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177 Utilization of glucose and cellobiose by Candida molischiana
S. N. Freer
Abstract: Some of the factors that influence the biosynthesis of the Candida 11101ischiana I)-glucosidase were investigated. The yeast produced maximal enzyme activity when grown at 28°C in a carbohydrate-containing complex medium (YM) in which the initial pH was adjusted to 6.0. The enzyme appeared to be produced constitutively, as activity was detected when either ethanol, glycerol, xylose, glucitoL mannitol, maltose, trehalose. cellobiose, cellodextrins, or soluble starch was used as the carbohydrate source. The presence of either glucose, mannose, or fructose (> 25 gIL) repressed I)-glucosidase expression: however, C. 11101ischiana did produce I)-glucosidase when the initial glucose concentration was <25 gIL. When the yeast was grown in YM medium containing glucose plus cellobiose, diauxic utilization of the carbon sources was observed, and I)-glucosidase activity was not detected until the glucose was depleted from the medium. Key words: I)-glucosidase, glucose repression, fennentation, yeast.
Resume: rai etudie certains facteurs qui influencent la biosynthese de la I)-glucosidase chez Candida 1110lischiana. Cette levure a presente une activite enzymatique maximale en culture a28°C dans un milieu complexe (YM) additionne d'hydrates de carbone dont Ie pH initial etait ajuste a6,0. La production de cette enzyme semblait constitutive car elle s'exprimait avec differentes sources d'hydrates de carbone, soit l'ethanol, Ie glycerol, Ie xylose, Ie glucitoL Ie mannitol, Ie maltose, Ie trehalose, Ie cellobiose, les cellodextrines ou I'amidon soluble. La presence de glucose, de mannose ou de fructose (> 25 gIL) reprimait l'expression de la I)-glucosidase mais par contre C. 1110lischiana produisait sa I)-glucosidase lorsque la concentration initiale en glucose etait inferieure a25 gIL. En culture dans un milieu YM contenant du glucose et du cellobiose, il y avait une utilisation diauxique des sources de carbone et l'activite de la I)-glucosidase n'etait pas detectable tant que Ie glucose n'avait pas ete elimine du milieu. Mots eNs: I)-glucosidase, repression par Ie glucose, fennentation, levure. [Traduit par la Redaction]
Introduction saccharification can also be increased by coupling Saccharomyces cerevisiae fermentations to the simultaneous enzymatic Cellulose has the potential to serve as a renewable carbon or saccharification (SSF) of cellulose (Blotkamp et al. 1978; energy source for the microbial production of fuels and Savarese and Young 1978). In these systems, glucose remains chemical feedstocks; however, few industrial microorganisms at subinhibitory concentrations because the yeast metabolizes utilize cellulose directly. If the cellulase enzyme complex is it to ethanol and cell constituents as the glucose is formed. used to saccharify cellulose to glucose, the rate-limiting enzymatic step is the conversion of cellobiose to glucose by When similar experiments were performed with yeasts that 13-1 A-glucosidase (EC 3.2.1.21) (Sternberg et al. 1977). Most ferment both glucose and cellobiose, 10-30% more ethanol l3-g1ucosidases are strongly inhibited by glucose (Fleming and was produced than in fermentations that employed Duerksen 1967; Hu et al. 1960; Duerksen and Halvorson 1958; Saccharomyces cerevisiae (Freer and Detroy 1983; Spindler Blondin et al. 1983; Tingle and Halvorson 1972; Drider et al. et al. 1992). 1993; Kohchi et al. 1985; Woodward and Wiseman 1982), The majority of the cellobiose-fermenting yeasts ferment while cellobiose has been shown to be a potent inhibitor of the only cellobiose and produce cytoplasmic l3-g1ucosidase(s) cellulase enzyme 1A-I3-g1ucan cellobiohydrolase (EC 3.2.1.91) (Freer 1991: Gonde et al. 1984). Thus, these yeasts transport (Ladisch et al. 1981). When Trichoderma reesie Simmons cellobiose across their cytoplasmic membranes prior to cellulase preparations are supplemented with fungal culture cleavage by l3-g1ucosidase (Freer and Greene 1990). In filtrates that are rich in l3-g1ucosidase, the amount of glucose contrast, only two yeasts, Candida wickerhamii and Candida produced is substantially increased (Sternberg et al. 1977: molischiana, ferment both cellobiose and cellodextrins and Desrochers et al. 1981). The efficiency of cellulose produce an extracytoplasmic l3-g1ucosidase that is active against both substrates (Freer and Detroy 1982; Gonde et al. Received August 10, 1994. Revision received November 15, 1984: LeClere et al. 1984; Freer 1985; Vasserot et al. 1991). 1994. Accepted November 30, 1994. The sugar transport systems of C. H'ickerhamii have been S.N. Freer Fennentation Biochemistry Research Unit. characterized (Freer and Greene 1990) and it was found that National Center for Agricultural Utilization Research. United C. wickerhamii transported glucose, but not cellobiose. Thus, States Department of Agriculture. Agricultural Research these yeasts cleave cellobiose and cellodextrins to glucose prior Service, 1815 North University Street. Peoria, IL 61604, U.S.A. to transport and metabolism.
