Biotechnol. Rog. 1991, 7, 315-322
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Regulation of Cellulase Synthesis in Acidothermus cellulolyticus Ming Shiang,t James C. Linden,+*$ Ali Mohagheghi,s Karel Grohmann,% and Michael E. Himmel'tf Departments of Microbiology and of Agricultural and Chemical Engineering, Colorado State University, Fort Collins, Colorado 80523, and Applied Biological Sciences Section, Biotechnology Research Branch, Solar Fuels Research Division, Solar Energy Research Institute, 1617 Cole Boulevard, Golden, Colorado 80401
The regulation of cellulase synthesis by induction and catabolite repression in the thermophilic, aerobic bacterium Acidothermus cellulolyticus was studied in batch fermentations. Various compounds, such as L-sorbose, cyclic AMP (CAMP),L-glucose, 2-deoxyglucose (2-DG), glucose l-phosphate (G-1-P), sophorose, salicin, sugar alcohols, and isopropyl thioglucoside (IPTGlu), were added along with Solka Floc to improve extracellular cellulase formation by the culture. When cAMP was added exogenously to A. cellulolyticus cultures in the concentration range of 0.01-0.2 g/L, cAMP did not affect cell growth; however, cellulase yields were increased with increasing levels of CAMP. The enzyme production rates with the different levels of CAMP addition during Solka-Floc fermentations were identical. L-Sorbose, L-glucose, 2-DG, G-1-P, sophorose, IPTGlu, and sugar alcohols enhanced cellulase activity produced in the medium, but the starting time and the time required to reach the maximum enzyme activity were different in each condition. All these substances may function as moderators of cellulase synthesis. From the experimental results, only cellobiose, xylose, sophorose, and unknown soluble derivatives from cellulose were considered as inducers. In a possible regulatory mechanism of cellulase synthesis, the repressor, inducer, CAMP,and moderator may be all involved in controlling the rate and the yield of enzyme production.
Introduction Cellulase synthesis is regulated by both induction and repression in fungi (Eriksson, 1981;Gong and Tsao, 1979; Gosh and Kundu, 1980; Montenecourt et al., 1979,1981; Okunev et al., 1983; Zhu et al., 1982). Cellulases can be induced by soluble derivatives from cellulose or several other low molecular weight carbohydrates, such as cellobiose, lactose, gentiobiose, sophorose, and L-sorbose. It has been shown that sophorose is a very powerful inducer of cellulase synthesis for Trichoderma reesei, being 2500 times as active as cellobiose (Montenecourt and Eveleigh, 1977; Montenecourt et al., 1981). However, cellulase synthesis is repressed by the presence of glucose or readily metabolized sugars. In the fungal system, especially in T. reesei, cellulase induction has been shown to occur in response to lactose, cellobiose, and sophorose (Mandels, 1982; Nisizawa et al., 1971). However, there is some disagreement as to whether CAMP is involved in controlling cellulase synthesis in fungi. Montenecourt et al. (1977, 1983)claimed that cAMP did not participate in regulating cellulase production by T. reesei. Goksoyr and Eriksen (1980) suggested the possibility that ATP, rather than CAMP, may be the small molecular regulator in some instances. Cellulase synthesis is also regulated by induction and repression in bacteria (Breuil and Kushner, 1976; Fennington et al., 1984;Kolankaya, 1980;Stewart and Leatherwood, 1976; Stoppok et al., 1982; Wood et al., 1984). Bacteria exhibit both positive and negative control of protein synthesis. An example of negative control in
* Corresponding author.
