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Induction of aldose reductase and xylitol dehydrogenase activities in Candida tenuis CBS 4435
FEMS Microbiology Letters 149 (1997) 31^37
Induction of aldose reductase and xylitol dehydrogenase activities in Candida tenuis CBS 4435 Martin Kern, Dietmar Haltrich *, Bernd Nidetzky, Klaus D. Kulbe Abteilung Biochemische Technologie, Institut fuër Lebensmitteltechnologie, Universitaët fuër Bodenkultur Wien (BOKU), Muthgasse 18, A-1190 Vienna, Austria Received 17 October 1996; revised 4 December 1996; accepted 4 December 1996
Abstract
In this study the ability of various sugars and sugar alcohols to induce aldose reductase (xylose reductase) and xylitol dehydrogenase (xylulose reductase) activities in the yeast Candida tenuis was investigated. Both enzyme activities were induced when the organism was grown on D-xylose or L-arabinose as well as on the structurally related sugars D-arabinose or D-lyxose. Mixtures of D-xylose with the more rapidly metabolizable sugar D-glucose resulted in a decrease in the levels of both enzymes formed. These results show that the utilization of D-xylose by C. tenuis is regulated by induction and catabolite repression. Furthermore, the different patterns of induction on distinct sugars suggest that the synthesis of both enzymes is not under coordinate control. Keywords : Candida tenuis ; Aldose reductase; Xylitol dehydrogenase; Induction; Repression
1. Introduction
Lignocellulosic biomass, comprising cellulose, hemicellulose, and lignin, is one of the most abundant renewable resources and has great potential as a feedstock for the production of value-added products including a number of useful chemicals and liquid fuels. Hemicellulose can constitute up to 39% of agricultural residues by dry weight, with the aldopentose D-xylose forming the major constituent of this fraction when derived from hardwood or agricultural residues, amounting up to 25% of the dry biomass [1]. For a potential utilization, this sugar has been primarily studied as a substrate for etha* Corresponding author. Tel.: +43 (1) 36006 6275; fax: +43 (1) 36006 6251; e-mail: [email protected]
nol fermentations [2,3]. In addition, the conversion of D-xylose to xylitol has recently received increased attention since this sugar alcohol ¢nds wide use as a sweetener due to its sweetness which is equal to that of sucrose and its anticariogenic properties. Xylitol can be produced microbiologically from D-xylose by a number of yeasts [1]. An alternative enzymatic approach employing isolated NAD(P)H-dependent aldose reductase (ALR) from the yeast Candida tenuis has been developed in our laboratory [4,5]. ALR from this organism is rather unspeci¢c and accepts, among other carbohydrates, D-xylose, L-arabinose, D-ribose and D-galactose as substrates [6]. Aldose (xylose) reductase catalyzes the initial step in D-xylose assimilation in yeasts and mycelial fungi, i.e., the reduction of this pentose to xylitol which is then reoxidized to D-xylulose by the enzyme xylitol
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 0 5 0 - 5
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M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
dehydrogenase (XDH). The synthesis of these two enzymes is both inducible and repressible as has been shown for several yeasts [7^11]. Typical inducers identi¢ed in these studies include D-xylose or L-arabinose whereas only low enzyme levels were formed when more readily metabolizable hexoses such as D-glucose or D-mannose were present. Since ALR from C. tenuis is not commercially available, a fermentation process ensuring high yields of this enzyme had to be established. The present study was undertaken to assess the e¡ect of di¡erent growth substrates on the synthesis of ALR and XDH in C. tenuis. 2. Materials and methods
2.1. Organism and culture conditions
The yeast Candida tenuis CBS 4435, which was obtained from the Centraalbureau voor Schimmelcultures (Baarn, The Netherlands), was used throughout this study. Shake £ask experiments were done on a basal medium (4.0 g l31 yeast extract and 4.0 g l31 peptone from casein) to which 20 g l31 of various carbohydrates were added as indicated. The initial pH of each medium was adjusted to 5.5. All cultures were incubated at 25³C on a rotary shaker at 110 rpm for 24 h. Precultures were grown in 100-ml Erlenmeyer £asks which were inoculated by transferring a loopful of cells from an agar plate to 25 ml of medium. These were then transferred to nonba¥ed 1000-ml Erlenmeyer £asks with 250 ml of medium containing the same carbon source as that used for pregrowth. 2.2. Bioprocess experiments
Batch cultivations were carried out in a 3-l laboratory bioreactor with a working volume of 2 l. The basal culture medium for these cultivations contained 16 g l31 yeast extract, 4 g l31 peptone from casein and 0.5 g l31 MgSO4 W7H2 O. This increase in the concentration of yeast extract as compared to the medium used for shake £ask experiments resulted in a signi¢cant increase in both biomass and enzyme formation, whereas the addition of Mg2 was found to be advantageous for growth of the yeast. The
initial pH of the medium was 5.5, the pH value was allowed to decrease to a minimum value of 4.5 and then held constant by the addition of NaOH (10%) or H3 PO4 (10%). Temperature was maintained at 25³C, the dissolved oxygen was held at 20% of air saturation by controlling air £ow and stirrer speed. Control of pH and dissolved oxygen was carried out by the digital control unit of the bioreactor. 2.3. Analytical methods
Sugars and polyols were determined by HPLC using an Aminex HPX-87P column (BioRad, Hercules, CA, USA). Water was used as the mobile phase at a £ow rate of 0.6 ml min31 at 85³C. Cell growth was estimated from the optical density at 600 nm. Cell dry weight was determined gravimetrically after drying at 105³C. 2.4. Preparation of cell extract