Can. J. Microbiol. 41: 177 -185 (1995) Printed in Canada /lmprime au Canada 178 Can. J. Microbiol. Vol. 41, 1995
Fig. I. Growth (e), l3-glucosidase activity (Ll.), ethanol production (0), and carbohydrate utilization by C. molischiGnG grown aerobically in YM medium containing different initial concentrations of glucose (0).
50 100 5 ogiL Glucose 10 giL Glucose 25 giL Glucose 40 80 4 :::::J -...... O'l 30 60 3 c:: 0 :.;::; ...... : C\S --I .---...... 20 40 2 --I c:: ...... O'l CJ.) - E c:: '-' ::::) c:: 0 ...... 0 :.;::; - 10 C\S 20 1 >, 0 ...... 0 c:: '> c:: CJ.) :.;::; C\S '-' '-' ..c:: c::
10 20 1
1234567 1 234 5 6 7 1234567 Time (days)
Some of the factors that regulate the biosynthesis of the p-Nitrophenyl-I3-D-glucopyranoside, 13-0-(+)-cellobiose, mannose, C. .vickerhamii l3-glucosidase have been previously fructose, maltose, trehalose, glucitol, mannitol, and soluble investigated and it was shown that the enzyme is under an potato starch were purchased from Sigma Chemical Co., unusual form of glucose repression (Freer and Detroy 1985). St. Louis, Missouri. All other chemicals were purchased from When this yeast is grown aerobically, glucose represses the Fisher Scientific Co., Fair Lawn, New Jersey. Cellodextrins expression of l3-glucosidase; however, anaerobiosis overcomes were prepared as previously described (Freer and Detroy 1982; this repression. Because of the uniqueness and importance of Miller et a!. 1960). The yeast Candida molisclziana (Zikes) these enzymes for the production of glucose from cellulose, Meyer et Yarrow (syn. Torulopsis molischiana Zikes) NRRL experiments were undertaken to determine the factors that Y-2237 was obtained from the Agricultural Research Service regulate the expression ofthe C. molischiana l3-glucosidase. In Culture Collection, National Center for Agricultural Utili this paper, I describe the effects that pH, temperature, aeration, zation Research, Peoria, Illinois. and carbohydrate composition and concentration have upon ethanol production and l3-glucosidase expression in C. moli Media and culture conditions sclziana. The basal medium consisted ofpeptone, yeast extract, and malt Materials and methods extract at concentrations of 5, 3, and 3 gIL, respectively. The medium was prepared at 2-fold concentration, the pH was Source of chemicals and yeast adjusted with HC!. and the basal medium was sterilized by Glucose, peptone, yeast extract. and malt extract were autoc!aving at 121°C for 20 min. The various sugars were purchased from Difco Laboratories, Detroit, Michigan. dissolved in deionized distilled water at concentrations that Freer 179
Fig. 2. Growth (e).13-glucosidase activity (L;,), ethanol production (0), and carbohydrate utilization by C. molischiana grown ferrnentatively in YM medium containing different initial concentrations of glucose (D). 50 100 5 agiL Glucose 10 giL Glucose 25 giL Glucose 40 80 4 ..--.. -l 8-- 30 60 3 0c +:::l ..--.. ~ -l "- ..--...... 20 0) 40 2 -l C ...... -- Q.) -- E c..:> c c .....0 ::::> 0 ~ -- 0 10 "- 20 1 >...... c .;:;..... 0 Q.) C c..:> +:::l ~ c c..:> ..c:..... 0100
1 2 345 6 7 1 234 567 1 234 5 6 7 Time (days) ranged from 5 to 20 gIL and sterilized by autoclaving as above, at 28°C. The reactions were terminated by the addition of except for xylose, which was filter sterilized. The experimental 0.5 mL of 0.4 M glycine-NaOH (pH 10.8), and the amount media were prepared by aseptically mixing appropriate ofp-nitrophenol produced was determined spectrophoto proportions of the concentrated basal medium and the sugar metrically at 400 nm (Freer and Detroy 1983). One unit of stocks. Inocula were prepared as described previously (Freer enzyme activity represents 1 fLmol of product (p-nitrophenol) and