Department of Microbiology, Colorado State University. Department of Agricultural and Chemical Engineering, Colorado State University. f Solar Energy Research Institute. f
t
8756-7938/9 113007-0315$02.50/0
prokaryotes is the lactose operon in Escherichia coli, and of positive control, the arabinose operon. Regulation of catabolite repressible enzymes in bacteria is through an additional control involving the levels of cAMP in the cell. Suzuki (1975) reported that cAMP was not involved in the case of Pseudomonas fluorescens var. cellulosa. On the contrary, Wood et al. (1984)showed that the addition of cAMP exogenously increased cellulase synthesis in toluene-treated Thermomonospora curvata. Moreover, T. curuata catabolite repression resistant mutants that expressed cellulase at elevated rates had 2-3 times the intracellular cAMP content of the parent strain (Wood et al., 1984). In Thermomonospora fusca, the repression effect was partially relieved when 5 mM cAMP was added concomitantlywith the repressing carbon source;however, cAMP never completely abolished repression (Lin and Wilson, 1987). Therefore, Stutzenberger suggested that cAMP is related to regulation of cellulase synthesis by Thermomonospora (Stutzenberger, 1985). Cellulase induction has been shown to occur in response to cellobiose, lactose, and sophorose in some bacteria but not in others (Saddler et al., 1980). Acidothermus cellulolyticus is a thermophilic,acidophilic,aerobic bacterium that produces filter paper degrading enzymes which have the highest thermostability reported to date (Tucker et al., 1989)and is thus of significant industrial interest. Cellulase productivity from the wild-type culture is rather low, however. In earlier work, addition of cellobiose, glucose,fructose,xylose,and sucrose to Solka Floc cellulose fermentations stimulated cellulase yields from 12% to69 % compared with the use of Solka Floc alone (Shiang et al., 1991a). However, only cellobiose and xylose were considered effective inducers. Understanding the control of induction may significantly contribute to the development of more efficient cellulase production methods. Not only elimination of catabolite
0 1991 American Chemical Scciety and American Institute of Chemical Engineers
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repression but also maximization of induction can improve enzyme yield and productivity. Hence, the objectives of the present study were (1) to screen several substances for their ability to induce cellulase production by A. cellulolyticus, (2) to examine catabolite repression and induction interactions in sugar analogue mixtures, and (3) to compare the cellulolytic enzyme productivities between cellobiose-grown and glucose-grown cultures.
Materials and Methods Microorganism. The microorganism used in this study was A. cellulolyticus 11B. A. cellulolyticus was isolated from the upper Norris Geyser basin in Yellowstone National Park (Mohagheghi et al., 1986) and has been deposited with the American Type Culture Collection, Rockville,MD (ATCC 43068). The strain was maintained frozen at -70 "C after addition of 77 pL of dimethyl sulfoxide/mL of culture suspension. Culture Media. The culture was prepared with a lowphosphate basal salts medium (LPBM) that contained the following, in grams per liter: NH4C1, 1.0; KHzP04, 1.0; NazHP0~7Hz0,0.1; MgS04.7Hz0, 0.2; and CaCL 2H20,0.2. The medium was also supplemented with the following: yeast extract (Difco Laboratories, Detroit, MI, 1.0 g/L; D-cellobiose, 5.0 g/L (unless noted otherwise); and Wolin trace mineral solution (Balch et al., 1979; Wolin, 1976), 1.0% (v/v). The cellulosic substrates used included (carboxymethy1)cellulose(Sigma Chemical Co., St. Louis, MO) and Solka Floc BW-200 (SF)(James River Co., Berlin, NH). Other chemicals from Sigma Chemical Co. included l-O-methyl8-D-ghcopyranoside (MG), isopropyl 8-D-thiogalacopyranoside(IPTGal),isopropyl 8-Dthioglucopyranoside (IPTGlu),a-glucose l-phosphate (G1-P),salicin, adenosine 3',5'-cyclic monophosphate (CAMP), L-glucose, 2-deoxy-~-glucose(2-DG), L-sorbose, D-cellobiose (CB), sucrose, D-xylose, maltose, mannitol, glycerol sorbitol, and D-glucose. a-D-Cellobiose octaacetate was from Aldrich Chemical Co., Inc. (Milwaukee, WI), and sophorose was from Koch-Light Ltd. (Haverhill, Suffolk, England). All media were adjusted to pH 5.2 and sterilized by autoclaving a t 121 "C. Flasks and tubes were processed for 30 min and fermentation vessels for 60 min. Inoculum. A l-mL aliquot of frozen culture was transferred directly to a shake flask containing 20 mL of medium. After overnight incubation a t 55 "C with rotary agitation at 120 rpm, a 10-mL aliquot was transferred to a 500-mL baffled shake flask containing 200 mL of medium. After growth under similar conditions, these inocula were transferred to fermenters at 8% (v/v) inoculum. Fermentations. Fermenters (B. Braun, Models Biostat V and Biostat s)of 1.0- or 2.5-L working volume were used. The medium was maintained at pH 5.2 during fermentation by the addition of 1.0 N NH40H and 1.0 N H3P04. The dissolved oxygen was maintained at 40 % of saturation by increasing the agitation rate and/or supplying pure oxygen as needed. The temperature was controlled at 55 "C. Assays. Filter paper activity (FPA),(carboxymethyl)cellulase (CMCase), and residual reducing sugar determinations were conducted with dinitrosalicylic acid (DNSA) reagent according to the recommendations on the measurement of cellulase activities prepared for the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987). In this study, international units of FPA and CMCase were reported. @-Glucosidaseactivity (pNPGase) was determined by hydrolysis of p-nitrophenyl P-D-glucoside, and xylanase activity was determined
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by the release of xylose from xylan. One unit of enzyme activity was defined as 1pmol of substance @-nitropheno1 or xylose) released from the substrate in 1min under the conditions of the assay. Cultures containing cellulose were extremely turbid; growth was estimated by a modified Lowry protein assay (Ghose, 1987). For this procedure, the cell pellets were suspended in Lowry reagent A and heated to 60 "C for 20 min to induce cell lysis. This suspension was then cooled and subjected to centrifugation a t 8000g for 5 min to pellet the cellulose and cell debris, and aliquots of the supernatants were mixed with Lowry reagents B and D. The chromophore was developed and measured as described by Ghose (1987). A very high correlation (1 g of cell dry weight = 0.62 f 0.03 g of cellular protein) was found for the relationship between cellular protein and cell dry weight determined in this manner. Specific growth rate was determined by dividing the maximum cell dry weight by the elapsed fermentation time required to achieve the maximum. Volumetric productivity was similarly calculated by dividing the maximum cellulase activity by the fermentation time required to reach maximum FPA; specific productivity was calculated by dividing the volumetric productivity by the maximum cell dry weight. Cell Growth for Adaptation Studies. The culture was grown to midexponential phase in LPBM containing either cellobiose or glucose as sole carbon and energy source at the concentration of 5 g/L in a 2.5-L working volume fermenter. Both sugar substrates provided rapid growth of cells to dry weight exceeding 2 g/L, the concentration of cells used for the adaptation studies. The bacterial cells were harvested by centrifugation at 1OOOOg. Cells were washed with phosphate buffer three times and resuspended in 0.05 M phosphate buffer (pH 5.2). Subsequently, the 2 g/L preadapted cells were transferred to either shake flasks or fermenters. Cultures were maintained at 55 "C and aliquots were taken during the respective incubations to determine FPA, CMCase, pNPGase, and xylanase activities.
Results and Discussion Effects of Various Presumed Inducers. The various levels of tested compounds that were mixed with 15 g/L Solka Floc for these studies were selected from representative experiments described in the literature. The compounds included L-glucose and 2-DG (0.1-0.5 g/L), CAMP (0.01-0.2 g/L), G-1-P (0.1-1.0 g/L), sophorose (0.02-0.2 g/L), L-sorbose (0.5-2.5 g/L), maltose, mannitol, and sorbitol (2.5 g/L), MG, salicin, CMC, and CB octaacetate (1g/L), IPTGlu and IPTGal(0.25 g/L), and glycerol (2.5 mL/L). The values were not optimized for this system. Supernatants from cultures were centrifuged and assayed for FPA across the entire fermentation period. The concentration effect of six of these is represented in time course plots of cellulase activity that resulted from the addition of L-glucose, CAMP,G-1-P, 2-DG, sophorose, and L-sorbose in Figure 1, panels a-f, respectively. Also, the kinetic parameters for cell growth, enzyme yields, and enzyme productivities are summarized in Table I. It should be noted a t this point that the control experiment, containing 15 g/L Solka Floc alone, had an enzyme yield of 0.065 unit/mL and productivities of 0.80 unit/(L*h) and 0.19 unit/ (g of dry wt-h). Maximum enzyme synthesis in this case occurred 81 h after inoculation. Maximum cell dry weight was 4.26 g/L and specific growth rate was 0.15 h-I (Shiang et al., 1991a). As a point of reference, the kinetics of enzyme formation for the control, i.e., with Solka Floc alone, were very similar to the curve referred to as 0.1 g/L 2-DG shown in Figure Id.
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TIME (hour)
TIME (hour)
TIME (hour)
Figure 1. Time fourse results of cellulase activity from the addition of (a) L-glucose, (b) CAMP, (c) G-1-P, (d) 2-DG, (e) sophorose, and (f) L-sorbose in Solka Floc fermentation.
