Characterization of Bacillus halodurans alpha-galactosidase Mel4A encoded by the mel4A gene (BH2228)

A family-4 alpha-galactosidase Mel4A of Bacillus halodurans was expressed in Escherichia coli and characterized. Recombinant enzyme rMel4A depended on NAD+, some divalent cations such as Mn2+, and reducing reagents such as dithiothreitol. rMel4A was active on small saccharides such as raffinose but not on highly polymerized galactomannan. Immunological analysis indicated that raffinose induced the production of Mel4A in B. halodurans.     

Melatonin affects the growth and cadmium accumulation of Malachium aquaticum and Galinsoga parviflora

Phytoremediation technology has become one of the main techniques for remediating soils polluted by heavy metals because it does not damage the environment, but heavy metal-tolerant plants have the disadvantages of low biomass and slow growth. A pot experiment was conducted to study the effects of melatonin (Mel) on growth and cadmium (Cd) accumulation in the Cd accumulator Malachium aquaticum and hyperaccumulator Galinsoga parviflora by spraying different concentrations of Mel on them. The results showed that shoot biomass, photosynthetic pigment content and antioxidant enzyme activity were increased in both species after Mel was sprayed on their leaves. Mel reduced the Cd content in shoots of M. aquaticum and increased it in those of G. parviflora. In general, Cd accumulation was greatest in M. aquaticum when Mel was 200 μmol L^?1 (120.71 μg plant^?1, increased by 15.97% than control) and in G. parviflora when Mel was 100 μmol L^?1 (132.40 μg plant^?1, increased by 68.30% than control). Our results suggest it is feasible to improve the remediation efficiency of lightly Cd-contaminated soil by spraying G. parviflora with100 μmol L^?1 Mel.     

Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism,growth, and protein synthesis of Pseudomonas halodurans and Alteromonas luteo-violaceus during sulfate limitation

Sulfate concentration in the growth medium exerted a strong influence on the sulfur content of protein in two marine bacteria, Pseudomonas halodurans and Alteromonasluteo-violaceus, but the distribution of sulfur in major biochemical fractions was not affected. 90% of the total cellular sulfur was contained in low molecular weight organic compounds and protein; inorganic sulfate was not an important component. The sulfur content of isolated protein and total cellular sulfur increased in proportion to the external sulfate concentration for both bacteria, reaching a maximum at about 100–250 M. The growth rate of P. halodurans only was dependent on the sulfate concentration.Sulfur starvation of cells labeled to equilibrium with ³⁵S-sulfate resulted in a rapid decrease in low molecular weight organic S with a concommitant increase in alcohol soluble (P. halodurans) or residue protein (A. luteo-violaceus). Although cell division was prevented, total protein increased in both bacteria, resulting in synthesis of sulfur-deficient protein. This effect was most pronounced in P. halodurans.Addition of ³⁵S-sulfate to sulfur-starved A. luteo-violaceus further demonstrated that sulfur metabolism was restricted primarily to the synthesis and utilization of sulfurcontaining protein precursors. The low molecular weight organic S pool was replenished rapidly, and the pool size per cell reached twice the normal value before cell division resumed. Incorporation into protein was very rapid.Abbreviations L.M.W. low molecular weight - TCA trichloroacetic acid     

Melatonin against acute ischaemic stroke dependently via suppressing both inflammatory and oxidative stress downstream signallings

This study tested the hypothesis that melatonin (Mel) therapy preserved the brain architectural and functional integrity against ischaemic stroke (IS) dependently through suppressing the inflammatory/oxidative stress downstream signalling pathways. Adult male B6 (n = 6 per each B6 group) and TLR4 knockout (ie TLR4^?/?) (n = 6 per each TLR4^?/? group) mice were categorized into sham control (SC^B6), SC^TLR4?/?, IS^B6, IS^TLR4?/?, IS^B6 + Mel (i.p. daily administration) and IS^TLR4?/? + Mel (i.p. daily administration). By day 28 after IS, the protein expressions of inflammatory (HMBG1/TLR2/TLR4/MAL/MyD88/RAM TRIF/TRAF6/IKK‐α/p‐NF‐κB/nuclear‐NF‐κB/nuclear‐IRF‐3&7/IL‐1β/IL‐6/TNF‐α/IFN‐γ) and oxidative stress (NOX‐1/NOX‐2/ASK1/p‐MKK4&7/p‐JNK/p‐c‐JUN) downstream pathways as well as mitochondrial‐damaged markers (cytosolic cytochrome C/cyclophilin D/SRP1/autophagy) were highest in group IS^B6, lowest in groups SC^B6 and SC^TLR4?/?, lower in group IS^TLR4?/? + Mel than in groups IS^TLR4?/? and IS^B6 + Mel and lower in group IS^B6 + Mel than in group IS^TLR4?/? (all P < .0001). The brain infarct volume, brain infarct area and the number of inflammatory cells in brain (CD14/F4‐88) and in circulation (MPO+//Ly6C+/CD11b+//Ly6G+/CD11b+) exhibited an identical pattern, whereas the neurological function displayed an opposite pattern of inflammatory protein expression among the six groups (all P < .0001). In conclusion, TLR inflammatory and oxidative stress signallings played crucial roles for brain damage and impaired neurological function after IS that were significantly reversed by Mel therapy.     