Detroy 1982) by growing the yeast aerobically for 24 h in formed/min. 20 mL ofbasal medium containing 20 g ofglucoselL. The cells To assay for medium carbohydrates, l-mL samples were were harvested aseptically by centrifugation, washed once with aseptically taken from the culture flasks at various times after water, and resuspended in 10 mL of sterile water. Thirty inoculation. The cells were removed by centrifugation at millilitres of experimental medium in 50-mL flasks was 8000 rpm for 10 min, and the residual medium carbohydrates inoculated with 0.225 mL of cell suspension. Flasks were were analyzed by high-performance liquid chromatography capped with cotton (aerobic growth) or they were capped with (HPLC) on a Spectrophysics P-4000 chromatograph fitted with serum stoppers and vented with 26-gauge sterile needles a Waters 717 autosampler and a Waters 410 refractive index (anaerobic growth). The cultures were incubated at prescribed monitor. A Bio-Rad HPX-87C column was employed using temperatures on a rotary shaker at 250 rpm. deionized distilled water (85°C) as the mobile phase.
Growth, carbohydrate, and 13-glucosidase analysis Results Growth was measured by the increase in optical density at
600 nm (A 60o ). To assay for 13-glucosidase activity, Initial optimization of the 13-glucosidase assay and appropriately diluted cells plus medium were incubated with production 0.5 mLof2.5 mMp-nitrophenyl-13-D-glucopyranoside (pNPG) Prior to performing extensive growth experiments with in 0.1 M acetic acid (adjusted to pH 3.5 with HCI) for 15 min C. molischiana, it was necessary to first define some factors 180 Can. J. Microbiol. Vol. 41, 1995
Fig. 3. Growth ce). l3-glucosidase activity C~). ethanol production CO). and carbohydrate utilization by C. molischiGnG grown aerobically in YM medium containing different initial concentrations of cellobiose C-). r------...,.------.5 25 giL Cellobiose
5 100 giL Cellobiose :=J E 4 :3 >, +-' 'S; 3 f5 1 234 5 6 7 1 234 5 6 7 1 2 345 6 7 Time (days) that affected both [3-g1ucosidase activity and production. The produced more total enzyme activity when grown aerobically; yeast was grown aerobically in YM medium containing 50 g however, the amount ofactivity per unit cell mass, as measured of cellobioselL as the carbon source. The enzyme assay. using by A600 , was about 40% higher in the cultures grown pNPG as the substrate, was performed over the pH range of2.5 anaerobically (0.20 U/A600 unit. anaerobic. versus 0.14 U/A 600 to 6.5 using the whole culture as the source of the enzyme. The unit, aerobic) (data not shown). When glucose was the carbon enzyme retained over 80% of its activity between pH 2.5 and source, maximal growth and ethanol production also occurred 4.5 and had maximal activity between pH 3 and 4. Upon between pH 4.0 and 7.0. However, as the initial pH increased fractionation of the culture into cells and culture broth, from 4.0 to 5.5, both the rates and final yields of ethanol approximately 85% of the activity remained associated with production increased. The rates of ethanol production for the the cells. Upon disruption of the cells by vortexing with cultures with an initial medium pH of 5.5-7.0 were 0.25-mm glass beads. little or no increase in activity was approximately equal. In all cases, the cultures lowered the pH observed (data not shown). Therefore, the assays were of the medium as they grew (data not shown). The optimal performed at pH 3.5 using appropriately diluted whole cultures initial pH of the medium for growth and production of ethanol as the enzyme source. and [3-g1ucosidase appeared to lie between pH 5.5 and 7.0. All The effect of the initial pH of the culture medium was subsequent experiments were