Table I. Effects of Various Compounds on Cell Growth and Cellulase Production in Solka Floc Fermentations max volumetric specific enzyme concn, time to enzyme productivity,' enzyme productivity, max cell specific growth condition unita/mL X 109 max FPA, h units/ (Leh) units/(g of dry wt-h) mass, g/L rate p, h-' 0.80 0.19 4.26 0.15 control: 15 g/L SF 65 81 0.1 g/L L-glucose + SF 91 120 0.76 0.17 4.81 0.12 168 47 3.57 0.72 5.19 0.13 0.2 g/L L-glucose + SF 1.42 0.33 4.37 0.15 0.5 g/L L-glucose + SF 136 96 1.37 0.31 4.54 0.17 0.01 g/L cAMP + SF 96 70 2.29 0.51 4.53 0.13 0.05 g/L cAMP + SF 119 52 1.90 0.37 5.29 0.15 0.10 g/L cAMP + SF 135 71 5.19 0.14 149 82 1.82 0.35 0.20 g/L cAMP + SF 118 56 2.11 0.42 5.02 0.12 0.1 g/L G-1-P + SF 5.43 0.14 201 71 2.83 0.57 0.5 g/L G-1-P + SF 5.37 0.15 141 53 2.66 0.54 1.0 g/L G-1-P + SF 1.03 0.29 3.64 0.10 0.1 g/L 2-DG + SF 73 71 2.17 0.49 4.56 0.13 0.2 g/L 2-DG + SF 167 77 0.44 0.19 2.76 0.09 0.5 g/L 2-DG + SF 42 96 4.63 0.12 99 95 1.04 0.23 0.02 g/L sophorose + SF 5.20 0.10 130 68 1.91 0.38 0.10 g/L sophorose + SF 1.98 0.50 4.40 0.12 0.20 g/L sophorose + SF 111 56 0.21 0.11 1.98 0.14 0.5 g/L L-sorbose + SF 25 120 2.32 0.58 4.19 0.12 1.0 g/L t-sorbose + SF 167 72 2.5 g/L L-sorbose + SF 115 68 1.69 0.34 5.17 0.13 3.17 0.76 4.18 0.10 0.25 g/L IPTGlu + SF 152 48 3.48 0.14 58 120 0.49 0.14 0.25 g/L IPTGal + SF 1.17 0.33 4.78 0.18 1.00 g/L MG + SF 140 120 0.86 0.21 4.76 0.12 2.5 mL/L glycerol + SF 86 100 5.10 0.11 165 82 2.01 0.39 2.5 g/L mannitol + SF 2.5 g/L sorbitol + SF 102 105 0.97 0.21 4.51 0.15 0.91 0.20 4.67 0.10 2.5 g/L maltose + SF 105 116 1.0 g/L salicin + SF 200 77 2.60 0.52 5.72 0.11 0.80 0.18 4.79 0.11 1.0 g/L CMC + SF 96 120 0.28 0.13 2.20 0.09 1.0 g/L CB octaacetate + SF 14 49 0
Volumetric productivities were calculated as follows: maximum FPA (unita/L)/fermentation time to achieve maximum FPA (h).
