Candida bombicola : production of novel alkyl glycosides based on glucose/2-dodecanol

Candida bombicola produces glycolipids containing sophorose and a glycosidically/esterically bound ω- or (ω−1)-hydroxy C₁₆₍₁₈₎ acid. Here we describe novel glycolipids from this source. Glucose and 2-dodecanol were used for the cultivation of the yeast, one part of the racemic secondary alcohol being connected directly with a glucose or a sophorose unit. A relatively high content of yeast extract, up to 4 g l⁻¹, and subsequently higher biomass concentrations favoured the production of novel products. The provision of 150 g l⁻¹ glucose and 15 g l⁻¹ 2-dodecanol resulted in maximum production of 22 g l⁻¹ novel alkyl glycosides (more than 90% novel products). The molecular structures were analysed by gas chromatography, fast atom bombardment/mass spectrometry, ¹H- and ¹³C-nuclear magnetic resonance and optical rotation studies. Sophorose and glucose were detected as carbohydrate moieties, (S)-(+)-2-dodecanol (88%) was found to be the major lipid moiety. The new glycolipids are suitable biosurfactants, reducing the surface tension of water from 72 mN m⁻¹ to 32–38 mN m⁻¹. Received: 8 December 1997 / Received revision: 19 March 1998 / Accepted: 20 March 1998     

Effect of soybean oil and glucose on sophorose lipid fermentation by Torulopsis bombicola in continuous culture

The effect of soybean oil and glucose on the growth of Torulopsis bombicola and sophorose lipid production in continuous culture was investigated. As the dilution rate in 100 g/l glucose and 100 g/l soybean oil medium was increased, the dry cell weight and sophorose lipid concentration decreased. Sophorose lipid productivity, however, was maximum at a dilution rate of 0.03 h⁻¹. The cell yield from glucose and the sophorose lipid production from soybean oil were approximately constant regardless of the dilution rate. The specific consumption rate of soybean oil was closely related to the specific production rate of sophorose lipid. These results suggest that soybean oil was used only for sophorose lipid production whereas glucose was used only for cell mass and maintenance. When the soybean oil concentration was varied at fixed dilution rate in 100 g/l glucose medium, a high concentration of soybean oil was found to inhibit sophorose lipid production. Received: 9 January 1997 / Received revision: 5 March 1997 / Accepted: 13 April 1997     

Glycolipids of the smut fungus Ustilago maydis from cultivation on renewable resources

When grown on vegetable oils and their derivatives, the smut fungus Ustilago maydis (DSM 4500 and ATCC 14826) produces several glycolipids under nitrogen-limiting conditions. With 45 g l⁻¹ sunflower oil fatty acids (technical grade) a yield of 30 g l⁻¹ glycolipid was achieved. The resulting mixture contained predominantly mannosylerythritol lipids together with smaller amounts of cellobiose lipids. The production of the more polar cellobiose lipids was enhanced when glucose was used as carbon source. The molecular structure of the main components of the glycolipid mixture were elucidated by a combination of NMR spectroscopic and mass-spectrometric techniques. Received: 22 June 1998 / Received revision: 11 September 1998 / Accepted: 13 September 1998     

An attempt to channel the transformation of vanillic acid into vanillin by controlling methoxyhydroquinone formation in Pycnoporus cinnabarinus with cellobiose

The effects of adding cellobiose on the transformation of vanillic acid to vanillin by two strains of Pycnoporus cinnabarinus MUCL39532 and MUCL38467 were studied. When maltose was used as the carbon source in the culture medium, very high levels of methoxyhydroquinone were formed from vanillic acid. When cellobiose was used as the carbon source and/or added to the culture medium of P. cinnabarinus strains on day 3 just before vanillic acid was added, it channelled the vanillic acid metabolism via the reductive route leading to vanillin. Adding 3.5 g l⁻¹ cellobiose to 3-day-old maltose cultures of P. cinnabarinus MUCL39532 and 2.5 g l⁻¹ cellobiose to 3-day-old cellobiose cultures of P. cinnabarinus MUCL38467, yielded 510 mg l⁻¹ and 560 mg l⁻¹ vanillin with a molar yield of 50.2 % and 51.7 % respectively. Cellobiose may either have acted as an easily metabolizable carbon source, required for the reductive pathway to occur, or as an inducer of cellobiose:quinone oxidoreductase, which is known to inhibit vanillic acid decarboxylation. Received: 24 July 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Propionic acid fermentation from glycerol: comparison with conventional substrates