Cells were harvested by centrifugation (9000Ug ; 40 min; 4³C). The pellet was washed twice (5 g l31 NaCl and 0.12 g l31 MgSO4 W7H2 O) and diluted 3fold in cold distilled water. Cells were broken by three passages through a French pressure cell (1380 bar; 4³C) and then cell debris was removed by ultracentrifugation (100 000Ug ; 45 min; 4³C). Enzyme activities and protein concentration were determined in this crude cell extract. 2.5. Enzyme assays
Aldose reductase (ALR, EC 1.1.1.21; alditol:NAD(P) 1-oxidoreductase) activity was assayed spectrophotometrically at 25³C by following the oxidation of the coenzyme NADPH at 340 nm with D-xylose as the substrate. Alternatively, NADH was used as coenzyme when speci¢ed. The assay system contained 925 Wl D-xylose (707 mM), 50 Wl appropriately diluted crude cell extract and 25 Wl NAD(P)H (220 WM). Each component was dissolved or diluted in 300 mM potassium phosphate bu¡er (pH 6.0). Xylitol dehydrogenase (XDH, EC 1.1.1.9; xylitol:NAD 2-oxidoreductase) activity was measured in a similar manner using 925 Wl xylitol (100 mM), 50 Wl appropriately diluted crude cell extract and 25 Wl NAD (1.5 mM). Each component was
FEMSLE 7478 1-5-97
M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
dissolved or diluted in 100 mM Tris-HCl bu¡er (pH 9.0). Reaction rates were corrected for endogenous coenzyme consumption or formation in crude extracts. These endogenous reactions accounted for less than 5% of the coenzyme consumed in the case of ALR determinations and less than 1% of the coenzyme formed in the XDH assays. One unit of enzyme activity is de¢ned as the amount of enzyme producing or consuming 1 Wmol of NAD(P)H per minute under the given experimental conditions. Speci¢c activities are based on protein determinations according to the method of Bradford [12] using bovine serum albumin as the standard and are expressed as units per mg of protein in the crude cell extract.
33
3. Results and discussion
Various carbohydrates, including several pentoses, hexoses and sugar alcohols, which were all employed in equal concentrations of 20 g l31 , were tested in growth experiments for their ability to induce the formation of ALR and XDH activities in C. tenuis. Results for growth of C. tenuis after cultivation for 24 h on these di¡erent substrates, expressed as optical density measured at 600 nm (OD600 ), are given in Fig. 1A. D-Glucose and D-galactose resulted in the highest concentrations of biomass formed. Of the pentoses used in this experiment only D-xylose was utilized signi¢cantly during the incubation period of 24 h for e¤cient growth. This resulted in concentrations of biomass which were markedly above the
Fig. 1. Growth and formation of NADPH-dependent aldose reductase (ALR) and xylitol dehydrogenase (XDH) activities in Candida tenuis CBS 4435 when grown on di¡erent substrates (20 g l31 ). The control medium contained no carbohydrate added to the basal medium. Results shown are the mean and standard deviation of at least two experiments. The enzyme activities corresponding to the reference values obtained for D-xylose are 3.04 and 4.18 U mg31 for ALR and XDH, respectively. In addition to the substrates shown here, the following carbohydrates were tested and resulted in enzyme activities corresponding to the values obtained for the control experiment: D-fructose, lactose, maltose, sucrose, maltodextrin, meso-erythritol, D-mannitol, and D-sorbitol.
FEMSLE 7478 1-5-97
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M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
value found for the control containing no carbohydrate substrate added to the growth medium. In a similar way, growth on various hexitols resulted in a considerable increase in biomass formed, whereas the cell concentrations obtained after growth on di¡erent pentitols were not signi¢cantly di¡erent from the value found for the control experiment. Relative speci¢c activities of NADPH-dependent ALR and NAD -dependent XDH formed by C. tenuis after growth on di¡erent carbohydrates are shown in Fig. 1B with the activities induced by growth on D-xylose being used as the reference values. They were arbitrarily expressed as 100%, with which the activities induced by other carbohydrates were compared. Data for NADH-dependent ALR activity are not shown in Fig. 1B, however, these values were always in the range of 60^70% of the ALR activities determined with NADPH as coenzyme. This compares very well with a puri¢ed xylose reductase from Pichia stipitis, which showed dual coenzyme speci¢city and for which a constant ratio of NADH- to NADPH-dependent activity of 0.65 has been reported [13]. Formation of ALR activity, both NADPH- and NADH-dependent, was considerably induced during growth of C. tenuis on several structurally related pentoses. D-Xylose and L-arabinose were identi¢ed as the best inducers, which is consistent with results previously reported for other yeasts [7,9^11]. In addition, the pentoses D-arabinose and D-lyxose, which do not commonly occur in nature, were found to provoke increased ALR activities. One structural similarity of these four aldopentoses, which seems to be important for induction of ALR, concerns the orientation of the hydroxyl groups at position C-3 and C-4 of the pentoses D-xylose and D-lyxose. In the energetically preferred 4 C1 conformation both of these groups are equatorially arranged. A change in either of these two positions, such as in D-ribose (axial at C-3) or L-xylose (axial at C-4), results in the loss of the inducing e¡ect of the molecule. A similar sterical arrangement can be found when the 1 C4 conformation of L-arabinose, in which the OH-groups again are equatorially orientated, is inverted (Fig. 2). Even when considering D-arabinose in this inverted 1 C4 conformation, equatorially arranged OH-groups can be found in positions corresponding to C-3 and C-4 of D-xylose (Fig. 2).