Effects on growth and production parameters of adding various concentrationsof D-glucose, cellobiose, xylose, and sucrose to the 15 g / L Solka Floc system have been published elsewhere (Shiang et al., l990,1991a,b). The
supplementation studies discussed below were not reproduced per se, except that, within an experimental set in which one variable was studied, the inoculum was identical and every batch fermenter was operated under the same
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control parameters. In the course of the overall study in which over 120 such experiments were conducted, fermentations with cellobiose and with cellobiose and Solka Floc were repeated from time to time. The reproducibility of the enzyme activity data was less than 5 7%,and specific growth values were reproduced to within 10%; productivities depended on cell dry weight and fermentation time, which were more variable. (19 L- Glucose Effects. Supplementation of L-glucose to A. cellulolyticus cultures increased cellulase enzyme yields at the concentrations of 0.1-0.5 g/L compared with those grown on Solka Floc alone (Table I). The fermentation time to reach maximum FPA was longer at the level of 0.1 and 0.5 g/L (Figure la). The optimum tested concentration of L-glucose for cellulase production was 0.2 g/L with a maximum enzyme titer of 0.168 unit/mL at 47 h of incubation. From the reducing sugar analysis using DNSA, L-glucose was shown to be partially utilized by this microorganism. Assuming this microorganism transported D-glucose by involvement of the phosphoeno1pyruvate:glucose phosphotransferase system (PTS), appropriate levels of L-glucose with Solka Floc may have turned on cellulase synthesis quickly without initiation of catabolite repression such as that observed with D-glucose. Of the presumed inducers tested, L-glucose exhibited induction as strongly and as early as that observed with IPTGlu and G-1-P. (ii)CAMPEffects. To determine whether cAMP was associated with the regulation of cellulase synthesis by A. cellulolyticus, exogenous cAMP was added to Solka Floc fermentations. The enzyme production rates with various concentrations of cAMP were nearly identical during the 35-50-h fermentation period. Although there is no direct evidence for cAMP penetration into A. cellulolyticus, the similarity in enzyme production kinetics may reflect an event that is independent of cAMP extracellular concentration. The amounts of CAMP addition were proportional to final cellulase yields (Figure lb). This CAMP-specific stimulation of cellulase production in A. cellulolyticus indicates that the synthesis of cellulase,like other inducible catabolic enzymes, may be regulated to some extent by intracellular cAMP levels. (ii19 G I - P Effects. Both aerobic and anaerobic cellulolytic bacteria, including Ruminococcus flauefaciens (Ayers, 1958),Celluibriogiluus (Schafer and King, 1965), Cellulomonusfimi (Schimz et al., 1988),and Clostridium thermocellum (Alexander, 1968), utilize cellobiose phosphorylase to convert cellobiose to glucose and G-1-P. This reaction normally occurs under intracellular conditions and preserves the energy of the glucosidic bond. In the case of A. cellulolyticus, adding G-1-P to Solka Floc increased cellulase yields and shortened the time required to reach maximum FPA (Figure IC).Enzyme yields and productivities with 0.5 g/L G-1-P and Solka Floc were almost 3 times as much as with Solka Floc alone. The maximum enzyme concentration produced after 71 h of inoculation was 0.201 unit/mL, and the volumetric and specific cellulase productivities were 2.83 units/ (Lsh) and 0.57 unit/ (g of dry wt-h), respectively. The significantly enhanced cellulase production by G-1-P may be as a result of direct enzyme induction. One might tentatively speculate that because G-1-P is one of the products from cellobiose by the cellobiose phosphorylase reaction, G-1-P may be acting as a basic inducer molecule to turn on cellulase enzyme synthesis in this culture. (iv)2-DG Effects. The antimetabolite glucose analogue 2-DG has been shown to affect cellulase secretion by inhibiting glycosylation (Merivuori et al., 1985). It