Instead of the conventional carbon sources used for propionic acid biosynthesis, the utilization of glycerol is considered here, since the metabolic pathway involved in the conversion of glycerol to propionic acid is redox-neutral and energetic. Three strains, Propionibacterium acidipropionici, Propionibacterium acnes and Clostridium propionicum were tested for their ability to convert glycerol to propionic acid during batch fermentation with initially 20 g/l glycerol. P. acidipropionici showed higher efficiency in terms of fermentation time and conversion yield than did the other strains. The fermentation profile of this bacterium consisted in propionic acid as the major product (0.844 mol/mol), and in minimal by-products: succinic (0.055 mol/mol), acetic (0.023 mol/mol) and formic (0.020 mol/mol) acids and n-propanol (0.036 mol/mol). The overall propionic acid productivity was 0.18 g l⁻¹h⁻¹. A comparative study with glucose and lactic acid as carbon sources showed both less diversity in end-product composition and a 17% and 13% lower propionic acid conversion yield respectively than with glycerol. Increasing the initial glycerol concentration resulted in an enhanced productivity up to 0.36 g l⁻¹h⁻¹ and in a maximal propionic acid concentration of 42 g/l, while a slight decrease of the conversion yield was noticed. Such a propionic acid production rate was similar or higher than the values obtained with lactic acid (0.35 g l⁻¹h⁻¹) or glucose (0.28 g l⁻¹h⁻¹). These results demonstrated that glycerol is a carbon source of interest for propionic acid production. Received: 15 July 1996 / Received revision: 11 November 1996 / Accepted: 11 November 1996     

Enhanced l-lysine production in threonine-limited continuous culture of Corynebacterium glutamicum by using gluconate as a secondary carbon source with glucose

In order to improve the production rate of l-lysine, a mutant of Corynebacterium glutamicum ATCC 21513 was cultivated in complex medium with gluconate and glucose as mixed carbon sources. In a batch culture, this strain was found to consume gluconate and glucose simultaneously. In continuous culture at dilution rates ranging from 0.2 h⁻¹ to 0.25 h⁻¹, the specific l-lysine production rate increased to 0.12 g g⁻¹ h⁻¹ from 0.1 g g⁻¹ h⁻¹, the rate obtained with glucose as the sole carbon source [Lee et al. (1995) Appl Microbiol Biotechnol 43:1019–1027]. It is notable that l-lysine production was observed at higher dilution rates than 0.4 h⁻¹, which was not observed when glucose was the sole carbon source. The positive effect of gluconate was confirmed in the shift of the carbon source from glucose to gluconate. The metabolic transition, which has been characterized by decreased l-lysine production at the higher glucose uptake rates, was not observed when gluconate was added. These results demonstrate that the utilization of gluconate as a secondary carbon source improves the maximum l-lysine production rate in the threonine-limited continuous culture, probably by relieving the limiting factors in the lysine synthesis rate such as NADPH supply and/or phosphoenolpyruvate availability. Received: 16 May 1997 / Received revision: 28 August 1997 / Accepted: 29 August 1997     

Nitrate removal from drinking water using a membrane-fixed biofilm reactor

Biological treatment of drinking water is a cost-effective alternative to conventional physico/chemical processes. A new concept was tested to overcome the main disadvantage of biological denitrification, the intensive post-treatment process to remove microorganisms and remnant carbon source. The biological reaction zone and carbon supply were separated from the raw water stream by a nitrate-permeable membrane. Denitrification takes place in a biofilm, which is immobilized at the membrane. In a series of bench-scale runs, different types of membranes and reactor configurations were investigated. The best denitrification rates achieved were 1230 mg NO₃ ⁻-N m⁻² day⁻¹. In one run, raw water containing 100 mg NO₃ ⁻ l⁻¹ was completely freed from nitrate. The membrane and the attached biofilm also represent a barrier against the passage of the C source and nutrients into the raw water. At concentrations of 20 mg l⁻¹ ethanol and 15 mg l⁻¹ phosphate in the bioreactor no diffusion through the membrane into the treated water was observed. Without any post-treatment, the effluent met nearly all the relevant criteria for drinking water; only the colony count was slightly increased. Received: 18 December 1996 / Received last revision: 14 April 1997 / Accepted: 19 April 1997     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Lipid storage compounds in marine bacteria