Fig. 2. Structure of pentoses which induce the synthesis of ALR and XDH in Candida tenuis CBS 4435.
Formation of XDH activity in C. tenuis was signi¢cantly induced by the pentoses D-xylose, L-arabinose and D-lyxose, while D-arabinose only had a low inducing e¡ect. Interestingly, growth on L-arabinose resulted in considerably higher XDH activities as compared to the D-xylose-based reference experiment, whereas only approximately 30% of the ALR activity of the reference was obtained. Xylitol, the substrate of XDH, did not speci¢cally induce the synthesis of the enzyme responsible for its assimilation in the cell. For a number of yeasts capable of metabolizing D-xylose a limited rate of xylitol transport across the plasma membrane has been shown [14,15] which results in poor growth of these organisms when cultivated on this polyol. However, XDH formation was notably provoked when L-arabitol was present in the growth medium and this polyol was also hardly utilized for growth as is evident from Fig. 1A. On the other hand, only low ALR activities comparable to the constitutive levels found for the control experiment were obtained when C. tenuis was cultivated on the L-arabitol-based medium. Only very low ALR and XDH activities were formed when C. tenuis was grown in media containing various hexoses or hexitols. The activities thus obtained are in the same range as those found for the control or even lower. From this di¡erent pattern of induction of both ALR and XDH synthesis when employing various inducing carbohydrates it can be concluded that the synthesis of these two enzymes is not under coordinate control in C. tenuis even though both en-
FEMSLE 7478 1-5-97
M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
35
zymes catalyze the two initial oxido-reductive steps in the assimilation of
D-xylose.
A separate control of
ALR and XDH formation has also been proposed for the yeast
Pachysolen tannophilus
[8].
In order to investigate whether the synthesis of ALR and XDH in
C. tenuis
is repressible in the
presence of easily metabolizable carbohydrates, the time course of enzyme formation and substrate utilization was studied in two laboratory fermentations. 1 A culture medium based on D-xylose (20 g l ) was
3
used as the control for the induction of enzyme activities. ALR and XDH formation on a mixture of D1 each) was compared xylose and D-glucose (10 g l
3
to this control experiment. Results are shown in Figs. 3 and 4. In
the
D-xylose-based
control fermentation the
substrate was depleted after approximately 13 h. Speci¢c ALR (both NADPH- and NADH-dependent) as well as XDH activities increased during the exponential growth phase while
D-xylose
was con-
Fig. 4. Time course of a laboratory cultivation of CBS 4435 using a mixture of
D-xylose
and
Candida tenuis
D-glucose
31
(10 g l
each) as a substrate. A 24 h old preculture (200 ml) grown on the same medium was used as an inoculum. Symbols as in Fig. 3;
a,
D-glucose.
sumed. While this increase was only gradual for ALR, XDH activity showed a distinct peak coinciding with
D-xylose
depletion. Following the consump-
tion of the inducing substrate, these enzyme activities started to decrease. The maximum values of 2.90, 1 determined in this fermenta1.81 and 3.90 U mg
3
tion for NADPH-dependent ALR, NADH-dependent ALR, and XDH, respectively, were found to be slightly lower than those activities obtained in shaken £ask cultivations (Fig. 1B). A possible explanation for this fact could be the reduced oxygen transfer in the unba¥ed Erlenmeyer £asks that were used in these cultivations. It is known that oxygen limitation can increase the levels of xylose-assimilating enzymes in several yeasts [2]. Fig. 3. Time course of a laboratory cultivation of CBS 4435 on a medium based on
D-xylose
Candida tenuis
31 ).
(20 g l
A 24 h old
preculture (200 ml) grown on the same medium was used as an inoculum. Symbols :
+,
D-xylose ;
8
, cell dry weight ;
box, NADPH-dependent aldose reductase ; aldose reductase ;
crossed
m, NADH-dependent
E, NAD -dependent xylitol dehydrogenase.
In the cultivation on mixed sugars D-xylose
D-glucose
and
were sequentially utilized (Fig. 4). During
the phase of
D-glucose
consumption the levels of
all three enzyme activities stayed almost constant. 1 for These values of 1.10, 0.71, and 1.15 U mg
FEMSLE 7478 1-5-97
3
M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
36
C. tenuis
NADPH-dependent ALR, NADH-dependent ALR,
of
and XDH, respectively, are, however, considerably
inhibited as has been shown for other yeasts [17,18].
higher than the maximum values which were ob-
As a matter of fact, the speci¢c rate of glucose con1 calculated sumption qS was reduced from 0.305 h
tained for these enzyme activities in a laboratory 1 fermentation using D-glucose (20 g l ) as the sole
3
carbohydrate substrate (Fig. 5). It is apparent that
D-
glucose represses the formation of ALR and XDH to a certain extent during the ¢rst growth phase of
D-
glucose utilization in the cultivation on mixed sugars.