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should be noted that two of the most useful T. reesei mutants, Rut-C30 (Montenecourt and Eveleigh, 1977)and RL-P37 (Sheir-Neiss and Montenecourt, 1984), were isolated by employing a 2-DG screeningmethod, However, Wood et al. (1984)found that 2-DG depressed cAMP levels in T. curuata and 2-DG was a much more effectiverepressor of cellulase synthesis than even glucose. When the culture of A. cellulolyticus was grown on 2-DG with Solka Floc, at the levels of 0.1 and 0.5 g/L, cell growth was inhibited slightly. However, 2-DG improved cellulase production a t the concentration of 0.2 g/L with maximum of FPA 0.167 unit/mL occurring at 77 h of fermentation (Figure Id). The transport of 2-DG into A. cellulolyticus seemed to be similar to that of the L-glucose system but different than that of D-glucose, because enzyme synthesis began earlier than observed with Solka Floc and D-glucose. ( v ) Sophorose Effects. The roles of glucose, cellobiose, sophorose, and other soluble sugars as repressors or inducers of cellulase synthesis varied from organism to organism. Sophorose was a good inducer of endoglucanase in T. reesei (Mandels et al., 1962), whereas in Acetiuibrio cellulolyticus (Saddler et al., 1980), it repressed endoglucanase production as efficiently as glucose. Three different concentrations of sophorose were used to study induction of cellulase in the A. cellulolyticus Solka Floc fermentation. The time requirement for initiation of cellulase synthesiswas reduced proportionately with addition of greater amounts of sophorose in Solka Floc (Figure le). Cellulase activity reached 0.111 unit/mL within 56 h and enzyme productivities were 1.98 units/(L-h) and 0.5 unit/ (g of dry wt-h), at a sophorose level of 0.2 g/L. Under these conditions of application of sophorose, there was no effect on cell growth. The results showed that induction by cellulase by sophorose in A. cellulolyticus was not as effective as that in T. reesei. (VI) L-Sorbose Effects. L-sorbose has been reported to be transported by the same carriers as those used for glucose transport in bakers’ yeast (Cirillo, 1961) and in Neurospora c r a m (Scarborough, 1970). Nevertheless, assimilation of L-sorbose affected the morphology of N. crassu (Trinci and Collinge, 1973) and Trichoderma pseudokoningii (Kubicek, 1983) because P-l,&glucan synthesis in the cell wall was inhibitied by this sugar. It was found also that L-sorbose induced cellulase in T. reesei (Kawamori et al., 1986). The experiments using various concentrations of L-sorbose in A. cellulolyticus fermentations for cellulase production (Figure If) showed that, at the level of 1.0g/L L-sorbose, maximum cellulaseactivity (0.167 unit/mL) was reached after a 72-h growth period. Volumetric and specific productivities were established to be 2.32 units/(L.h) and 0.58 unit/(g of dry wt-h), respectively. The onset of enzyme synthesiswas later than, and the volumetric productivity under the 1.0 g/L m o r bose condition was less than, those values observed for the L-glucose (0.2 g/L) and G-1-P (0.5 g/L) experiments. As in the latter two cases, higher concentrations of morbose (2.5 g/L) did not inhibit cell growth, but cellulase synthesis was repressed somewhat. (vi4 ITPGlucose and ITPGalactose. From the pioneering work of Jacob and Monod (1961), it is known that some substrate analogues, those that cannot be enzymatically cleaved, are effective inducers for glycosidases. For example, alkyl 0-D-l-thiogalactoside derivatives, such as the commonly used isopropyl form, IPTGal, have been shown to induce the lac operon in E. coli. When IPTGal was mixed with Solka Floc for the inducer study with A. cellulolyticus, cellulase activity and cell growth were inhibited slightly. A. cellulolyticus could not utilize
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Table 11. Effwts of Sugar and Sugar Analogue Mixtures on Cell Growth and Cellulase Production volumetric specific enzyme enzyme max specific max enzyme concn, time to productivity: productivity, cell dry growth sugar and sugar analogue mixture unita/mL X 108 max FPA, h unite/(L.h) unita/(g of dry wtsh) weight, g/L rate p , h-1 2.5 g/L CB 21 23 0.92 0.63 1.46 0.18 5.0 g/L CB 35 25 1.40 0.51 2.73 0.19 2.5 g/L CB + 2.5 g/L glucose 29 47 0.62 0.30 2.68 0.17 2.5 g/L CB + 2.5 g/L sucrose 43 25 1.72 0.57 3.01 0.17 2.5 g/L CB + 1.0g/L L-sorbose 58 59 0.98 0.79 1.71 0.13 34 72 2.5 g/L CB + 1.0 g/L xylose 0.47 0.66 1.55 0.13 3 71 0.04 0.04 1.12 0.16 2.5 g/L G + 5.0 g/L methyl glucoside 2.5 g/L G + 1.0 g/L L-sorbose 13 48 0.27 0.21 1.39 0.13 2.5 g/L G + 1.0 g/L salicin 1 48 0.03 0.02 1.64 0.18 2.5 g/L sucrose + 1.0 g/L L-sorbose 9 48 0.19 0.13 1.54 0.14 Volumetric productivities were calculated aa follows: maximum FPA (unita/L)/fermentation time to achieve maximum FPA (h).