Enhanced production and characterization of a β-glucosidase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ∼40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A ₆₀₀ 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. K _m and k _cat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec⁻¹, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase from B. halodurans.     

Cytotoxic Activity of Cunila angustifolia Essential Oil

The Cunila angustifolia essential oil was obtained from fresh leaves by hydrodistillation and analyzed by GC‐FID and GC‐MS to determine its chemical composition. The essential oil presented pulegone (29.5 %) and isomenthol (27.0 %) as major components, and other compounds such as menthone (8.6 %), neomenthol (7.2 %), menthyl acetate (2.5 %) and caryophyllene oxide (2.0 %) were identified. The cytotoxic activity of the essential oil was evaluated by MTS assay, with the human cancer cell lines of the lung (A549), breast (MCF‐7) and skin melanoma (SK‐Mel‐28). The assay showed the highest selectivity, to MCF‐7 cell lines, with IC₅₀ equal to 34.0 μg mL^?1, low selectivity for SK‐Mel‐28 cell lines, with IC₅₀ equal to 279.9 μg mL^?1, and no mortality to A549 cell lines.     

Stachyose synthesis in leaves of Cucurbita pepo

An enzyme synthesizing stachyose, galactinol-raffinose galactosyltransferase (EC2.4.1.67), has been purified ca 40-fold from mature leaves of Cucurbita pepo using ammonium sulphate precipitation, Sephadex gel filtration and DEAE-Sephadex gel chromatography. The purified enzyme fraction was separated from all but 2 % of the total,α-galactosidase activity extracted from the tissue. The enzyme was optimally active at pH 6.9 and was stable for at least a month at 4° in the presence of 20 mM 2-mercaptoethanol. The enzyme displayed high specificity for the donor galactinol (K_m 7.7 mM) and the acceptor raffinose (K_m 4.6 mM) and was unable to effect synthesis of any other member of the raffinose series of galactosyl-sucrose oligosaccharides. Co²⁺, Hg²⁺, Mn²⁺ and Ni²⁺ ions were particularly inhibitory; no metal ion promotion was observed and 5 mM EDTA was ineffective. Myo-inositol was strongly inhibitory (K_i 2 mM), melibiose weakly so. Tris buffer (0. 1 M) was also inhibitory. Galactinol hydrolysis occurred in the absence of the acceptor raffinose but there was no hydrolysis of either raffinose or stachyose in the absence of the donor galactinol. The reaction was readily reversible and exchange reactions were detected between substrates and products. It is proposed that the synthesis of stachyose in mature leaves ofC. pepo proceeds via this galactosyltransferase and not via α-galactosidase.     

Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism,protein synthesis,and growth of Pseudomonas halodurans and Alteromonas luteo-violaceus during unperturbed batch growth

Analysis of the distribution of ³⁵S-sulfate and ¹⁴C-glutamate in major biochemical components of the two marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus, was compared with cell density and total cellular protein during exponential growth in batch culture. For both organisms, the sulfur distribution was restricted principally to the low molecular weight organic and protein fractions, which together accounted for over 90% of the total sulfur. Carbon was more widely distributed, with these two fractions containing only 70% of the total label.Growth rate constants calculated from increases in cell numbers, protein, and ³⁵S and ¹⁴C in the various fractions indicated nearly balanced growth in A. luteo-violaceus, with constants derived from all biosynthetic parameters agreeing within 5% during the exponential phase. In contrast, protein synthesis and ³⁵S incorporation into residue protein constants were 30% higher than constants derived from cell counts and incorporation of ¹⁴C in P. halodurans. Therefore the cellular protein content P. halodurans varied over a two-fold range, with maximum protein per cell in the late exponential phase. A distinct reduction in the rate constants for total protein and ³⁵S incorporation into residue protein foreshadowed entry into the stationary phase more than one generation before other parameters.Incorporation of ³⁵S-sulfate into residue protein paralleled protein synthesis in both bacteria. The weight percent S in protein agreed well with the composition of an average protein derived from the literature. Sulfur incorporation into protein may be a useful measurement of marine bacterial protein synthesis.Abbreviations L.M.W. low molecular weight - TCA trichloroacetic acid - CFU colony forming unit     