D-glucose
is characteristic of ca-
by the transport systems of
is
3
for the fermentation on only D-glucose to a value of 1 , which was obtained for the cultivation on
3
0.248 h
a mixture of
D-glucose
and
D-xylose.
This reduced
D-
glucose uptake and availability might lead to reduced glucose repression of the enzyme activities.
This repression of the synthesis of inducible enzymes in the presence of
D-glucose
In the fermentation on mixed sugars, lization started only when
D-glucose
D-xylose
uti-
was almost de-
tabolite repression or the glucose e¡ect [16]. How-
pleted in the medium (Fig. 4). Consumption of this
ever, ALR and XDH formation was not completely
inducing sugar was again accompanied by a consid-
repressed during the phase of
utilization.
erable increase in ALR and XDH activities. With the
in the growth medium
depletion of
seems to induce a certain level of the enzyme activ-
to decrease.
The presence of
D-xylose
D-glucose
ities necessary for its assimilation even when this
D-xylose,
these enzyme activities started
The repression of both ALR and XDH during D-glucose
is also evident from Fig. 5
pentose is not consumed. A possible, alternative ex-
growth on
planation for the formation of elevated enzyme ac-
showing the time course of a laboratory cultivation 1 glucose as the substrate. Lowest employing 20 g l
tivities during the phase of
D-glucose
could be that in the presence of
consumption
D-xylose
the uptake
3
enzyme levels were obtained during the phase of
D-
glucose consumption. With the depletion of this substrate, enzyme activities started to increase signi¢cantly. The formation of ethanol or polyols could not be observed during all of these bioprocess cultivations which were well aerated. The increase in biomass after the depletion of the carbohydrate substrates in the growth medium is presumably caused by the utilization of the organic nitrogen sources yeast extract and peptone from casein which were employed in relatively high concentrations. The results obtained in our study are in good agreement with results previously reported for several yeasts [7^11]. The synthesis of ALR and XDH in
C. tenuis
as
D-xylose
presence
of
is both inducible by pentose sugars such and
L-arabinose
D-glucose.
and repressible in the
This
repression,
however,
was not complete during the initial phase of
D-glu-
cose consumption and signi¢cant levels of ALR and XDH were obtained during the second growth phase in which
D-xylose
was utilized. For a possible fer-
mentation process aiming at producing ALR for the enzymatic production of xylitol these results are quite encouraging. The use of cheap hydrolysates Fig. 5. Time course of a laboratory cultivation of CBS 4435 on a medium based on tions and symbols as in Fig. 3 ;
a,
D-glucose D-glucose.
Candida tenuis
(20 g l
31 ).
Condi-
from mainly
renewable D-xylose
agricultural and
D-glucose,
strates should thus be feasible.
FEMSLE 7478 1-5-97
residues,
containing
as fermentation sub-
M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
Acknowledgments
37
[8] Bicho, P.A., Runnals, P.L., Cunningham, J.D. and Lee, H. (1988) Induction of xylose reductase and xylitol dehydrogen-
This research was supported by a grant from the ë sterreichischen Nationalbank', `Jubila ë umsfonds der O
ase activities in
Pachysolen tannophilus
and
Pichia stipitis
on
mixed sugars. Appl. Environ. Microbiol. 54, 50^54. è , E. (1992) Induction of aldose reductase and polyol [9] Machova
project number 4846. We would like to thank Miss
dehydrogenase activities in
Marianne Prebio for corrections of the manuscript.
lose,
L-arabinose
and
Aureobasidium pullulans
D-galactose.
by
D-xy-
Appl. Microbiol. Biotech-
nol. 37, 374^377. [10] Sugai, J.K.
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[1] Singh, A. and Mishra, P. (1995) Microbial Pentose Utiliza-
xylose reductase and xylitol dehydrogenase activities on mixed
Candida guilliermondii.
tion, Progress in Industrial Microbiology, Vol. 33, 401 pp.
sugars in
Elsevier, Amsterdam.
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[2] Hahn-Ha ë gerdal, B., Jeppsson, H., Skoog, K. and Prior, B.A.
[12] Bradford, M.M. (1976) A rapid and sensitive method for the
(1994) Biochemistry and physiology of xylose fermentation by
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yeasts. Enzyme Microb. Technol. 16, 933^943. [3] du Preez, J.C. (1994) Process parameters and environmental factors a¡ecting
D-xylose
fermentation by yeasts. Enzyme Mi-
[4] Nidetzky, B., Neuhauser, W., Haltrich, D. and Kulbe, K.D. (1996) Continuous enzymatic production of xylitol with simultaneous coenzyme regeneration in a charged membrane reactor. Biotechnol. Bioeng. 52, 387^396.
coenzyme conversions economically. Chemtech 26, 31^36. [6] Kulbe, K.D., Schmidt, H., Schmidt, K. and Scholze, H.A. Continuous synthesis reductase
in
a
of xylitol by NAD(P)H-linked
charged
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ultra¢ltration
membrane-en-
zyme reactor. In : Xylans and Xylanases (Visser, J., Beldman,
[14] McCracken, L.D. and Gong, C.-S. (1983) lism by mutant strains of
Candida
metabo-
[15] Xu, J. and Taylor, K. (1993) E¡ect of nystatin on the metabolism of xylitol and xylose by
Pachysolen tannophilus.