galactose, as shown in earlier work by Shiang et al. (1990). However, addition of IPTGlu stimulated cellulase yields. Cellulase synthesis occurredearlier with IPTGlu than most other tested substances; maximum enzyme activity (0.152 unit/mL) was achieved within 48 h. Therefore, the greatest specific enzyme productivity observed with A. cellulolyticus [0.76unit/(gof drywt-h)]wasobtainedwhen 0.25 g/L IPTGlu was mixed with Solka Floc. Detectable levels of reducing sugar remained in the broth throughout the entire fermentation when IPTGal was used but not when IPTGlu was used. (viii)Poly01 Compounds. The effects of the sugar alcohols glycerol, mannitol, and sorbitol on cell growth and cellulase production were also investigated. Although these sugar alcohols acted as repressors of cellulase synthesis, enzyme activities were improved from 32 5% to 1545% compared to fermentations using Solka Floc as the only substrate. The resulting repression was manifested in the longer fermentation time required to complete enzyme synthesis. Thus, enzyme productivities were not improved by using sugar alcohols with Solka Floc. (ix) Oligosaccharides. In a previous study using sucrose and Solka Floc as substrates, it was possible to reduce the time to achieve maximum FPA and produce the high cellulase activity found from this organism (Shiang et al., 1991a,b). It was presumed that cytosolic sucrose phosphorylase may play a role in initiation of cellulase protein synthesis. Maltose is the a-1,Clinked disaccharide containing two glucose moieties that is analogous to the structure of @-l,l-linkedcellobiose. In addition to sucrose phosphorylase, maltose phosphorylase also has been found in many bacteria. Comparing cellulase production from maltose and Solka Floc to that from sucrose and Solka Floc, it seemsthat maltoseacts more like glucose, as a repressor delaying cellulase synthesis and still stimulating enzyme yield in A. cellulolyticus. Some fungi grow on cellobiose octaacetate because this insoluble substrate can be utilized by organisms capable of producing an esterase that can hydrolyze the acetyl groups. Such organisms produce cellulase when grown on cellobiose octaacetate, often in higher yield than when grown on cellobiose and sometimes in higher yield than when grown on cellulose (Mandels and Reese, 1960). A. cellulolyticus grew poorly on cellobiose octaacetate, although it was solubilized by the culture. Moreover, cellulase production was completely repressed by a large quantity of reducing sugar (about 2 g/L) released into the medium during the fermentation using 1.0 g/L cellobiose octaacetate. Addition of CMC to Solka Floc did not improve cellulase productivities compared to Solka Floc alone. Salicin contains a /3-1,4-linked phenol group and can act as an inducer and energy source to induce cellulase
production in mesophilic, anaerobic bacteria such as Acetiuibrio cellulolyticus (Saddler et al., 1980). Likewise, Acidothermus cellulolyticus produced high concentrations of cellulase enzyme (0.2 unit/mL) in a short time period (77 h) with a mixture of salicin (1g/L) and Solka Floc. When methylo-glucoside(MG)was mixed with Solka Floc, cellulose activity and cell growth were inhibited slightly. Detectable levels of reducing sugar remained throughout the fermentation. Sugar and Sugar Analogue Mixture Effects. To understand whether the cellulolytic enzyme system of A. cellulolyticus was catabolite sensitive or inducible by some sugars and sugar analogues, media containing mixtures of cellobiose, glucose, sucrose, sorbose, xylose, methyl glucoside, or salicin as substrates for enzyme production were further studied. The effects of sugar and sugar analogue mixtures on cell growth and cellulase production are summarizedin Table 11. The time courses of enzyme yields in the various conditions are also shown in Figure 2. The results of an earlier report (Shiang et al., 1990) demonstrated that cellulase activities were related to the levels of cellobiose and cell mass. In the current study, the same cell mass was obtained from medium containing 2.5 g/L cellobiose and 2.5 g/L glucose and medium containing 5 g/L cellobiose alone. As shown in Figure 2, there was a lag of 4-6 h to initiate cellulase protein synthesis in the mixture of cellobiose and glucose compared with 2.5 g/L cellobiose. Obviously, the process initiating cellulase synthesis was delayed by glucose catabolite repression. Moreover, maximum enzyme activity produced from cellobiose and glucose was lower than that from 5 g/L cellobiose but higher than that from 2.5 g/L cellobiose. This is consistent with results of experiments in which glucose addition stimulated cellulase yield in Solka Floc fermentation (Shiang et al., 1991a,b). In the 2.5 g/L cellobiose and 2.5 g/L sucrose system, cellulase production increased immediately after the sugar concentration had been reduced to a certain level. Furthermore, sucrose enhanced cellulase yield and enzyme productivities. Sucrose is an easily metabolizable aubstrate that is considered a repressor, like glucose, for cellulase synthesis in A. cellulolyticus. For an explanation of increasing enzyme activity without delay in the presence of sucrose and cellobiose, there must be some factors, other than inducer and repressor, existing in the cellulolytic enzyme system of this organism. Identical phenomena were observed in cultures with medium containing sucrose and Solka Floc (Shiang et al., 1991b). These factors may be moderators as discussed below. Addition of the proper concentration of L-sorbose to Solka Floc has been shown to bring forth high cellulase titer within a short time period (Table I). In the case of 2.5 g/L cellobiose and 1.0 g/L L-sorbose cultures, enzyme
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t 2 I-
4 o
a: W a
2 a
2 W
TIME (hour)
TIME (hour)
t-2 h - + t 4 h - + c 6
,
Figure 2. Time course results of cellulase yields in the sugar mixtures.