Improved genetic transformation methods for the model alkaliphile Bacillus halodurans C-125

Aims: Bacillus halodurans C‐125 is a Gram‐positive bacterium that was the first alkaliphilic species to have its genome completely sequenced. Despite its many years as a model for alkaliphily and source of industrially important enzymes, genetic manipulation of B. halodurans C‐125 remains difficult, and therefore, we sought to develop a robust method to allow routine transformation of this organism. Methods and Results: A plasmid artificial modification system (PAM system, Yasui et al. 2008 ) for B. halodurans C‐125 was created that increases transformation efficiency by 10‐ to 1000‐fold. Also, recovering transformed protoplasts on succinate nutrient agar (SNA) yields faster, more robust colony recovery than on the traditional recovery medium. Combining these two techniques often allows recovery of transformants in as little as 48 h. Conclusions: Use of the B. halodurans C‐125 PAM system and SNA greatly improves the efficiency and speed of protoplast transformation of B. halodurans C‐125. Significance and Impact of the Study: These techniques allow routine genetic manipulation of B. halodurans C‐125, a model alkaliphilic bacterium with important industrial properties.     

A cloned Bacillus halodurans multicopper oxidase exhibiting alkaline laccase activity

The gene product of open reading frame bh2082 from Bacillus halodurans C-125 was identified as a multicopper oxidase with potential laccase activity. A homologue of this gene, lbh1, was obtained from a B. halodurans isolate from our culture collection. The encoded gene product was expressed in Escherichia coli and showed laccase-like activity, oxidising 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid), 2,6-dimethoxyphenol and syringaldazine (SGZ). The pH optimum of Lbh1 with SGZ is 7.5–8 (at 45°C) and the laccase activity is stimulated rather than inhibited by chloride. These unusual properties make Lbh1 an interesting biocatalyst in applications for which classical laccases are unsuited, such as biobleaching of kraft pulp for paper production.     

Studies on the Decomposition of Raffinose by α-Galactosidase of Actinomycetes

α-Galactosidase was isolated from the culture broth of Streptomyces olivaceus and was partially purified by chromatography on a DEAE-sephadex column. The optimum pH of the preparation was found to be 5.2 for raffinose and the preparation was inactivated completely by maintaining it at 60°C for 15 minutes. p-Chloromercuribenzoate, HgCl₂ and AgNO₃ caused complete inhibition of the enzyme activity at 2 × 10^?5 M concentration. The preparation showed transglycosylase activity. A sugar spot, chromatographically identical with that of stachyose, appeared in the digest of raffinose. However, the preparation hydrolyzed raffinose completely into galactose and sucrose after a prolonged incubation.

A simple raffinose estimation method was developed using the enzyme preparation, and it was found that the method allowed to estimate 125~500 μg of raffinose with an accuracy of ±5%. The method was applied to the estimation of raffinose in beet molasses.     

Degradation of stachyose,raffinose, melibiose and sucrose by different tempe-producing Rhizopus fungi

Forty-six strains of tempe-forming Rhizopus species were screened for their ability to grow on raffinose as the sole carbon source. Six of the strains showed good growth and sporulation. These isolates were one Rhizopus oligosporus, one Rhizopus microsporus var. chinensis, three Rhizopus oryzae and one Rhizopus stolonifer. These six moulds and R. oligosporus strain NRRL 2710 were investigated for their metabolism of the raffinose family of -galactoside carbohydrates. Degradation experiments were performed in submerged culture in a medium containing soybean -protein, sodium phytate and either stachyose, raffinose or melibiose. R. oryzae and R. stolonifer completely consumed the tested carbohydrates as carbon source. R. microsporus var. chinensis failed to hydrolyse the -galactosidic bonds of raffinose, stachyose or melibiose, whereas it was able to use sucrose and the fructose moiety of raffinose or stachyose for growth. R. oligosporus NRRL 2710 was unable to hydrolyse any of the tested carbohydrates. The results of the oligosaccharide degradation experiments could be verified during tempe production from soybeans with the selected fungal species.     