[16] Gancedo, J.M. and Gancedo, C. (1986) Catabolite repression mutants of yeast. FEMS Microbiol. Rev. 32, 179^187. [17] Lucas, C. and van Uden, N. (1986) Transport of hemicellulose monomers in the xylose-fermenting yeast Appl. Microbiol. Biotechnol. 23, 491^495.
sterdam.
Appl.
Environ. Microbiol. 59, 1049^1053.
Progress in Biotechnology, Vol. 7, pp. 565^571. Elsevier, Am-
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[18] Spencer-Martins, I. (1994) Transport of sugars in yeasts : Im-
[7] Bolen, P.L. and Detroy, R.W. (1985) Induction of NADPHD-xylose
reductase and NAD-linked xylitol dehydro-
genase activities in binose, or
D-Xylose
sp. Adv. Biochem. Eng.
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linked
Appl.
Microbiol. Biotechnol. 29, 148^154.
Biotechnol. 27, 33^85.
[5] Nidetzky, B., Haltrich, D. and Kulbe, K.D. (1996) Carry out
aldose
(1988) Xylose fermentation by yeasts. 4. Puri¢cation and kinetic studies of xylose reductase from
crob. Technol. 16, 944^956.
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[13] Rizzi, M., Erlemann, P., Bui-Thanh, N.-A. and Dellweg, H.
Pachysolen tannophilus
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D-xylose, L-ara-
Biotechnol. Bioeng. 27, 302^307.
FEMSLE 7478 1-5-97
Induction of aldose reductase and xylitol dehydrogenase activities in Candida tenuis CBS 4435 Martin Kern, Dietmar Haltrich *, Bernd Nidetzky, Klaus D. Kulbe Abteilung Biochemische Technologie, Institut fuër Lebensmitteltechnologie, Universitaët fuër Bodenkultur Wien (BOKU), Muthgasse 18, A-1190 Vienna, Austria Received 17 October 1996; revised 4 December 1996; accepted 4 December 1996
Abstract
In this study the ability of various sugars and sugar alcohols to induce aldose reductase (xylose reductase) and xylitol dehydrogenase (xylulose reductase) activities in the yeast Candida tenuis was investigated. Both enzyme activities were induced when the organism was grown on D-xylose or L-arabinose as well as on the structurally related sugars D-arabinose or D-lyxose. Mixtures of D-xylose with the more rapidly metabolizable sugar D-glucose resulted in a decrease in the levels of both enzymes formed. These results show that the utilization of D-xylose by C. tenuis is regulated by induction and catabolite repression. Furthermore, the different patterns of induction on distinct sugars suggest that the synthesis of both enzymes is not under coordinate control. Keywords : Candida tenuis ; Aldose reductase; Xylitol dehydrogenase; Induction; Repression
1. Introduction
Lignocellulosic biomass, comprising cellulose, hemicellulose, and lignin, is one of the most abundant renewable resources and has great potential as a feedstock for the production of value-added products including a number of useful chemicals and liquid fuels. Hemicellulose can constitute up to 39% of agricultural residues by dry weight, with the aldopentose D-xylose forming the major constituent of this fraction when derived from hardwood or agricultural residues, amounting up to 25% of the dry biomass [1]. For a potential utilization, this sugar has been primarily studied as a substrate for etha* Corresponding author. Tel.: +43 (1) 36006 6275; fax: +43 (1) 36006 6251; e-mail: [email protected]
nol fermentations [2,3]. In addition, the conversion of D-xylose to xylitol has recently received increased attention since this sugar alcohol ¢nds wide use as a sweetener due to its sweetness which is equal to that of sucrose and its anticariogenic properties. Xylitol can be produced microbiologically from D-xylose by a number of yeasts [1]. An alternative enzymatic approach employing isolated NAD(P)H-dependent aldose reductase (ALR) from the yeast Candida tenuis has been developed in our laboratory [4,5]. ALR from this organism is rather unspeci¢c and accepts, among other carbohydrates, D-xylose, L-arabinose, D-ribose and D-galactose as substrates [6]. Aldose (xylose) reductase catalyzes the initial step in D-xylose assimilation in yeasts and mycelial fungi, i.e., the reduction of this pentose to xylitol which is then reoxidized to D-xylulose by the enzyme xylitol
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 0 5 0 - 5
FEMSLE 7478 1-5-97
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M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
dehydrogenase (XDH). The synthesis of these two enzymes is both inducible and repressible as has been shown for several yeasts [7^11]. Typical inducers identi¢ed in these studies include D-xylose or L-arabinose whereas only low enzyme levels were formed when more readily metabolizable hexoses such as D-glucose or D-mannose were present. Since ALR from C. tenuis is not commercially available, a fermentation process ensuring high yields of this enzyme had to be established. The present study was undertaken to assess the e¡ect of di¡erent growth substrates on the synthesis of ALR and XDH in C. tenuis. 2. Materials and methods
2.1. Organism and culture conditions
