avicel Agar, Picked to Cellobiose Broth for One Transfer, and Reinoculated to Fructose. Growth and Cel- lulase was Assayed During Sequential Cycles of Growth in Fructose Medium. The Different Symbols Represent Individual Isolates. -126-

glucose-l-phosphate (204). However, fructose or sorbitol adapted cultures were not "induced" for cellulase synthesis by one cycle of growth in cellobiose (Table 14); after 15 mass doub-

lings (3 cycles) the cellulase titer gradually returned to the

level on serial transfer in cellobiose (2.1 units/ml) (data not

shown). Fructose-adapted cultures were also not induced by growth in a cell-free medium preconditioned with cellobiose- adapted cells. Although the fructose-adapted cells grew well

in the conditioned medium with fresh cellobiose added as the carbon source, they did not rapidly increase their production of cellulase. Finally, the addition (0.1 g/l) of cellulose derivatives (including glucose, glucose-l-phosphate, cellobiose, cellotriose, cellotetraose, a mixture of soluble dextrins), to

fructose or cellobiose medium failed to boost the production of cellulase from a fructose inoculum in a single transfer (- 5 mass doublings). Cellobiose analogues including lactose, salicin and thiocellobiose were also inactive. Taken together,

the above experiments argue against the requirement for a cellu-

lose-derived inducer in cellulase synthesis.

Analysis of soluble fermentation products from fructose

and cellobiose adapted cultures revealed significantly differ-

ent patterns (Table 16). In fructose, the formation of lactic

acid was almost completely turned off, possibly due to a decrease in fructose-1,6-diphosphate, which activates lactic de-

hydrogenase. The diminished lactic acid formation leaves more

pyruvate available for oxidative decarboxylation, with possible -127-

Table 16

Difference in Product Formation by Cellobiose- or Fructose-Adapted Cells in Cellobiose and Fructose, Respectively

Final Cells EtOH Acetate Lactate C Source pH (g/l) (MM) (mM) (MM)

cb 6.4 0.56 7.6 5.6 4.6

fru 6.9 0.34 4.7 3.3 0.3 -128-

increased ATP yield (Figure 1 ). This was shown directly by measurement of the ATP levels in the cells with firefly luciferase; ATP was elevated in the tructose-adapted cells (Table 17).

The increased decarboxylation of pyruvate also requires that

NADH be reoxidized by a mechanism not involving lactic dehydrogenase (LDH). In the clostridia, this is mainly accomplished by NADH:ferredoxin oxidoreductase (j9) and formation of molecular hydrogen. The reduction of ferredoxin (E0 = -398 mV) by reduced

NAD (E0 = -320 mV) is thermodynamically unfavorable, and is inhibited by the accumulation of low partial pressures of hydrogen gas (77,118Y. Indeed, fructose-adapted cultures did not grow in an atmosphere of hydrogen (Table 18), unless supplemented with cellobiose which probably allowed the formation of lactate. These data suggest that the stronger repression of cellulase synthesis in fructose than in cellobiose adapted cells is related to the increased formation of ATP from decarboxylation of pyruvate. This was supported by the action of inhibitors which block this pathway of pyruvate metabolism. The addition of potassium cyanide, known to shift an alcoholic fermentation to a homolactic fermentation in saccharolytic clostridia (123), caused increased formation ot lactate and significantly increased cellulase production in the culture (Table 19). Similarly, the addition of methyl viologen, which reacts with hydrogenase, blocking the decarboxylation pathway, caused an increase in cellulase formation (Table 19). -129-

Table 17

ATP Levels in C. thermocellum Grown on Soluble Carbon Sources

1 nmoles ATP/ Inoculum C Source mg DCW Growth Phase

cb cb 2.1 + 0.35 exponential

fru fru 3.25 + 0.17 exponential

cb none 1.30

1 Carbon sources were at 5 g/l. -130-

Table 18

Influence of Gas Atmosphere on Growth of Fructose-Adapted Cells

Carbon +0.5 mg/ml Final DCW Atmosphere Source Cellobiose pH (g/l)

