Towards a cost effective strategy for cutinase production by a recombinant ... The costs, in terms of hexoses, incurred with this fermentation strategy were ...
Appl Microbiol Biotechnol (2003) 61:69–76 DOI 10.1007/s00253-002-1196-0
ORIGINAL PAPER
B. S. Ferreira · C. R. C. Calado · F. van Keulen · L. P. Fonseca · J. M. S. Cabral · M. M. R. da Fonseca
Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects Received: 27 July 2002 / Revised: 4 November 2002 / Accepted: 8 November 2002 / Published online: 24 January 2003 Springer-Verlag 2003
Abstract Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell – required for the induction of cutinase production – and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.
Introduction Cutinases are hydrolytic enzymes that degrade cutin, a cuticular polymer of higher plants with numerous and very diverse potential commercial applications (Carvalho et al. 1999). In order to obtain an efficient and low cost production system for cutinase from Fusarium solani pisi, this enzyme was overproduced in the recombinant B. S. Ferreira ()) · C. R. C. Calado · F. van Keulen · L. P. Fonseca · J. M. S. Cabral · M. M. R. da Fonseca Centro de Engenharia Biolgica e Qumica, Instituto Superior Tcnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal e-mail: bsf@ ist.utl.pt Tel.: +351-21-8417233 Fax: +351-21-8480072
Saccharomyces cerevisiae strain SU50. For heterologous expression of cutinase cDNA, the mature fragment of the gene was cloned into a yeast expression vector. The cutinase gene is fused to the invertase signal sequence to achieve efficient cutinase secretion. Expression is controlled by the strong inducible Gal7 promoter (van Gemeren et al. 1995). The productivity of fermentations employing recombinant micro-organisms depends on multiple factors, namely interacting relationships between microbial physiology, copy number and stability of the plasmid, and gene expression. S. cerevisiae offers several advantages as a host for the production of heterologous proteins: (1) availability of a large amount of information concerning its molecular biology and physiology; (2) ease of genetic manipulation; (3) ability of performing post-translational modifications; (4) long history of utilisation in traditional industrial processes, being generally regarded as safe. Furthermore, S. cerevisiae holds advantages with regard to product secretion, such as a well characterised secretory pathway. Also, it normally secretes only a very small number (5%) of its own proteins to the medium, which is advantageous for product recovery (Smith et al. 1985; Bitter et al. 1987; Das and Shultz 1987; Calado et al. 2001). Product loss due to degradation of cutinase by proteases secreted by S. cerevisiae is negligible (Verrips et al. 2000). Nevertheless, as a host strain, S. cerevisiae still poses several problems, namely: 1. Glucose repression: common industrial carbon sources are composed of a mixture of sugars that are used sequentially. A glucose derepressed strain would use all the sugars of the complex medium simultaneously, shortening the production time and avoiding environmental problems. In addition, glucose is known to repress the GAL-mediated expression system. 2. Crabtree effect: alcoholic fermentation may occur even under fully aerobic conditions, negatively affecting biomass yields and also the cutinase yield as heterologous cutinase produced during fermentation is correlated to biomass produced (Verrips et al. 2000).
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In addition, high ethanol concentrations decrease the amount of recombinant protein produced (Park et al. 1995; Kapat et al. 2000) and proteases that degrade the recombinant protein may be expressed during growth on ethanol (Gimenez et al. 2000). The strain used in this work is known to exhibit a strong Crabtree effect (Giuseppin et al. 1993). Alcoholic fermentation is currently minimised during aerobic yeast growth by operating in the fed-batch mode under conditions where the carbon source supply limits the growth rate and the full aeration capacity of the vessel is utilised. The strain used in this work is able to metabolise the inducer, galactose, which is much more expensive than glucose. Thus, an optimised fermentation strategy should enhance cutinase productivity while minimising galactose consumption. The experiments performed were aimed at providing fundamental knowledge to allow a better understanding of the physiological aspects involved in recombinant cutinase production by yeast. The first fermentation consisted of a batch phase on glucose, followed by a fed-batch period during which galactose was added as carbon source. To better understand the effects of culturing the strain on galactose, a batch fermentation was performed with galactose as carbon source. Next, a third fermentation was carried out, including a batch and a fed-batch period, using a mixture of glucose and galactose as carbon source in both periods, to study glucose repression and the possibility of combining the higher biomass productivity obtained on glucose with the inductive action of galactose. Complex media were used, aiming at mimicking the conditions of the industrial process.