The yeast Candida tenuis CBS 4435, which was obtained from the Centraalbureau voor Schimmelcultures (Baarn, The Netherlands), was used throughout this study. Shake £ask experiments were done on a basal medium (4.0 g l31 yeast extract and 4.0 g l31 peptone from casein) to which 20 g l31 of various carbohydrates were added as indicated. The initial pH of each medium was adjusted to 5.5. All cultures were incubated at 25³C on a rotary shaker at 110 rpm for 24 h. Precultures were grown in 100-ml Erlenmeyer £asks which were inoculated by transferring a loopful of cells from an agar plate to 25 ml of medium. These were then transferred to nonba¥ed 1000-ml Erlenmeyer £asks with 250 ml of medium containing the same carbon source as that used for pregrowth. 2.2. Bioprocess experiments
Batch cultivations were carried out in a 3-l laboratory bioreactor with a working volume of 2 l. The basal culture medium for these cultivations contained 16 g l31 yeast extract, 4 g l31 peptone from casein and 0.5 g l31 MgSO4 W7H2 O. This increase in the concentration of yeast extract as compared to the medium used for shake £ask experiments resulted in a signi¢cant increase in both biomass and enzyme formation, whereas the addition of Mg2 was found to be advantageous for growth of the yeast. The
initial pH of the medium was 5.5, the pH value was allowed to decrease to a minimum value of 4.5 and then held constant by the addition of NaOH (10%) or H3 PO4 (10%). Temperature was maintained at 25³C, the dissolved oxygen was held at 20% of air saturation by controlling air £ow and stirrer speed. Control of pH and dissolved oxygen was carried out by the digital control unit of the bioreactor. 2.3. Analytical methods
Sugars and polyols were determined by HPLC using an Aminex HPX-87P column (BioRad, Hercules, CA, USA). Water was used as the mobile phase at a £ow rate of 0.6 ml min31 at 85³C. Cell growth was estimated from the optical density at 600 nm. Cell dry weight was determined gravimetrically after drying at 105³C. 2.4. Preparation of cell extract
Cells were harvested by centrifugation (9000Ug ; 40 min; 4³C). The pellet was washed twice (5 g l31 NaCl and 0.12 g l31 MgSO4 W7H2 O) and diluted 3fold in cold distilled water. Cells were broken by three passages through a French pressure cell (1380 bar; 4³C) and then cell debris was removed by ultracentrifugation (100 000Ug ; 45 min; 4³C). Enzyme activities and protein concentration were determined in this crude cell extract. 2.5. Enzyme assays
Aldose reductase (ALR, EC 1.1.1.21; alditol:NAD(P) 1-oxidoreductase) activity was assayed spectrophotometrically at 25³C by following the oxidation of the coenzyme NADPH at 340 nm with D-xylose as the substrate. Alternatively, NADH was used as coenzyme when speci¢ed. The assay system contained 925 Wl D-xylose (707 mM), 50 Wl appropriately diluted crude cell extract and 25 Wl NAD(P)H (220 WM). Each component was dissolved or diluted in 300 mM potassium phosphate bu¡er (pH 6.0). Xylitol dehydrogenase (XDH, EC 1.1.1.9; xylitol:NAD 2-oxidoreductase) activity was measured in a similar manner using 925 Wl xylitol (100 mM), 50 Wl appropriately diluted crude cell extract and 25 Wl NAD (1.5 mM). Each component was
FEMSLE 7478 1-5-97
M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
dissolved or diluted in 100 mM Tris-HCl bu¡er (pH 9.0). Reaction rates were corrected for endogenous coenzyme consumption or formation in crude extracts. These endogenous reactions accounted for less than 5% of the coenzyme consumed in the case of ALR determinations and less than 1% of the coenzyme formed in the XDH assays. One unit of enzyme activity is de¢ned as the amount of enzyme producing or consuming 1 Wmol of NAD(P)H per minute under the given experimental conditions. Speci¢c activities are based on protein determinations according to the method of Bradford [12] using bovine serum albumin as the standard and are expressed as units per mg of protein in the crude cell extract.
33
3. Results and discussion
Various carbohydrates, including several pentoses, hexoses and sugar alcohols, which were all employed in equal concentrations of 20 g l31 , were tested in growth experiments for their ability to induce the formation of ALR and XDH activities in C. tenuis. Results for growth of C. tenuis after cultivation for 24 h on these di¡erent substrates, expressed as optical density measured at 600 nm (OD600 ), are given in Fig. 1A. D-Glucose and D-galactose resulted in the highest concentrations of biomass formed. Of the pentoses used in this experiment only D-xylose was utilized signi¢cantly during the incubation period of 24 h for e¤cient growth. This resulted in concentrations of biomass which were markedly above the
Fig. 1. Growth and formation of NADPH-dependent aldose reductase (ALR) and xylitol dehydrogenase (XDH) activities in Candida tenuis CBS 4435 when grown on di¡erent substrates (20 g l31 ). The control medium contained no carbohydrate added to the basal medium. Results shown are the mean and standard deviation of at least two experiments. The enzyme activities corresponding to the reference values obtained for D-xylose are 3.04 and 4.18 U mg31 for ALR and XDH, respectively. In addition to the substrates shown here, the following carbohydrates were tested and resulted in enzyme activities corresponding to the values obtained for the control experiment: D-fructose, lactose, maltose, sucrose, maltodextrin, meso-erythritol, D-mannitol, and D-sorbitol.