N 2 Fructose no 6.7 0.42

" yes 6.8 0.40

H 2 no 7.3 0.14

yes 6.8 0.40

Gas mix no 6.7 0.38

yes 6.7 0.39

1 Glove box atmosphere (90% N 2: 5% H 2 : 5% CO 2 ). -131-

Table 19

Derepression of Cellulase Synthesis in Fructose-Adapted Cells by Inhibitors of Pyruvate Decarboxylation

DCW Cellulase Lactic Additioni (mg/ml) U/ml U/mg DCW Acid (mrM)

None 0.44 0.7 1.6 0.2

10 mM KCN 0.36 2.6 7.2 2.7 2 5 mM KCN 0.33 2.7 8.2 nd

1 iM methyl viologen 0.31 2.9 9.4 nd

2.5 PM methyl viologen 0.30 3.4 11.3 nd

1 mM dinitrophenol 0.37 1.8 4.9 nd

The carbon source was fructose (5 g/1). Additions were made to freshly prepared media before inoculation with a log phase culture in fructose medium.

Not determined. -132-

The results presented above indicate that cellulase is constitutively produced on the different carbon substrates, but that its differential rate of synthesis is controlled by catabolite repression (141). This was further supported by the increased cellulase synthesis observed when the cells were treated with various energy metabolism inhibitors (Table 20). For example, treatments which are known to dissipate pH gradients resulted in increased cellulase formation, due to the dissipation of cellular energy levels (Table 20). For example, an uncoupler

(carbonylcyanide m-chlorophenylhydrazone, CCCP), an ATPase inhibitor (N,N'-dicyclohexylcarbodimide, DCCD), and organic acids, all which dissipate the pH gradient (193, 12b) and decrease ATP levels (126), stimulated cellulase accumulation. On the other hand, imposing a pH gradient by adding a strong acid (HCl) or a membrane-permeable weak base (Tris) reduced the production of cellulase. These treatments are tigntly linked to the phosphorylation potential, and either increase (e.g. HCl) or decrease (e.g. CCCP) the energy sufficiency of the cells.

In conclusion, cellulase in C. thermocellum is a constitutively produced enzyme system, whose rate of formation is regulated by the rate of catabolism and energy sufficiency of the cells. The molecular mechanism of this regulation is not known; the addition of 2-10 mM cAMP or cGmP had no effect on growth or cellulase formation in C. thermocellum (data not shown), although the cAMP dibutyryl derivative did stimulate cellulase formation in cellobiose medium (Table 21). -133-

Table 20

Changes in Cellulase Synthesis from Treatments which Affect ATP Accumulation and Formation of ApH

Final DCW C Source Addition pH (mg/ml) U/ml U/mg

Expt. I:

fru None 6.6 0.42 1.0 2.4 10 PM CCCP 6.7 0.25 2.0 8.0 100 PM CCCP 6.8 0.17 4.1 24 10 mM HCi 6.3 0.34 0.25 0.8 20 mM KOH 6.9 0.50 1.7 3.4 5 pM DCCD 6.9 0.37 3.2 8.7

cb None 6.25 0.39 2.5 6.4 10 PM CCCP 6.3 0.25 2.4 9.6 10 mM HCi 6.2 0.36 0.13 0.36

Expt. II:

fru None 0.42 0.7 1.6 Sodium pyruvate, 2.5 mM 0.38 4.6 12.1 Sodium fumarate, 12.5 mM 0.34 3.1 9.1 Sodium acetate, 4 mM 0.46 1.7 3.7 Tris, 4 mM 0.52 0.4 0.8

1 Additions were made to freshly prepared media before inoculation with a log phase culture growing on the same carbon source used in the experiments. -134-

Table 21

Effect of Cyclic Nucleotides on Cellulase Synthesis in Cellobiose Medium

D.C.W. Cellulase (mg/ml) (U/ml) U/mg

1 None 0.53 0.80 1.50

2 0.025 mM dibutyryl cGMP 0.33 0.45 1.30

3 0.025 mM dibutyryl cAMP 0.50 1.50 3.00

4 0.1 mM " " 0.50 1.70 3.40 -135-

4. DISCUSSION

The present study shows clearly the presence of true cellulase activity in a bacterial extracellular preparation. C. thermocellum produces an extracellular enzyme which, in the presence of Ca2+ and DTT, has the ability to solubilize native and derived forms of cellulose (cotton, filter paper and Avicel) at a rate and to an extent comparable with T. reesei cellulase.