Materials and methods
Bioreactor A 5 l bioreactor (Biostat MD; Braun, Melsungen, Germany), equipped with a jacketed glass vessel, height/diameter ratio of 2, and three equally spaced Rushton turbines, was used. The air flow rate, dissolved oxygen, pH, temperature, stirrer speed and volume of acid and base added were computer logged. Dissolved oxygen was monitored with a polarographic electrode (Ingold, Urdorf, Switzerland). Zero and 100% calibration of the electrode were performed at 30C and 500 rpm by sparging nitrogen and air, respectively, until stable signals were obtained. Anti-foam (silicon anti-foaming agent; Merck) was added manually. Fermentation A: fed-batch fermentation with galactose feeding Initially, the bioreactor contained 2 l culture medium: 20 g/l yeast extract (Difco), 10 g/l peptone (BDH, Poole, Dorset, UK), 20 g/l d(+)-glucose (Merck) and 10% (v/v) inoculum. A solution of 200 g/ l d(+)-galactose (Sigma, Munich, Germany), 35 g/l yeast extract (Difco) and 45 g/l peptone (BDH, UK) started being fed with a peristaltic pump (Watson Marlow 505Di, Falmouth Cornwall, UK) immediately after the depletion of the ethanol produced during the batch period. The feed rate was constant and set to 116 ml/h until 1.68 l medium (density =1,110 kg/m3) had been added. The amount of medium added was monitored gravimetrically (PB1502 balance; Mettler-Toledo, Greifensee, Switzerland). The fermentation was carried out at 30C and the pH was maintained at 6.0€0.5 with 2 M NaOH and 2 M HCl. Dissolved oxygen was kept above 60% and 30% saturation during the batch and the fed-batch periods, respectively, at a constant air flow rate of 2.0 lN/min, by variation of the stirring speed. A condenser was coupled to the fermenter air outlet and cooled with water at 4C to minimise losses by evaporation. Fermentation B: batch fermentation on galactose The bioreactor contained 4.0 l culture medium: 10 g/l yeast extract (Difco, Detroit Mich.), 10 g/l peptone (BDH), 20 g/l d(+)-galactose (Sigma) and 10% (v/v) inoculum. Temperature and pH were controlled as before. Dissolved oxygen was kept above 30% saturation at a constant air flow rate of 3.9 lN/min by varying the stirring speed.
Strain The S. cerevisiae strain SU50 (MATa, ciro, leu2-3,112, his4-519, can1) containing the expression vector pUR7320 was used. The strain was constructed and kindly provided by the Unilever Research Laboratory, Vlaardingen, The Netherlands. The plasmids contained ribosomal DNA sequences for chromosomal integration and a LEU2d gene for selection on leucine-lacking plates (van Gemeren et al. 1995). The stock cultures were maintained in a 50% (v/v) mixture of selective medium (Leu agar) and glycerol (Merck, Darmstadt, Germany) at 80C. Inoculum preparation The composition of the selective medium for inoculum preparation was: 6.7 g/l yeast nitrogen base without free amino acids (Difco, Detroit, Mich.), 20 g/l d(+)-glucose (Merck), supplemented with 20 mg/l l-histidine (Merck). The inoculum was grown overnight in shake flasks at 30C and 200 rpm in an orbital shaker (Agitorb 160E, Aralab, Oeiras, Portugal) until a dry cell weight between 1.1 and 1.8 g/l was obtained (Calado et al. 2002a).