performed at an initial medium examined by growing C. molischiana in YM medium pH of6.0. containing either 50 g of glucose or cellobioselL. The initial The effects of incubation temperature were examined by pH values ranged from 2.5 to 7.0 and the cultures were grown incubating both glucose and cellobiose cultures either both aerobically and anaerobically. When cellobiose was the aerobically or anaerobically at 28,32, or 37°C (Table I). When carbon source, maximal growth and ethanol and [3-g1ucosidase glucose was the carbon source, the anaerobic cultures grown production occurred between pH 4.0 and 7.0. Cultures at 28 and 32°C produced the most ethanol (24.1 and 24.6 gIL. Freer 181 Table 1. Effect of temperature upon growth and production of Table 2. Effect of various carbon sources upon growth and ethanol and l3-glucosidase by C. molischiana grown in YM l3-glucosidase production by C. molischiana. medium containing either glucose (G I) or cellobiose (G2). I3-Glucosidase Growth I3-Glucosidase" Ethanol Substrate 02 A600 (U/mL) Ratio conditions A600 (U/mL) (gIL) 2.5% ethanol + 30.8 4.12 0.14 28 5%GI +02 31.1 1.57 0.65 (4) 5% glycerol + 31.1 4.63 0.15 -02 11.8 (4) 0.28 2.41 (3) 5% xylose + 28.1 4.42 0.16 5% G2 +02 36.4 4.83 0.52 (3) 5% glucose + 32.3 (6) 2.83 0.09 -02 10.3 (2) 2.80 (4) 2.36 (5) 5% glucose 8.7 (6) 0.20 0.02 5%GI +02 32.6 2.48 1.52 (2) 5% mannose + 30.7 (4) 1.84 0.06 -02 7.7 (I) 0.20 (4) 2.46 (5) 5% mannose 7.4 (6) 0.10 0.01 5% G2 +02 33.7 3.77 1.00 (2) 5% fructose + 29.6 (5) 1.40 0.05 -02 9.1 (4) 1.91 2.35 (4) 5% fructose 7.2 (5) 0.06 0.01 37 5%GI +02 21.1 0.88 2.03 (2) 5% glucitol + 31.2 5.27 0.17 -02 7.4(1) 0.26 (5) 2.02 (5) 5% mannitol + 30.4 5.50 0.18 5% G2 +02 21.2 4.56 1.47 (2) 5% cellobiose + ::).)" ..7 5.10 0.15 -02 7.3 (I) 1.53 (2) 2.01 (5) 5% cellobiose 7.7 1.78 0.23 5% maltose + 34.1 5.40 0.16 NOTE: Values in parentheses represent the day on which the maximum 5% trehalose + 33.1 3.27 0.10 occurred. If parentheses are absent. the maximum occurred on day 7. 5% trehalose 10.7 2.41 0.23 a None of the glucose-grown cultures produced any l3-glucosidase until 2% cellodextrins + 18.6 3.50 0.19 after 3 days of incubation. 2% celodextrins 7.3 1.80 0.25 5% soluble starch + 7.46 respectively); however, the culture grown at 28°C required only NOTE: Values in parentheses represent the day on which the 3 days to produce the maximal concentration of ethanol, while maximum occurred. If parentheses are absent, the maximum the culture grown at 32°C required 5 days. When cellobiose occurred on day 7. was the carbon source, the cultures grown at 28 and 32°C produced similar amounts of ethanol. The 28°C culture grew to a slightly greater density and produced more l3-g1ucosidase anaerobiosis (Fig. 2). Significant amounts of l3-g1ucosidase than did the 32°C culture. In all cases, the cultures incubated at were produced in anaerobically grown cultures that either 37°C grew more poorly and produced less l3-g1ucosidase and lacked or initially contained only 10 g of glucoselL. However, ethanol than the other cultures. A general trend among all of l3-g1ucosidase production did not occur until 24-48 h after the the aerobic cultures, whether grown on glucose or cellobiose. glucose had been exhausted from the medium. If the initial was that the Crabtree effect (the fermentative utilization of glucose concentration was greater than 25 gIL. little or no sugars under aerobic conditions) was stimulated at the higher l3-glucosidase was produced. temperatures. Aerobic, glucose-grown C. molischiana cultures Candida molischiana produced l3-g1ucosidase when grown produced 6.5. 