FEMSLE 7478 1-5-97
34
M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
value found for the control containing no carbohydrate substrate added to the growth medium. In a similar way, growth on various hexitols resulted in a considerable increase in biomass formed, whereas the cell concentrations obtained after growth on di¡erent pentitols were not signi¢cantly di¡erent from the value found for the control experiment. Relative speci¢c activities of NADPH-dependent ALR and NAD -dependent XDH formed by C. tenuis after growth on di¡erent carbohydrates are shown in Fig. 1B with the activities induced by growth on D-xylose being used as the reference values. They were arbitrarily expressed as 100%, with which the activities induced by other carbohydrates were compared. Data for NADH-dependent ALR activity are not shown in Fig. 1B, however, these values were always in the range of 60^70% of the ALR activities determined with NADPH as coenzyme. This compares very well with a puri¢ed xylose reductase from Pichia stipitis, which showed dual coenzyme speci¢city and for which a constant ratio of NADH- to NADPH-dependent activity of 0.65 has been reported [13]. Formation of ALR activity, both NADPH- and NADH-dependent, was considerably induced during growth of C. tenuis on several structurally related pentoses. D-Xylose and L-arabinose were identi¢ed as the best inducers, which is consistent with results previously reported for other yeasts [7,9^11]. In addition, the pentoses D-arabinose and D-lyxose, which do not commonly occur in nature, were found to provoke increased ALR activities. One structural similarity of these four aldopentoses, which seems to be important for induction of ALR, concerns the orientation of the hydroxyl groups at position C-3 and C-4 of the pentoses D-xylose and D-lyxose. In the energetically preferred 4 C1 conformation both of these groups are equatorially arranged. A change in either of these two positions, such as in D-ribose (axial at C-3) or L-xylose (axial at C-4), results in the loss of the inducing e¡ect of the molecule. A similar sterical arrangement can be found when the 1 C4 conformation of L-arabinose, in which the OH-groups again are equatorially orientated, is inverted (Fig. 2). Even when considering D-arabinose in this inverted 1 C4 conformation, equatorially arranged OH-groups can be found in positions corresponding to C-3 and C-4 of D-xylose (Fig. 2).
Fig. 2. Structure of pentoses which induce the synthesis of ALR and XDH in Candida tenuis CBS 4435.
Formation of XDH activity in C. tenuis was signi¢cantly induced by the pentoses D-xylose, L-arabinose and D-lyxose, while D-arabinose only had a low inducing e¡ect. Interestingly, growth on L-arabinose resulted in considerably higher XDH activities as compared to the D-xylose-based reference experiment, whereas only approximately 30% of the ALR activity of the reference was obtained. Xylitol, the substrate of XDH, did not speci¢cally induce the synthesis of the enzyme responsible for its assimilation in the cell. For a number of yeasts capable of metabolizing D-xylose a limited rate of xylitol transport across the plasma membrane has been shown [14,15] which results in poor growth of these organisms when cultivated on this polyol. However, XDH formation was notably provoked when L-arabitol was present in the growth medium and this polyol was also hardly utilized for growth as is evident from Fig. 1A. On the other hand, only low ALR activities comparable to the constitutive levels found for the control experiment were obtained when C. tenuis was cultivated on the L-arabitol-based medium. Only very low ALR and XDH activities were formed when C. tenuis was grown in media containing various hexoses or hexitols. The activities thus obtained are in the same range as those found for the control or even lower. From this di¡erent pattern of induction of both ALR and XDH synthesis when employing various inducing carbohydrates it can be concluded that the synthesis of these two enzymes is not under coordinate control in C. tenuis even though both en-
FEMSLE 7478 1-5-97
M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
35
zymes catalyze the two initial oxido-reductive steps in the assimilation of
D-xylose.
A separate control of
ALR and XDH formation has also been proposed for the yeast
Pachysolen tannophilus
[8].
In order to investigate whether the synthesis of ALR and XDH in
C. tenuis
is repressible in the
presence of easily metabolizable carbohydrates, the time course of enzyme formation and substrate utilization was studied in two laboratory fermentations. 1 A culture medium based on D-xylose (20 g l ) was
3
used as the control for the induction of enzyme activities. ALR and XDH formation on a mixture of D1 each) was compared xylose and D-glucose (10 g l
3
to this control experiment. Results are shown in Figs. 3 and 4. In
the
D-xylose-based
control fermentation the
substrate was depleted after approximately 13 h. Speci¢c ALR (both NADPH- and NADH-dependent) as well as XDH activities increased during the exponential growth phase while
D-xylose
was con-
Fig. 4. Time course of a laboratory cultivation of CBS 4435 using a mixture of
D-xylose
and
Candida tenuis
D-glucose
31
(10 g l
each) as a substrate. A 24 h old preculture (200 ml) grown on the same medium was used as an inoculum. Symbols as in Fig. 3;
a,
D-glucose.
sumed. While this increase was only gradual for ALR, XDH activity showed a distinct peak coinciding with
D-xylose
depletion. Following the consump-
tion of the inducing substrate, these enzyme activities started to decrease. The maximum values of 2.90, 1 determined in this fermenta1.81 and 3.90 U mg
3
tion for NADPH-dependent ALR, NADH-dependent ALR, and XDH, respectively, were found to be slightly lower than those activities obtained in shaken £ask cultivations (Fig. 1B). A possible explanation for this fact could be the reduced oxygen transfer in the unba¥ed Erlenmeyer £asks that were used in these cultivations. It is known that oxygen limitation can increase the levels of xylose-assimilating enzymes in several yeasts [2]. Fig. 3. Time course of a laboratory cultivation of CBS 4435 on a medium based on
D-xylose
Candida tenuis
31 ).