Fermentation C: fed-batch fermentation with glucose and galactose feeding To start with, the bioreactor contained 2 l culture medium: 20 g/l yeast extract (Difco), 10 g/l peptone (BDH), 15 g/l d(+)-glucose (Merck), 5 g/l d(+)-galactose (Sigma) and 10% (v/v) inoculum. Feeding with a solution of 10 g/l yeast extract (Difco), 20 g/l peptone (BDH), 45 g/l d(+)-glucose (Merck) and 10 g/l d(+)galactose (Sigma) through a peristaltic pump (Watson Marlow 202 U1) was switched on at 19.3 h. The feed rate was exponential until 645 ml medium had been added. Thereafter, between 26 h and 32 h, the flow rate was constant and equal to 233 ml/h. Temperature and pH were controlled as before and the total amount of medium fed, 1.85 l, was monitored gravimetrically as in fermentation A. Dissolved oxygen was set at 30% saturation at a constant air flow rate of 1.9 lN/min. Analytical procedures Determination of cell dry weight Cell dry weight (cdw) was obtained by filtering a known volume of culture through 0.20 mm pore size filters (Whatman, Clifton, N.J.) predried in an infrared dryer (Mettler LP16) at 105C until constant
71 weight. The wet cell mass obtained was washed with distilled water and dried on the filter in the infrared drier to constant weight. Analysis of sugars and metabolites Samples taken from the fermentation medium were centrifuged for 10 min at 3,000 g (Sigma 201; Braun). The supernatants were analysed for glucose, galactose, acetate and ethanol with enzymatic kits (d-glucose kit 716251, lactose/d-galactose kit 176303, acetic acid kit 148261 and ethanol kit 176290, all from Boehringer Mannheim, Germany). Determination of extracellular protein concentration The protein concentration was determined by the method of Bradford (1976). Cutinase activity assay The cutinase estereolytic activity was determined spectrophotometrically, following the hydrolysis of p-nitrophenylbutyrate at 400 nm (Calado et al. 2002a). One unit of activity was defined as the amount of enzyme required to convert 1 mol p-nitrophenylbutyrate to p-nitrophenol per minute. Exhaust gas analysis On-line exhaust gas analysis was carried out with a quadrupole mass spectrometer (Spectra International, Edgeware, Middlesex, UK) after appropriate calibration with gaseous mixtures (Ferreira et al. 1998). The exhaust gas was dried through a Dimroth condenser cooled with water at 4C, before entering the mass spectrometer. The data for the calculation of oxygen and carbon dioxide concentrations were logged by computer.
Results Fermentation A: fed-batch fermentation with galactose feeding Production of cutinase by S. cerevisiae was first studied by adopting a typical fed-batch fermentation strategy: a first period of biomass growth on glucose, followed by a fed-batch period on galactose for cutinase production (Fig. 1). A typical profile of a batch fermentation of S. cerevisiae on glucose was obtained in the initial batch period: a first stage in which glucose is consumed with simultaneous production of biomass and ethanol, followed by a second stage, after glucose depletion, when the culture grows by metabolising the ethanol produced during the earlier stage. The presence of ethanol at time zero is due to the ethanol introduced with the inoculum. Oxygen limitation never occurred throughout the fermentation, given its relatively low volume and good mixing. Thus, ethanol production was a result of the Crabtree effect. Indeed, the specific glucose uptake rate, qS, was 1.75 g g1 h1, higher than the reported threshold qS (0.54 g g1 h1; Kppeli 1986; Cortassa and Aon 1998), above which the respiratory pathway is saturated and ethanol is produced as a result of the onset of the
Fig. 1 Fermentation A, a fed-batch fermentation of Saccharomyces cerevisiae on galactose, after a batch period on glucose for biomass growth. Dry cell weight, and glucose, galactose, ethanol, acetate and protein concentrations over the time course are shown. F Substrate feed rate, RQ respiratory quotient, OUR oxygen uptake rate, CPR carbon dioxide production rate
fermentative pathway. When the metabolism is not exclusively oxidative, the respiratory quotient (RQ) is greater than 1, confirmed by measurements at the beginning of fermentation (Fig. 1). Ethanol and acetate concentrations followed a similar profile during the batch glucose consumption period. Next, both ethanol and acetate concentrations decreased until almost full depletion of acetate and, later, ethanol. The theoretical RQ during the aerobic oxidation of ethanol is 0.67, in good agreement with the RQ of 0.66 obtained experimentally. Upon ethanol exhaustion, the RQ increased to a fairly constant value close to 1. At 17.5 h of fermentation, galactose began to be fed at a constant rate (116 ml/h). From this time on, acetate accumulation was observed until complete exhaustion of ethanol, at which time acetate started to be consumed until its exhaustion. During the first 10 h of feeding, most of the galactose simply accumulated. Nevertheless, both biomass growth and protein production still occurred (Fig. 1). Acetate accumulated both at the start of induction and later at the start of galactose uptake.