Forty psychrophile or psychrotrophic crude-oil-utilizing marine bacteria were investigated for their ability to accumulate lipid storage compounds in the cytoplasm during cultivation under nitrogen-limiting conditions. Most of them (73%) were able to accumulate specialized lipids like polyhydroxyalkanoic acids (PHA) while other lipids such as wax esters occurred in two isolates. Accumulation of PHA occurred predominantly at low temperatures (4–20 °C) as demonstrated for three isolates. Electron microscopy revealed polyphosphate inclusions occurring in two isolates in addition to PHA. Cells of the isolate Acinetobacter sp. 211 were able to synthesize and accumulate lipid inclusions during growth on acetate, ethanol, olive oil, hexadecanol and heptadecane. The composition of the lipid inclusions depended on the compounds provided as carbon source. Wax esters and acylglycerols occurred mainly during the cultivation on olive oil; in contrast, wax esters and free alcohols occurred during cultivation on hexadecanol. Total fatty acids in cells of the Acinetobacter sp. 211 amounted to 25% of the cellular dry weight in olive-oil-grown cells. Palmitic acid was the main fatty acid in the lipids when the cells were cultivated on acetate or ethanol (44% and 32% of total fatty acids respectively). In contrast, fatty acids occurring in the lipids during cultivation on hexadecanol, heptadecane or olive oil were related to the carbon source. The fatty acids present in the accumulated lipids consisted predominantly of saturated and unsaturated straight-chain fatty acids with a chain length ranging from 12 to 18 carbon atoms. Analysis of the lipid-granule-associated proteins in cells of Acinetobacter sp. 211 revealed a protein of 39 kDa as the predominant protein species. Received: 2 July 1996 / Received revision: 3 September 1996 / Accepted: 28 September 1996     

Batch and continuous cultivation of Anaerobiospirillum succiniciproducens for the production of succinic acid from whey

Batch and continuous cultivation of Anaerobiospirillum succiniciproducens were systematically studied for the production of succinic acid from whey. Addition of 2.5 g l⁻¹ yeast extract and 2.5 g l⁻¹ polypeptone per 10 g l⁻¹ whey was most effective for succinic acid production from both treated and nontreated whey. When 20 g l⁻¹ nontreated whey and 7 g l⁻¹ glucose were used as cosubstrates, the yield and productivity of succinic acid reached at the end of fermentation were 95% and 0.46 g (l h)⁻¹, respectively. These values were higher than those obtained using nontreated whey alone [93% and 0.24 g (l h)⁻¹ for 20 g l⁻¹ whey]. Continuous fermentation of A. succiniciproducens at an optimal dilution rate resulted in the production of succinic acid with high productivity [1.35 g (l h)⁻¹], high conversion yield (93%), and higher ratio of succinic acid to acetic acid (5.1:1) from nontreated whey. Received: 23 July 1999 / Received revision: 17 November 1999 / Accepted: 24 December 1999     

Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris

We tested 10 different Chlorella and Parachlorella strains under lipid induction growth conditions in autotrophic laboratory cultures. Between tested strains, substantial differences in both biomass and lipid productivity as well as in the final content of lipids were found. The most productive strain (Chlorella vulgaris CCALA 256) was subsequently studied in detail. The availability of nitrates and/or phosphates strongly influenced growth and accumulation of lipids in cells by affecting cell division. Nutrient limitation substantially enhanced lipid productivity up to a maximal value of 1.5 g l⁻¹ day⁻¹. We also demonstrated the production of lipids through large-scale cultivation of C. vulgaris in a thin layer photobioreactor, even under suboptimal conditions. After 8 days of cultivation, maximal lipid productivity was 0.33 g l⁻¹ day⁻¹, biomass density was 5.7 g l⁻¹ dry weight and total lipid content was more than 30% dry weight. C. vulgaris lipids comprise fatty acids with a relatively high degree of saturation compared with canola oil offering a possible alternative to the use of higher plant oils.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Improvement of ferulic acid bioconversion into vanillin by use of high-density cultures of Pycnoporus cinnabarinus