The crude clostridial cellulase works effectively at protein concentrations fifty times less than the Trichoderma enzyme, and thus appears to have a much higher specific activity. Fur- thermore, the clostridial cellulase comprises only 30-35% of the crude broth protein, as shown by partial purification, and therefore probably has a specific activity at least 100-fold improved over the Trichoderma cellulase. The protein secreted by T. reesei is known to be at least 85% cellulase (7). It should be stressed, however, that low concentrations (< 3 g/l) of cellulose were used in the present study, and saccharification of concentrated slurries of cellulose (e.g. 15%) have not yet been accomplished with the bacterial enzyme.

The Clostridium cellulase displays an unusual preference for highly crystalline substrates. Filter paper was the preferred substrate for the Trichoderma cellulase but was a poor substrate

for the bacterial cellulase in the initial stages of hydrolysis.

In contrast, cotton presented less of a problem to the Clostridium -136-

enzyme then to the Trichoderma cellulase. This high specific activity on resistant substrates reflects C. thermocellum's ability to proliferate under thermophilic, anaerobic conditions on partially digested plant tissues. As an anaerobe depending solely on glycolysis for its cellular energy, C. thermocellum cannot afford to produce high quantities of extracellular protein.

I observed two biochemical properties of the C. thermocellum cellulase which probably contribute to its effective solubilization of cellulose. The crude enzyme contains sulfhydryl groups essential for activity and may also employ iron in cellulose depolymerization. The participation of sulfhydryl groups has been demonstrated in hydrolytic enzymes including papain and ficin (224) in which the sulfhydryl acts directly as a nucleo- philic catalyst. Sulfhydryls also have a strong affinity for metals, such as Ca 2+, Cu2+ and Fe3+ (224, 250). The lecithinase from Bacillus cereus and the a-toxin from Clostridium welchii are activated by Ca2+ (32), possibly due to protection of SH groups from oxidation. The binding of ferric iron to protein may provide a strong acid catalyst (250). I found that the cellulase from C. thermocellum was inhibited by o-phenanthroline, and this inhibition was reversed by the addition of iron. Fur- thermore, clostridial cellulase purified by dialysis and column chromatography contained a high concentration of ferric ion.

These results suggest iron as a component in the cellulase. -137-

Iron is not commonly found in hydrolytic enzymes, although fer-

ric iron was recently reported to stimulate the acid phosphatase

from Saccharomyces rouxii (10). It is interesting that the aldo-

lase isolated from clostridia is a thiol enzyme which requires

iron (14), unlike the aldolase from enteric bacteria.

The sensitivity of the Clostridium cellulase to oxidation clearly differentiates the bacterial enzyme complex from the

cellulases of aerobic fungi including Sporotrichum pulverulen-

turn, Polyporus adustus, Myrothecium verrucaria and Trichoderma

viride, whose cellulases work most effectively in the presence of oxygen (56). The cellulase from T. reesei is not reported

to have essential sulfhydryls and is inactivated by 3.2 mM DTT

(190). This is probably due to the disruption of stabilizing disulfide bonds. It is not surprising that T. reesei does not