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galactose concentration, although oxygen limitation did not occur. Acetate was present at a fairly constant concentration throughout the fermentation, despite the very low flux through glycolysis. Fermentation C: fed-batch fermentation with glucose and galactose feeding
Fig. 2 Fermentation B, a batch fermentation of S. cerevisiae strain SU50 on galactose. OUR, CPR, dry cell weight, concentrations of glucose, galactose, ethanol, acetate, and protein, and cutinase activity during the time course are shown. Due to the noise of oxygen measurements, the OUR values presented were filtered with a Butterworth filter
Growth on galactose showed two different stages: an exponential phase, followed by almost linear growth. Fermentation B: batch fermentation on galactose A batch fermentation on galactose was performed to clarify some observations during fermentation A, namely the limited specific galactose uptake rate and the apparent importance of acetate to interpret the time course of the fermentation (Fig. 2). Initially, ethanol carried over from the inoculum was consumed. Acetate concentration was apparently increasing at the time of the first sample. The carbon dioxide production rate (CPR) shows a peak in the first 5.5 h that was not accompanied by any detectable peak in oxygen uptake rate (OUR), suggesting that the culture was fermenting glucose present in the inoculum. A large peak is observed in both OUR and CPR profiles during the period in which the culture was growing on ethanol. The abrupt drop of OUR and CPR at about 16 h coincides with the exhaustion of ethanol. Due to a problem in the mass spectrometer calibration, the OUR was consistently higher than the CPR, and thus no quantitative information can be drawn from the exhaust gas analysis. The results clearly show that growth was linear and was accompanied by a linear decrease in the
The galactose uptake rate is much lower than the glucose uptake rate, leading to increased times for growth and production (i.e. low volumetric productivities) when galactose is used as the sole carbon source. It would be advantageous if the strain could grow on glucose and, simultaneously, produce cutinase due to the presence of galactose. The transport of galactose into the cell, necessary for galactose induction to occur, is repressed by the presence of glucose (zcan and Johnston 1999). Additionally, wild strains of S. cerevisiae continuously growing on galactose stop metabolising galactose upon addition of a glucose pulse, but restart metabolising galactose when the glucose concentration drops (Sierkstra et al. 1992). To investigate the influence of glucose on galactose uptake and protein production, a fed-batch with a mixed feed of glucose and galactose was performed (Fig. 3). A batch growth was carried out for biomass build-up prior to the feeding period, on a medium containing glucose and galactose so that when the fedbatch phase was started the cells were already able to metabolise galactose. The behaviour of the culture during the first 12 h closely followed that obtained when growing the strain batchwise on glucose. Glucose was readily consumed, with concomitant production of ethanol and acetate. Upon glucose exhaustion, no immediate galactose consumption was observed, the carbon source being ethanol. The synthesis of enzymes necessary for galactose utilisation explains this delay in galactose uptake. Acetate concentration increased until about 15 h of fermentation, 3 h after glucose depletion, and only then did galactose consumption start. At this stage, galactose and ethanol were consumed simultaneously. A slight increase in the protein concentration was also observed upon glucose exhaustion, i.e. when the culture became unrepressed and was able to take up galactose. At the beginning of the exponential feeding period, the concentration of galactose decreases until about 20 h and a slight accumulation of glucose is detected. Galactose starts accumulating thereafter, while practically all the glucose fed is consumed with consequent ethanol formation in addition to cell mass. The galactose concentration increases sharply until a fairly constant concentration is reached between 4 and 4.5 g/l, despite the increasing galactose input. This concentration level is also maintained when the feeding rate is kept constant. During the feeding period, both ethanol and acetate build up. The CPR increases sharply while feeding is exponential, indicating that the carbon source is being metabolised mostly through the fermentative pathway. During the
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although some galactose remained in the broth, both growth and protein production ceased.