activity increased continuously throughout the 72 h of growth. By comparisonwith other kinetic data presented, this is a unique phenomenon. A small amount of detectable enzyme activity was present in the cultures grown on either glucose with sorbose or sucrose with sorbose. Possibly, sorbose is not a real inducer for cellulase synthesis but acts as a moderator in A. cellulolyticus fermentations, such as sucrose. Enzyme concentration in the fermentation broth was also improved when cellobiose and xylose were used as substrates, but the cellulase production rate was less efficient. Because both cases in which glucose with methyl glucoside and glucose with salicin resulted in low enzyme production, these glucose analogues may also act as moderators of protein synthesis. Adaptation Effects. Adaptation tocultural conditions through induction is an important mechanism in several well-studied systems (Canevascini et al., 1979;Mandels and Reese, 1960). In T.curuata, while the cellobiosegrown cells produced measurable amounts of cellulase within 1 day and reached maximum activity within 1 week, the maltose-growncells failed to produce detectable levels of cellulase even after 15 days of incubation (Stutzenberger, 1985). The effects of adaptation through induction using cellobiose as a cellulase inducer in the A. cellulolyticus system are addressed in the following two sets of experiments. (4Shake Flask Induction Studies. Cells that were pregrown in fermenters with either cellobiose or glucose as substrates were washed, mixed with selected carbon sources and inducers, and incubated for 22 h. A final concentration of 2 g/L cell dry weight was inoculated into flasks under one of two conditions. One condition was the control group having either glycerol (2.5mL/L), sucrose (2.5g/L), or no carbon source without inducer. The other condition was the test group, which contained either glycerol or sucrose as a carbon source and 80 pg/mL sophorose as an inducer. The time course of filter paper degrading activities from cellobiose-grown and glucosegrown cells are shown in Figure 3, panels a and b, respectively. Generally speaking, the cellobiose-adapted cultures released more enzyme into the broth than did the glucoseadapted cultures. Even after growth for 22 h under each condition, cellulase synthesis from glucose-grown cells did not approach the levels from cellobiose-grown cells. Also, the control groups without carbon source showed trace levels of enzyme activities, possibly indicating constitutive levels of cellulase that remained in the cells from the cell growth phase of the experiment. Cellobiose-growncultures incubated in the control medium with sucrose as a substrate did release cellulase promptly without a lag phase
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(Figure 3a). Supplementation of sophoroseto bothglycerol and sucrose substrates resulted in decreased cellulase yields, not induction. However, the effects of sucrose, glycerol, and sophorose on cellulase activities were distinctly different in the two cultures pregrown on glucose or cellobiose. In the glucose-pregrown culture system, sophorose in the glycerol medium induced more enzyme activity than in the control groups (Figure 3b). Presumably, sophoroseis an inducer of cellulase protein synthesis and can relieve the repression of glucose-grown cultures. As mentioned previously, sucrose may act as a moderator to initiate enzyme synthesis or modulate cellulase secretion. As above, sophorose did not induce cellulase formation from the cellobiose-adapted cells. Under both adaptation conditions, similar trends were observed for CMCase, pNPGase, and xylanase activities in the control and inducer groups (data not shown). (ii)Solka Floc Fermentations. The cellobiose-grown or glucose-grown cells were collected as described above. Each of the preadapted cultures (1.5-2.0 g/L cell dry weight) was then transferred separately to two fermenters containing LPBM with Solka Floc as the onlysubstrate. The time courses of cell growth, cellulase formation, and reducing sugar content of the cellobiose-adapted and glucose-adapted cultures are shown in Figure 4, panels a and b, respectively. A long growth lag phase occurred with the cellobiosegrown cells after being transferred to the Solka Floc medium. Once cell growth resumed, cellulase formation paralleled growth between 28 and 60 h of fermentation. The maximum enzyme concentration (0.130unit/mL) was established 70 h after inoculation (Figure 4a). Cellulase
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&techno/. Rog., 1991, Vol. 7, No. 4 CB-GROWN CULTURE IN SF
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