15.2, and 20.3 g ofethanollL at 28,32, and 37°c' either aerobically or anaerobically in YM medium containing respectively. At 37°C, the aerobic and anaerobic glucose-grown cellobiose as the carbon source (Figs. 3 and 4). The total cultures produced equivalent amounts of ethanol. All amount of l3-glucosidase produced by the various aerobic subsequent experiments were performed at 28°C, cultures (Fig. 3) was approximately the same (3-5 U/mL); however, the amount of growth (A 600) increased with Effect of varying glucose and cellobiose concentrations increasing initial cellobiose concentrations. As the initial To further examine the effects that glucose and cellobiose have cellobiose concentration was increased, the amount of upon l3-g1ucosidase production, C. molischiana was first l3-g1ucosidase per A600 unit (at 7 days) decreased from about grown either aerobically or anaerobically in YM medium 0.17 to 0.09 U/A 600 unit. The synthesis of l3-glucosidase containing varying amounts of glucose (Figs. 1 and 2). When appeared to be biphasic, especially in the cultures containing the yeast was grown in YM medium lacking a carbon source, the higher initial cellobiose concentrations. The rate of growth was limited; however, the cultures did produce between l3-g1ucosidase synthesis appeared to decrease after 3 days and 1.5 (aerobic) and 3.0 (anaerobic) units of enzyme/mL. The then accelerate after 4 days. Ethanol stopped accumulating in aerobic cultures (Fig. 1) that initially contained 75 or 100 g of the medium after 4-5 days and, in most cultures, was glucoselL produced no detectable l3-g1ucosidase activity. In the metabolized. As in the aerobic, glucose-grown cultures, cultures that initially contained less than 50 g of glucoselL, C. molischiana appeared to be utilizing ethanol to effect this l3-glucosidase activity was not produced until the medium second phase of l3-g1ucosidase synthesis. glucose concentration was reduced to less than 10 gIL. The anaerobic, cellobiose-grown cultures produced from Concurrent with this production of l3-g1ucosidase activity was 1.5 to 4.7 U/mL of l3-g1ucosidase activity. The highest a decrease in the ethanol concentration. This suggests that after l3-glucosidase activity (4.7 U/mL at 3 days) was observed in the glucose was consumed by the culture, C. molischiana was the culture initially containing 10 g of cellobioselL. The able to utilize the ethanol present in the culture beer for growth cultures initially containing 50-100g ofcellobioselL produced and enzyme production. about 1.5 U/mL of activity after 3 days, while the culture The above experiment was repeated under conditions in initially containing 25 g of cellobioselL produced about which the yeast was unable to metabolize ethanol. i.e., 2.4 U/mL ofactivity after 5 days. The amount of l3-g1ucosidase 182 Can. J. Microbial. Vol. 41, 1995 Fig. 4. Growth (e).I3-glucosidase activity (.6), ethanol production (0), and carbohydrate utilization by C. molischiana grown fermentatively in YM medium containing different initial concentrations of cellobiose (_). 50 100 5 ..--...... J 25 giL Cellobiose E 40 :::::> 4 --...... --...... >...... J 'S: OJ :;::; --...... 30 3 c..> c c:::( 0 Q) :;::; Cf) C1:l ..--.. ('ij ...... ~ .....J c 20 2 "C Q) en...... Cf) c..> 0 c c..> c 0 0 :;::; ::::3 0 10 ('ij 20 1 (!) ...... ~ I 0 c CCl. c Q) ('ij c..> .c...... c:: Ll.J 50 0 100 5 "C 0 c:: Q) 50 giL Cellobiose 75 giL Cellobiose 100 giL Cellobiose ..--.. ('ij ...... ('ij .....J ~ E :::::J "C 40 >. 80 4 :::::> E .c --...... a 0 a ..c ...... >. -- ~ to 'S: 30 ('ij 3 :;::; S- O c..> .c...... c:::( $ Q) 0 Cf) ~ 20 40 2 C1:l (!) "C Cf) 0 c..