Discussion
Fig.3 Fermentation C, a fed-batch fermentation of S. cerevisiae strain SU50 on glucose and galactose. Feeding rate profile, OUR, CPR, dry cell weight, concentrations of glucose, galactose, ethanol, acetate, and protein, and cutinase activity during the time course are shown. Dotted vertical lines Times at which the feeding regime was changed
period of constant feeding, the CPR decreases as a result of a lower specific carbon uptake rate due to a constant volumetric uptake at increasing biomass concentrations. This means that the carbon flux to the individual cell decreases and thus its fermented fraction also decreases. During this period, an increase in the protein concentration is observed. From 32 h to about 52 h, both ethanol and galactose concentration decrease and the RQ remains close to 1, similar to the situation towards the end of the batch phase. At the end of this period, the galactose uptake rate is very low, although the galactose concentration is still ca. 1 g/l. Between 52 h and 63.5 h, the remaining ethanol is totally consumed, resulting in a significant increase in biomass concentration from 12.7 g/l to 19.2 g/l. The protein concentration, however, remained constant. During this period, the RQ decreases (to an average value of 0.65), as observed previously when the culture was growing on ethanol. Once ethanol was exhausted, acetate was consumed, until its depletion at 67 h. Subsequently,
The production of cutinase from F. solani pisi by a recombinant strain of S. cerevisiae was addressed as a contribution to the future design of a fermentation strategy to enhance the productivity of the fermentation process, while minimising the cost of carbon sources and inducer. During fermentation A, galactose was fed to the culture after a batch phase on glucose for biomass growth. Surprisingly, most of the galactose accumulated during the first 10 h of the feeding period. Nevertheless, both biomass growth and protein production still occurred, though at low rates. After induction, the cell starts to assemble the machinery necessary for cutinase production, thus the biosynthetic pathways will be very active and will consume NADPH (Mathews and van Holde 1990). Upon exhaustion of NADPH produced by, e.g. the pentose-phosphate pathway, the NADPH required will come from the production of acetate, a key source of NADPH supply in yeast (Stephanopoulos et al. 1998). Thus, the peak of acetate production observed between 17.5 h and 22.5 h is probably related to the production of NADPH required for protein biosynthesis. The peak of acetate between 27.5 h and 35 h, the period during which galactose uptake started and attained its maximum rate, probably corresponds to induction of genes encoding the Leloir pathway enzymes. This would also explain the apparent increase in acetate concentration at the time of the first sample in fermentation B. Acetate was present at a fairly constant concentration throughout fermentation B, despite the very low flux through glycolysis, suggesting that the cells are producing NADPH required for biosynthesis via production of acetate. The coincidence between acetate production and initiation of cutinase production or galactose consumption was also observed during fermentation C. How galactose utilisation in the presence of glucose is carried out is a key, and still controversial, issue in the understanding of the regulation of hexose metabolism in S. cerevisae (Horak and Wolf 1997, 2001; Meijer et al. 1998; Rohde et al. 2000). It is well known that glucose is able to exert catabolite repression, and it is widely accepted that galactose cannot be transported inside the cell or metabolised in the presence of glucose. In fermentation A, during the period in which galactose accumulated, cutinase was produced, indicating that galactose induction did occur, i.e. some galactose was being transported into the cell, although another carbon source, probably from the yeast extract (which contains 17.5% of carbohydrates that might be able to repress and inhibit galactose consumption), was used. Galactose transport was perhaps achieved through a constitutive low-affinity transport system present in cells grown on glucose (Lagunas 1993). This suggests that it might be
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Fig. 5 Specific cutinase activity with respect to biomass in fermentation A: taking into account total biomass (l) or biomass produced only after induction (m)
Fig. 4 Glucose concentration (l) and glucose (—) and galactose (– –) specific uptake rates during fermentation C. The oscillations observed on the graph are mainly a result of the cubic splines used to interpolate the experimental concentrations
possible to achieve galactose induction while growing the strain on glucose. This possibility was studied during fermentation C. The glucose- and galactose-specific uptake rates during the feeding period are shown in Fig. 4. While the initial galactose was still being metabolised, the feeding period was started with an instantaneous glucose concentration increase and concomitant drop of galactose uptake rate. When the glucose concentration decreased to values lower than 0.20 g/l, galactose consumption resumed, although glucose continued to be metabolised. These results agree with the findings of Meijer et al. (1998), according to which glucose repression in S. cerevisiae seems to be related to the glucose concentration rather than the glucose flux. This suggests the feasibility of using cheaper feeding solutions containing both glucose and galactose, which will convey sufficient galactose for efficient induction, the remaining carbon being supplied by the much cheaper sugar, glucose. No cutinase was produced prior to induction with galactose, but it was produced as soon as galactose was added, except in the batch phase of fermentation C due to the repressive effect of glucose. Extracellular cutinase activity and extracellular protein were linearly correlated. The slope of the linear regression gives the specific activity of cutinase: 226€13, 179€9 and 414€13 U/mg protein for fermentations A, B and C, respectively. As reported in the literature (Verrips et al. 2000; Calado et al. 2002a, 2002b), cutinase and biomass production were linearly correlated. The slope of these lines gives the specific cellular activity, (7.90€0.39) 103, (2.62€0.16) 104 and (1.40€0.60) 104 U/g cdw for fermentations A, B and C, respectively. This can be converted into a cutinase to biomass yield with the