contain sulfhydryl groups in its extracellular cellulase, since

it operates in an aerobic environment. The presence of free

sulfhydryls (i.e., cysteine) in extracellular proteins of aero-

bic organisms is rare (57), and nearly all the cysteine residues

are present as the disulfide cystine (222). The presence of

cystine residues in aerobic extracellular proteins is generally

conservative, without much variation in positioning of the cys-

tines or their frequency of appearance (222). In contrast, free

cysteine residues appear sporadically, and are usually elimina-

ted by natural selection, since their exposure in aerobic or-

ganisms leads to drastic oxidation and polymerization (222). -138-

The occurrence of cysteine or cystine in extracellular proteins in anaerobic, facultative and aerobic bacteria was considered by Fahey et al. (57) to be determined by the oxidation state of the environment in which the protein functions. For example, clostripain from the anaerobe Clostridium histolyticum is a sulfhydryl protein, serine protease from the aerobe Streptomy- ces griseus is a disulfide-containing protein, and most extracellular proteins from facultative bacteria contain neither cysteine nor cystine (57). The cellulase from the rumen anaerobe Ruminococcus albus is sensitive to oxidation (211) but it has not been reported whether this is caused by sulfhydryl inactivation. The reactivity of thiols (113) and their ability to combine with essential metals (250) might provide anaerobic bacteria with catalytic abilities not observed with aerobes.

It is likely that the component of C. thermocellum cellulase which contains essential sulfhydryl is a cellobiohydrolase (exo-

-1,4-glucanase), since CMCase activity was found to be unaffected by oxidation or sulfhydryl reagents. Two distinct endo-6-

(1,4)-glucanase (CMCases) have been purified from C. thermocellum

(168, 181) and neither of these enzymes are affected by sulfhydryl reagents. The CMCase purified by Ng and Zeikus (168) completely lacks cysteine. The further purification and characterization of the C. thermocellum cellulase complex should help de-

termine the role of sulfhydryls and metal ions in its catalytic

activity. -139-

Despite earlier observations that hydrolysis of dyed-CMC and dyed-Avicel by C. thermocellum cellulase was not inhibited by cellobiose or glucose (68), I have found in the present work that hydrolysis of crystalline cellulose is inhibited by cellobiose. While this thesis was in preparation, Petre et al.

(181) reported that purified endo- -glucanase of C. thermocellum is relatively insensitive to cellobiose using TNP-CMC as substrate. The effect of cellobiose is therefore dependent on the nature of the substrate. Reese et al, (188) first showed that cellobiose may have anomolous effects on cellulase activity depending on the substrate used and the incubation conditions; inhibition or even stimulation may occur with CMC or derived celluloses.

An important limitation in cellulose hydrolysis is enzyme inhibition by cellobiose, especially when the substrate is highly crystalline and the enzyme preparation is low in - glucosidase. With respect to cellobiose inhibition, our studies point to a similarity between C. thermocellum and Tricho- derma. The inhibition of Trichoderma cellulase is competitive

(84), and increases with resistance of the cellulose to breakdown (84). Addition of -glucosidase preparation of high specific activity to cellulose saccharification mixtures leads to cellobiose hydrolysis and thus alleviates the inhibition of the cellulase by its product (215).

The sensitivities of the fungal and bacterial cellulases to glucose inhibition are strikingly different (Figure 17); this -140-

may be the result of different -glucosidase concentrations in the broths. S-Glucosidase is known to greatly enhance cellulose hydrolysis (215) and is present in low concentrations in

T. reesei RUT-C30 (215). On the other hand, C. thermocellum is not known to produce an extracellular -glucosidase although it possesses a periplasmic -glucosidase (2) and a periplasmic cellobiose phosphorylase (4), which together convert cellobiose to glucose and glucose-l-phosphate. The improvement in cellulose saccharification observed in the presence of added -glucosidase (Table 7) suggests that it would be useful to develop strains which secrete cellobiase.

A potentially important finding in this study is that cellobiose analogs such as salicin, arbutin and lactose also inhibit

C. thermocellum cellulase (Table 8 ). It was previously reported that the cellulase of C. thermocellulaseum is inhibited by cellobiose and lactose (53). Since salicin, arbutin and lactose are not carbon sources for growth of C. thermocellum, it may be possible to use these analogs as selective agents for isolation of strains of C. thermocellum affected in cellulase synthesis or activity.

In this study, I have shown that C. thermocellum requires only four growth factors (biotin, vitamin B 6, p-aminobenzoic

acid and vitamin B1 2) in a chemically defined medium. Its vitamin requirements are similar to the cellulose digesters in the

rumen of cattle and sheep (107). Growth of these anaerobes is -141-

frequently stimulated by vitamin B1 2, biotin and p-amonibenzoic acid. C. thermocellum has an unusual requirement for vitamin

B 6 ' which is generally not required by cellulolytic anaerobes

(24, 107) or soil bacteria (137).