> 10 20 1 ::::3 (!) c::o..I 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Time (days) produced by the anaerobic, cellobiose-grown cultures When C. molischiana was grown fermentatively, similar, but appeared to decrease as the initial cellobiose concentration not identical, results were obtained (Fig. 6). As with the aerobic increased to 50 gIL. The reason for this is unknown; however, cultures, no l3-glucosidase activity or cellobiose utilization was it does not appear to be due to the production of free glucose observed until the glucose had been completely utilized. in the medium by the l3-glucosidase. No glucose was detected The major differences seen between the aerobic and in the medium of any of the cellobiose cultures. anaerobic cultures were that (i) the anaerobic cultures initially containing the two higher glucose concentrations produced Growth of C. molisclziana in glucose-cellobiose mixtures little l3-glucosidase « 0.5 U/mL), (ii) little increase in If glucose acts as a repressor of l3-glucosidase synthesis, then l3-glucosidase activity occurred after 3 days, and (iii) less total cultures that are grown in medium initially containing both l3-glucosidase was produced. These differences can be glucose and cellobiose should not produce l3-glucosidase until attributed to the cultures' inability to utilize ethanol in an the glucose has been metabolized. Candida molischiana was anaerobic environment. grown aerobically in medium initially containing various amounts of glucose and cellobiose (Fig. 5). When cellobiose Effect of various carbon sources on 13-glucosidase was the sole carbon source, l3-glucosidase activity was expression detectable 12 h after inoculation. When glucose was also To determine if the C. molischiana l3-glucosidase is either initially present in the medium, l3-glucosidase activity was not induced by cellobiose or produced constitutively, the yeast was detected until the glucose had been completely utilized. grown in media containing carbon sources other than glucose Likewise, no cellobiose utilization could be detected until the and cellobiose. Enzyme levels comparable to those obtained glucose was exhausted from the medium. when the yeast was grown aerobically in medium containing Freer 183 Fig. 5. Growth (e).13-glucosidase activity (6). ethanol production (0). and carbohydrate utilization by C. molischiGnG grown aerobically in YM medium containing different initial concentrations of glucose (0) and cellobiose (.). 50 giL Glucose 40 giL Glucose 5 .--.. 10 giL Cellobiose .....J E ::::> :=J 40 4 - >...... O'l .::;..... -0) :;::; ..... C,.) C'I:l 30 3 .... 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Time (days) 50 g of cellobioselL were obtained when either ethanol. expression of l3-glucosidase in Kluyveromyces lactis (Herman glycerol, xylose. glucitol. mannitol. maltose. trehalose. or and Halvorson 1963a. 1963b) and Kluyveromyces jragilis X cellodextrins were used as the carbon source (Table 2). Kluyveromyces dobzharskii (MacQuillan and Halvorson 1962; Maximall3-glucosidase activity (7.46 U/mL) was expressed in MacQuillan et al. 1960). These cytoplasmic enzymes are medium containing soluble starch. Both mannose and fructose produced constitutively when the yeasts are grown on carbon appeared to repress the expression of l3-glucosidase in a sources other than glucose. fructose. or mannose (MacQuillan manner similar to glucose. and Halvorson 1962; MacQuillan et al. 1960). The regulation of l3-glucosidase expression by C. wickerhamii is similar. but Discussion not identical. to that described in the above organisms. When grown aerobically. C. wickerhamii produces l3-glucosidase Glucose or catabolite repression of microbial glucosidases. constitutively; however. glucose. at concentrations greater than resulting in diauxic utilization of carbohydrate mixtures. is a 50 gIL. represses its expression. When grown anaerobically. general regulatory mechanism among fungi. The expression of l3-glucosidase is produced even in the presence of 100 g of l3-glucosidases has been shown to be repressed by glucose in glucoselL (Freer and Detroy 1985). Candida molischiana several filamentous fungi. e.g.