specific activity of purified cutinase, 630 U/mg protein (Calado 2001), resulting in 13, 42 and 22 mg cutinase/g cdw for fermentations A, B and C, respectively. The highest cutinase cellular specific activity obtained in fermentation B, where the growth rate was the lowest, is consistent with the results of Verrips et al. (2000). Brown and co-workers (2000) discussed the hypothesis that, upon induction, existing cells are unlikely to form cutinase to any significant extent because they may continue in a repressed state and that, on the other hand, daughter cells are likely to be less repressed, and galactose induction will occur in these cells. Thus, the specific cellular activity was calculated according to two distinct approaches: (1) on the basis of the total biomass (Eq. 1) and (2) taking into account only the cells produced after induction (Eq. 2): SA ¼ At =Xt S
ð1Þ
SA ¼ ðAt Ai Þ=ðXt Xi Þ
ð2Þ
where SA is the specific cellular activity, A is the extracellular cutinase activity, X is cdw, and the subscripts t and i refer to the sampled time and the induction time, respectively. The specific cellular cutinase activity calculated with the total biomass increases during the first hours after induction (Fig. 5), i.e. either a lag phase exists corresponding to the time required for the cells to assemble the machinery for cutinase formation, or only the daughter cells are capable of producing cutinase. When the specific cellular activity was calculated taking only the cells formed after induction into account, it immediately attained the levels observed throughout the fermentation, suggesting that only the daughter cells are capable of producing cutinase. Understanding which situation best reflects reality will require further study. The highest specific cellular activity and the lowest growth rate observed during fermentation B indicate that cells have directed most of their resources to cutinase production. This, together with the hypothesis that two different populations exist (induced and non-induced), is crucial to the development and optimisation of a fed-
75 Table 1 Comparison of the performance of the fermentation strategies tested. The values refer to pure cutinase, calculated by dividing the cutinase activity by the specific activity of purified cutinase, 630 U/mg protein (Calado et al. 2001). Results for fermentation C were calculated at 50 h fermentation time Fermentation Cutinase on total hexoses (mg cutinase/g hexoses) Cutinase volumetric productivity (mg cutinase l1 h1) Cutinase concentration (mg cutinase/l) Hexose cost (/g cutinase)
A
B 6.36
11.0 546 18.8
C
22.6 4.45 416 6.05
8.25 4.68 338
tional fermentation strategy, i.e. a fed-batch fermentation with galactose feeding. These results could aid development of a highly efficient cutinase production process based on the appropriate design of the feeding regime (rate and glucose/galactose ratio) and time of induction. Acknowledgements This work was supported by Funda ¼o para a CiÞncia e a Tecnologia, PRAXIS XXI programme (grant GGP XXI/BD/2936/96 awarded to B.S. Ferreira and grant GGP XXI/BD/ 18276/98 awarded to C.R.C. Calado). The authors wish to thank Dr. Maarten Egmond, Dr. Maurice Mannessi and Dr. Arthur Fellinger and Unilever Research Laboratory for providing the transformed S. cerevisiae strain.
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References batch strategy, specifically with regard to the optimum time for induction. Table 1 compares the cutinase/sugar yield, the volumetric productivity, the cutinase concentration and the cutinase production cost in terms of hexoses for the three fermentations. The prices of glucose and galactose were obtained by averaging values of pharmacopoeia grade reagents from various manufacturers, sold in packs of 1 kg (12.6/kg for glucose and 137/kg for galactose). These costs will obviously be lower for bulk quantities but their ratio will remain roughly the same, making the comparison meaningful. Fermentation B gave the highest cutinase yield on total hexoses, but since it was carried out on galactose only, it was not the most cost effective in terms of hexoses. Since both growth and galactose uptake rates were low, this fermentation was the longest, giving a relatively low cutinase volumetric productivity. Fermentation A exhibited the highest productivity, which can be ascribed to both the short fermentation duration and the high cutinase production. The short duration of fermentation A has the additional benefit of lower energy expenditure. However, the cutinase yield on total hexose was the lowest and cutinase was only produced when galactose was used as the only hexose, resulting in fermentation A being the least cost effective option in terms of hexose consumption. Fermentation C was evaluated at 50 h fermentation time, i.e. when the extracellular cutinase activity levelled off. This fermentation was the most cost effective in terms of hexose consumption. However, cutinase concentration in fermentation C was the lowest (Table 1), implying more costly downstream processing. However, the specific activity of cutinase was the highest, possibly leading to lower costs in the final purification steps, often the most significant in terms of overall production costs. To define an efficient production strategy, both the composition of the cultivation medium, especially the glucose/galactose ratio, and the medium feeding rate, have to be optimised. Fermentation C, carried out with a mixed feed of glucose and galactose, was shown to be the most cost effective with regard to hexose consumption (Table 1). Sugar costs were only 23% of those of fermentation A, which was carried out with a conven-
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