There are stiking similarities in the nutrition and physiology of rumen cellulose digesters (Ruminococcus and Bacteroides), free-living Sporocytophaga and C. thermocellum. These organisms rapidly use cellobiose, but only reluctantly use the hexoses fructose, glucose and mannose (12, 68, 210). Other energy sources fail to support growth. The organisms characteristically produce a yellow-orange pigment when growing on cellulose. The ruminococci and Bacteroides appear to have an obligate requirement for CO 2 or bicarbonate, and characteristically produce high concentrations of succinic acid. In contrast, the cellulolytic clostridia generally don't produce much succinate. Of particu- lar interest is the preference among these organisms for cellobiose, confirming this disaccharide as the main product of cellulose digestion. The cells employ a phosphorolytic cleavage of cellobiose (4, 12, 107). This is an energy-saving mechanism in these cells. C. thermocellum possesses more than one pathway for the dissimilation of cellobiose. It reportedly has a cellobiokinase (166) and -glucosidase (2) for the catabolism of cellobiose, and also possesses a cellodextrin phosphorylase

(204) which may function in the cells to synthesize cellodextrins from cellobiose, possibly as a reserve polysaccharide (105). -142-

Although the regulation of cellodextrin formation has not been studied in the clostridia, the formation of glycogen in bacteria and its phosphorolytic cleavage is regulated by allosteric mechanisms, by catabolite repression, and by covalent modification

(31), suggesting an important role for this process in microorganisms.

As demonstrated in the present study, the degradation of cellulose by C. thermocellum appears to be well-coordinated with the growth and energy needs of the cell. Formation of cellulase occurs at the most rapid differential rate when the cells are growing slowly on cellulose or on fructose. Little cellulase is made during rapid growth on cellobiose or when cellobiose-grown cells are exposed to glucose, in which even slow growth does not occur possibly due to severe decline in ATP levels. The synthesis of extracellular cellulase occurs in highest quantity when cells are presented with an environment in which they are capable of growth, but are deprived of carbon substrate and energy.

Cellulase is repressed during growth on rapidly metabolized carbon sources. The severity of repression appears to be related to the degree of pyruvate decarboxylation and the level of ATP available to the cells (48, 172). Oxidative decarboxylation of pyruvate supplies more energy than does reduction to lactate, and lowers the specific titer of cellulase. Other studies have established that production of xylanases and cellulases is constitutive in the rumen bacteria (67, 183). The rate of their -143-

formation is inversely related to the growth rate of the cells in carbon-limited continuous culture.

Very little work has been done on the control of catabolic pathways in the clostridia, and my review of the literature found only mechanisms for the inhibition or activation of key catabolic enzymes. In Clostridium tetanomorphum, the first enzyme of threonine degradation, threonine deaminase, is activated by ADP,

GDP or IDP (164, 223, 247)). The enzyme is inhibited by sulfhydryl inhibitors (164). Purification and characterization of the enzyme has shown that it has an allosteric site responsible for activation (164). In the same organism, glucose fermentation was shown by Anthony and Guest (8) to be delayed in preference for more favorable energy sources (probably amino acids). They concluded that glycolytic enzymes necessary for glucose fermentation are inhibited, but not repressed, by a catabolite of amino acid metabolism.

Evidence is presented in this thesis that C. thermocellum is able to control the synthesis of cellulase by catabolite repression. Since C. thermocellum is only able to dissimilate cellulose, cellobiose, glucose, fructose and sorbitol as carbon sources, it seems unlikely that catabolite repression evolved to enable the bacterium to select its most desired energy source.

Instead, the regulatory mechanism may provide protection from excess energy metabolism (.1, 13, 63, 192). In both gram-positive and gram-negative bacteria, excessive metabolism through -144-

Embden-Meyerhof pathway, in which the terminal steps are over- loaded due to deficiency of electron acceptors, results in the formation of glycolyticbyproducts (e.g. methylglyoxal) (38) which are toxic to the cells. Formation of lethal methylglyoxal has been demonstrated in Clostridium acetobutvlicum (182),

Clostridium pasteurianum (38), and Streptococcus faecalis (180), especially at high cell densities and high temperatures.