High-density cultures of Pycnoporus cinnabarinus were tested with a view to optimisation of ferulic acid bioconversion into vanillin. The dry weight was increased fourfold by using glucose, fructose or a mixture of glucose and phospholipids as carbon source instead of maltose, the carbon source previously used. 5 mmol l⁻¹ vanillin, i.e. 760 mg l⁻¹, was produced over 15 days with glucose-phospholipid medium. In contrast, formation of vanillin was lower using glucose or fructose compared to the maltose control. A bioreactor (2 l) with a glucose-phospholipid medium gave a molar yield of vanillin of 61% (4 mmol l⁻¹). An alternative strategy was to grow the fungus on a glucose or fructose medium for 3 days, then switch to maltose during the bioconversion phase: this method allowed 3.3 mmol l⁻¹ vanillin to be obtained in 10 days. Many by-products such as methoxyhydroquinone and vanillyl alcohol were also produced. Received: 19 February 1999 / Received revision: 4 June 1999 / Accepted: 4 June 1999     

Growth-associated production of poly(hydroxybutyric acid) by Azotobacter beijerinckii from organic nitrogen substrates

Poly(hydroxybutyric acid) (PHB) was produced by a selectant of Azotobacter beijerinckii in media containing only organic nitrogen sources such as N substrates. The chosen compounds were casein peptone, yeast extract, casamino acids and urea, each combined with carbon substrates glucose or sucrose. The PHB was synthesized under growth-associated conditions. The concentrations amounted to more than 50% of cell dry mass on casein peptone/glucose as well as urea/glucose medium within 45 h fermentation time. Corresponding to these yields, productivities of about 0.8 g PHB l⁻¹ h⁻¹ were discovered. The highest values increased to 1.06 g PHB l⁻¹ h⁻¹ on casein peptone/glucose medium and 1.1 g PHB l⁻¹ h⁻¹ on yeast extract/glucose medium after a period of 20 h. It was found that oxygen limitation was essential for successful product formation, as demonstrated earlier. These data from basic research may support further investigations into the use of technical proteins from renewable sources as substrates for PHB production by a strain of A. beijerinckii. Received: 3 June 1997 / Received revision: 29 August 1997 / Accepted: 15 September 1997     

Phytoplankton and particulate matter at the Weddell / Scotia Confluence (47°W) in summer 1989, as a final step of a temporal succession (EPOS project)

During January 1989, phytoplankton biomass and species composition were studied in a north / south transect at the Weddell / Scotia Confluence (47°W), between 57° and 61°30′S. Results showed a diatom bloom in the Scotia Sea (chlorophyll a 1.9 μg l⁻¹, particulate organic carbon 239 μg l⁻¹), dominated by Fragilariopsis cylindrus, Dactyliosolen antarcticus and Chaetoceros dichaeta. Low chlorophyll a / phaeopigments ratios (about 1.4) and silicate concentrations (15 μmol l⁻¹) suggested that this was an advanced bloom phase, probably linked to high grazing pressure. Minimum chlorophyll a values of 0.1–0.2 μg l⁻¹ and particulate organic carbon 46 μg l⁻¹ were found at the Weddell / Scotia Front and in a subsurface layer of the Weddell Sea Water. In the southern part of the transect (61°30′S), in the Weddell Sea, a second surface maximum was found (chlorophyll a 0.9 μg l⁻¹, particulate organic carbon 120 μg l⁻¹), but with a different species composition, with Cryptomonas sp. dominant. Our results show a succession within the diatom community in the Weddell / Scotia Confluence Waters when comparing the three EPOS legs. In the Weddell Sea from spring to summer, nanoflagellates, with only a minor contribution from diatoms, persist over a long period with little change in the community structure. We suggest that the frontal system, together with the receding ice edge and the grazing pressure of either krill or protozooplankton, are mainly responsible for the phytoplankton distribution patterns found. Received: 3 July 1996 / Accepted: 3 November 1996     

Growth of Photorhabdus luminescens in batch and glucose fed-batch culture

Photorhabdus luminescens, a bacterial symbiont of entomopathogenic biocontrol nematodes, was grown in batch and glucose fed-batch culture. The cell density, bioluminescence, production of antibiotic substances, number of cells with inclusion bodies, glucose concentration and oxygen uptake rate were recorded. The addition of 12.4 g l⁻¹ glucose prolonged the growth, and the yield almost doubled, from 6.85 g l⁻¹ to 12.45 g l⁻¹ dry mass. The production of antibiotic substances increased by 140%. Bioluminescence was higher in the batch culture. A shift of P. luminescens to phase II variants was not detected. Received: 21 January 2000 / Received revision: 3 April 2000 / Accepted: 7 April 2000     