(20 g l
A 24 h old
preculture (200 ml) grown on the same medium was used as an inoculum. Symbols :
+,
D-xylose ;
8
, cell dry weight ;
box, NADPH-dependent aldose reductase ; aldose reductase ;
crossed
m, NADH-dependent
E, NAD -dependent xylitol dehydrogenase.
In the cultivation on mixed sugars D-xylose
D-glucose
and
were sequentially utilized (Fig. 4). During
the phase of
D-glucose
consumption the levels of
all three enzyme activities stayed almost constant. 1 for These values of 1.10, 0.71, and 1.15 U mg
FEMSLE 7478 1-5-97
3
M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
36
C. tenuis
NADPH-dependent ALR, NADH-dependent ALR,
of
and XDH, respectively, are, however, considerably
inhibited as has been shown for other yeasts [17,18].
higher than the maximum values which were ob-
As a matter of fact, the speci¢c rate of glucose con1 calculated sumption qS was reduced from 0.305 h
tained for these enzyme activities in a laboratory 1 fermentation using D-glucose (20 g l ) as the sole
3
carbohydrate substrate (Fig. 5). It is apparent that
D-
glucose represses the formation of ALR and XDH to a certain extent during the ¢rst growth phase of
D-
glucose utilization in the cultivation on mixed sugars.
D-glucose
is characteristic of ca-
by the transport systems of
is
3
for the fermentation on only D-glucose to a value of 1 , which was obtained for the cultivation on
3
0.248 h
a mixture of
D-glucose
and
D-xylose.
This reduced
D-
glucose uptake and availability might lead to reduced glucose repression of the enzyme activities.
This repression of the synthesis of inducible enzymes in the presence of
D-glucose
In the fermentation on mixed sugars, lization started only when
D-glucose
D-xylose
uti-
was almost de-
tabolite repression or the glucose e¡ect [16]. How-
pleted in the medium (Fig. 4). Consumption of this
ever, ALR and XDH formation was not completely
inducing sugar was again accompanied by a consid-
repressed during the phase of
utilization.
erable increase in ALR and XDH activities. With the
in the growth medium
depletion of
seems to induce a certain level of the enzyme activ-
to decrease.
The presence of
D-xylose
D-glucose
ities necessary for its assimilation even when this
D-xylose,
these enzyme activities started
The repression of both ALR and XDH during D-glucose
is also evident from Fig. 5
pentose is not consumed. A possible, alternative ex-
growth on
planation for the formation of elevated enzyme ac-
showing the time course of a laboratory cultivation 1 glucose as the substrate. Lowest employing 20 g l
tivities during the phase of
D-glucose
could be that in the presence of
consumption
D-xylose
the uptake
3
enzyme levels were obtained during the phase of
D-
glucose consumption. With the depletion of this substrate, enzyme activities started to increase signi¢cantly. The formation of ethanol or polyols could not be observed during all of these bioprocess cultivations which were well aerated. The increase in biomass after the depletion of the carbohydrate substrates in the growth medium is presumably caused by the utilization of the organic nitrogen sources yeast extract and peptone from casein which were employed in relatively high concentrations. The results obtained in our study are in good agreement with results previously reported for several yeasts [7^11]. The synthesis of ALR and XDH in
C. tenuis
as
D-xylose
presence
of
is both inducible by pentose sugars such and
L-arabinose
D-glucose.
and repressible in the
This
repression,
however,
was not complete during the initial phase of
D-glu-
cose consumption and signi¢cant levels of ALR and XDH were obtained during the second growth phase in which
D-xylose
was utilized. For a possible fer-
mentation process aiming at producing ALR for the enzymatic production of xylitol these results are quite encouraging. The use of cheap hydrolysates Fig. 5. Time course of a laboratory cultivation of CBS 4435 on a medium based on tions and symbols as in Fig. 3 ;
a,
D-glucose D-glucose.
Candida tenuis
(20 g l
31 ).
Condi-
from mainly
renewable D-xylose
agricultural and
D-glucose,
strates should thus be feasible.
FEMSLE 7478 1-5-97
residues,
containing
as fermentation sub-
M. Kern et al. / FEMS Microbiology Letters 149 (1997) 31^37
Acknowledgments
37
[8] Bicho, P.A., Runnals, P.L., Cunningham, J.D. and Lee, H. (1988) Induction of xylose reductase and xylitol dehydrogen-
This research was supported by a grant from the ë sterreichischen Nationalbank', `Jubila ë umsfonds der O
ase activities in
Pachysolen tannophilus
and
Pichia stipitis
on
mixed sugars. Appl. Environ. Microbiol. 54, 50^54. è , E. (1992) Induction of aldose reductase and polyol [9] Machova
project number 4846. We would like to thank Miss
dehydrogenase activities in
Marianne Prebio for corrections of the manuscript.
lose,
L-arabinose
and
Aureobasidium pullulans
D-galactose.
by
D-xy-
Appl. Microbiol. Biotech-
nol. 37, 374^377. [10] Sugai, J.K.
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