From the results presented above, it appears that cellulose degradation in C. thermocellum is controlled by cellulase inhibition and oxidative inactivation, and by catabolite repression. The mechanism of catabolite repression was not elucida- ted; it may involve phosphorylated glycolytic intermediates and highly phosphorylated nucleotides, which are involved in catabolite repression in the spore-forming bacilli (64, 139). The effector signalling energy deficiency is probably not cyclic

AMP(21,179) which has not been isolated from anaerobes (21, 111,

179) or sporeformers (21).

In conclusion, this study shows that the thermophilic anaerobe, C. thermocellum, synthesizes a true cellulase of an anaerobic nature and high specific activity on crystalline cellulose.

C. thermocellum possesses controls to regulate both the activity and formation of its efficient extracellular enzyme. Judging

from the primitiveness of the saccharolytic clostridia, thought

(174, 241) to closely resemble the first organisms present on earth some 3.5 billion years ago, further elucidation of the -145-

control of metabolism in C. thermocellum might provide funda- mental insights into the development of higher organisms. -145a-

5. RECOMMENDATIONS FOR FUTURE RESEARCH

A. Activity of the Cellulase

Compared to the Trichoderma cellulase, the crude Clos- tridium cellulase appears to have novel biochemical requirements and an exceptionally high specific activity on crystalline substrates. The bacterial cellulase complex active on Avicel and cotton should be purified and characterized for its specific activity, molecular weight, peptide composition, sulfhydryl and metal content, and substrate preferences. In addition, the possible roles of sulfhydryls and iron in catalysis and aggregation should be examined using the purified enzyme. The individual reactions involved in the degradation of insoluble cellulose could be separated and characterized. It would be useful to carry out hydrolysis studies on thick slurries of cellulose, and then on agricultural residues. The sensitivity of the

Clostridium cellulase to oxygen inactivation suggests that a novel, non-oxidative mechanism might be active in the depolymerization of lignin, which could be investigated in the extracellular protein preparation.

B. Formation of the Cellulase

Evidence is presented in this thesis for the regulation of cellulase synthesis by the energy requirements of the cells. -145b-

The differential rates of cellulase synthesis (units cellulase synthesized per unit protein synthesis) on various carbon sources should be determined using the uptake of a radioac- tive amino acid as an indicator of protein synthesis and turnover. The changes in the levels of the polypeptides in the cellulase complex could be determined on the different carbon sources using electrophoresis or antibodies with purified cellulase as standard.

To understand the regulation of cellulase synthesis in

Clostridium thermocellum, the relationship between energy production by glycolysis and cellulase synthesis should be studied.

Specifically, the control of the branch resulting in pyruvate reduction or oxidative decarboxylation should be investigated.

The oxidative branch employs iron sulfur proteins (ferredoxin and hydrogenase) whose levels and activities might be influenced by the availability of iron, hydrogen gas and electron acceptors in the medium. The simple, fermentative metabolism of C. thermocellum suggests that it may be possible to monitor energy production and catabolite repression by a simple and non- destructive method, e.g. by the uptake of inorganic phosphate.

C. thermocellum experiences a sharp decline in ATP levels during the stationary phase of growth and when adapting to glucose. Probably the extremely low levels of ATP prevent the synthesis of cellulase under these conditions. The role of proton ATPase, energy-dissipating futile cycles (e.g. glucokinase/phosphatase), and energy storage as polysaccharide -145c-

should be investigated as causes for the lack of growth on glucose.

C. Selection of Mutants Affected in Extracellular Cellu- lase Formation

An understanding of the regulation of cellulase activity and synthesis and its functions for C. thermocellum (e.g. cellulose hydrolysis and metal scavenging) would suggest methods for the enrichment of mutants changed in cellulase formation. For example, limiting energy production by availability of carbon source, by including a non-metabolizable inhibitor of cellulase in the medium, or by limiting the iron supply would provide a selective pressure for increased cellulase formation. These mutants could be isolated and tested for cellulase formation and changes in composition of the enzyme. -146-

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