• Phanerochaete clu}'sosporium produced l3-glucosidase constitutively. however. glucose. (Despande et al. 1978; Smith and Gold 1979). Trichoderma fructose. and mannose. at concentrations greater than 25 gIL. viride (Berg and Pettersson 1977; Sternberg 1976). repressed its expression (Table 2). This yeast also produced Schizophyllum commune (Wilson and Niederpruem 1967). and l3-glucosidase when grown in medium initially containing 109 Mucor racemosus (Borgia and Sypherd 1977). In yeasts. of glucoselL; however. enzyme synthesis did not commence glucose. at concentrations greater than 2 gIL. represses the until the glucose had been depleted from the medium (Figs. 1 184 Can. J. Microbiol. Vol. 41, 1995 Fig. 6. Growth (e), l3-glucosidase activity (L.I), ethanol production (0), and carbohydrate utilization by C. molischiana grown fermentatively in YM medium containing different initial concentrations of glucose (0) and cellobiose (_). 50 giL Glucose 40 giL Glucose .--... 5 --l 10 giL Cellobiose E :::::> 4 ---...- >. .:;;+-' :;:::; 3 «u Q.) en ctl "Cl 2 en 0 u :::J c.!J I C!l. 10 giL Glucose 50 giL Cellobiose 5 .--... 40 giL Cellobiose --l E 40 4 -:::::> >. .:;;+-' :;:::; 3 «u Q.) en ctl 2 "Cl en 0 u :::J 1 c.!J I C!l. 1 234 5 6 7 1 2 3 4 5 6 7 2 3 4 5 6 7 Time (days) and 2), and regardless of the aeration state, diauxic utilization culture was about 4.7 at this time (data not shown), the enzyme of glucose-cellobiose mixtures was observed (Figs. 5 and 6). was functioning at about 50% of its maximal rate. Thus, this Thus, the C. molischiana l3-glucosidase appears to be regulated culture appeared to ferment 2-4 times more cellobiose per day similarly to the l3-glucosidases of Kluyveromyces spp. than the l3-glucosidase could hydrolyze. Although these Candida molischiana fermented glucose slightly faster than calculations are only rough approximations, they indicate that cellobiose. If one compares the rate of sugar usage of the amount of l3-glucosidase produced might be the comparable cultures that initially contained 75 g of rate-limiting step in the fermentation of cellobiose, as is the carbohydratejL (Figs. 2 and 4), the glucose-grown culture case for C. ,vickerhamii (Freer 1993). An alternative possibility fermented an average of 14 g (0.078 mmol/mL) of is that, unlike C. wickerhamii (Freer and Greene 1990), sugar' L-I . day-I, while the cellobiose-grown culture C. molischiana might transport both glucose and cellobiose. Ifthis fermented an average of 11.25 g (0.03 mmol/mL of cellobiose were true, then one would observe greater cellobiose usage than or 0.062 mmol/mL of glucose equivalents)' L-I . day-I. could be accounted for solely by the action of the extracellular During day 3 through day 6, the A600 and l3-glucosidase activity l3-glucosidase. Experiments are currently in progress to try to (1.5 U/mL, as measured with the substrate pNPG) remained differentiate between the two possibilities. constant. Therefore, this culture could utilize 2.16 mmol of pNPG' mL-I . day-I. However, since the purified References C. molischiana l3-glucosidase is only 0.68% as active against cellobiose as pNPG (Vasserot et al. 1991), the Berg, B., and Pettersson, G. 1977. Location and formation of cellobiose-grown culture should be able to hydrolyze only cellulases in Trichoderma viride. J. Appl. Bacteriol. 42: 0.014 mmol/mL of cellobiose/day. Also, since the pH of the 65-75. 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