The action of antibiotics on the anaerobic digestion process

Antibiotics can disturb the production of biogas during anaerobic digestion. This study shows a systematic approach to understanding how the different bacterial populations involved in the final conversion of organic matter into methane are inhibited by 15 antimicrobial agents with different specificities and modes of action. The results obtained show the following trends: (i) some inhibitors, such as the macrolide erythromycin, lack any inhibitory effect on biogas production; (ii) some antibiotics, with different specificities, have partial inhibitory effects on anaerobic digestion and decrease methane production by interfering with the activity of propionic-acid- and butyric-acid-degrading bacteria,␣(e.g. antibiotics that interfere with cell wall synthesis, RNA polymerase activity and protein synthesis, especially the aminoglycosides); (iii) the protein synthesis inhibitors chlortetracycline (IC₅₀ 40 mg l⁻¹) and chloramphenicol (IC₅₀ 15–20 mg l⁻¹) are very powerful inhibitors of anaerobic digestion. The majority of the antibiotics tested lacked activity against acetoclastic methanogens, being active only on the acetogenic bacteria. However, chloramphenicol and chlortetracycline could cause the complete inhibition of the acetoclastic methanogenic archaea. Received: 6 February 1996 / Received revision: 24 July 1996 / Accepted: 5 August 1996     

The microbial production of poly(hydroxyalkanoates) from tallow

The bacteria Pseudomonas oleovorans, P. resinovorans, P. putida, and P. citronellolis were evaluated for their ability to grow and produce poly(hydroxyalkanoates) (PHA) using tallow free fatty acids and tallow triglyceride as carbon substrates. Tallow free fatty acids supported cell growth and PHA production for all four organisms, yielding PHA contents of 18%, 15%, 19% and 3% of their cell dry weights for P. oleovorans, P.␣resinovorans, P. putida, and P. citronellolis respectively. Only P. resinovorans, however, was able to grow and produce PHA polymer, with cells attaining a PHA content of 15% of their cell dry weight, using unhydrolyzed tallow as the substrate. Extracts from 46-h cultures of P. resinovorans were found to have a higher esterase activity (12.80 units μl⁻¹min⁻¹) compared to the activities found for cultures of P. oleovorans, P. citronellolis, and P. putida ( < 0.03 units μl⁻¹min⁻¹). Polymer repeat-unit compositions were determined by GC analysis of the β-hydroxymethyl esters of hydrolyzed PHA, and ranged in carbon-chain lengths from C₄ to C₁₄, with some mono-unsaturation in the C₁₂ and C₁₄ side-chains. PHA compositions were similar for the polymers obtained from all four organisms, with repeat units of chain lengths C₈ and C₁₀ predominating. Received: 16 February 1996 / Received revision: 23 May 1996 / Accepted: 10 June 1996     

Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens

Three white-rot basidiomycetes, Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens, were cultivated on a liquid medium supplemented with l-phenylalanine, a precursor for benzaldehyde (bitter almond aroma) and benzyl alcohol. Remarkable amounts of benzaldehyde (587 mg l⁻¹) were found in cultures of B. adusta. Immobilization of this fungus on polyurethane foam cubes allowed an 8.3-fold increase of the production of benzaldehyde and a 15-fold increase of the productivity as compared with non-immobilized cells. Aryl-alcohol oxidase activity was only detected in B. adusta. This activity was also significantly enhanced in immobilized cells, suggesting that it plays an important role in benzaldehyde biosynthesis. Conversely, consistent amounts of benzyl alcohol (340 mg l⁻¹ for B. adusta and I. benzoinum and 100 mg l⁻¹ for D. squalens) were produced by the three fungi when immobilized. Laccase activity was found only in the strains I. benzoinum and D. squalens. This activity was markedly enhanced in free cells cultures. Immobilization of the fungi did not promote benzyl alcohol production by comparison with free cell cultures (500 mg l⁻¹). Received: 10 December 1996 / Received revision: 17 February 1997 / Accepted: 22 February 1997     

Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system

A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg l⁻¹ of the fusion protein (420 mg l⁻¹ of the recombinant hCT precursor) within 14 h, reaching 45 g l⁻¹ cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor l⁻¹ h⁻¹) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-l scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g⁻¹ yeast extract). Received: 18 January 2000 / Received revision: 14 April 2000 / Accepted: 14 April 2000