Analysis of the genome of an alkaliphilic Bacillus strain from an industrial point of view

Bacillus species and other microbes with pH optima for growth higher than pH 9 are defined as alkaliphiles. A large number of alkaliphilic Bacillus strains producing useful enzymes, have been isolated from various environments. Some of these enzymes, such as proteases and cellulases from alkaliphilic Bacillus strains, have been commercialized and have brought great advantages to industry and domestic life. To support further development of the enzyme industry, we initiated analysis of the genome of Bacillus halodurans C-125, which is 4.25 Mb in size, and constructed a physical and genetic map for comparison with the Bacillus subtilis chromosome. Systematic sequencing of the whole genome of Bacillus halodurans C-125 has been automated since the beginning of May 1998, and sequencing of 98% of the whole genome has been done so far. Through genome analysis, it became apparent that the genome organization of alkaliphilic Bacillus halodurans C-125 is totally different from that of B. subtilis orthologues. Received: July 11, 1999 / Accepted: December 27, 1999     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

Formation of Levan from Raffinose by Levansucrase of Zymomonas mobilis

Levansucrase (EC 2.4.1.10.) of Zymomonas mobilis 113S can perform the polymerisation of fructose moiety from raffinose to levan concomitantly with a release of non‐catabolised melibiose into the medium. The kinetic parameters of the levansucrase‐catalysed reaction provide even higher reaction velocities on raffinose as compared to sucrose, particularly at low substrate concentrations. A decreased value in the number of the average molecular mass (M_n = 1693 kDa), an increased intrinsic viscosity (η = 49.47 cm³/g), and a diminished Huggin's constant (K^' = 0.67) are intrinsic to the levan synthesis from raffinose, indicating certain structural peculiarities compared to a polysaccharide obtained from sucrose (M_n = 1851 kDa, [η] = 42.47 cm³/g, K' = 1.21).     

Sugar synthesis and phloem loading in Coleus blumei leaves

Sugar-synthesis and -transport patterns were analyzed in Coleus blumei Benth. leaves to determine where galactinol, raffinose, and stachyose are made and whether phloem loading includes an apoplastic (extracellular) step or occurs entirely within the symplast (plasmodesmata-connected cytoplasm). To clarify the sequence of steps leading to stachyose synthesis, a pulse (15 s) of ¹⁴CO₂ was given to attached leaves followed by a 5-s to 20-min chase: sucrose was rapidly labeled while galactinol, raffinose and stachyose were labeled more slowly and, within the first few minutes, to approximately the same degree. Leaf tissue was exposed to either ¹⁴CO₂ or [¹⁴C]glucose to identify the sites of synthesis of the different sugars. A 2-min exposure of peeled leaf tissue to [¹⁴C]glucose resulted in preferential labeling of the minor veins, as opposed to the mesophyll; galactinol, raffinose and stachyose were more heavily labeled than sucrose in these preparations. In contrast, when leaf tissue was exposed to ¹⁴CO₂ for 2 min for preferential labeling of the mesophyll, sucrose was more heavily labeled than galactinol, raffinose or stachyose. We conclude that sucrose is synthesized in mesophyll cells while galactinol, raffinose and stachyose are made in the minorvein phloem. Competition experiments were performed to test the possibility that phloem loading involves monosaccharide uptake from the apoplast. Two saturable monosaccharide carriers were identified, one for glucose, galactose and 3-O-methyl glucose, and the other for fructose. Washing the apoplast of peeled leaf pieces with buffer or saturating levels of 3-O-methyl glucose, after providing a pulse of ¹⁴CO₂, did not inhibit vein loading or change the composition of labeled sugars, and less than 0.5% of the assimilated label was recovered in the incubation medium. These and previous results (Turgeon and Gowan, 1991, Plant Physiol. 94, 1244–1249) indicate that the phloem loading pathway in Coleus is probably symplastic.Abbreviations 3-OMG 3-O-methyl glucose - PCMBS p-chloromercuribenzenesulfonic acid - SE-CCC sieve-element-companion-cell complex This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds.     

Thalassococcus lentus sp. nov., an alphaproteobacterium isolated from seawater of a seaweed farm

A Gram-negative, non-motile and rod- or ovoid-shaped bacterial strain, designated YCS-24^T, was isolated from seawater of a seaweed farm in the South Sea, South Korea. Strain YCS-24^T grew optimally at 25–28 °C, at pH 7.0–7.5 and in the presence of 2 % (w/v) NaCl. Strain YCS-24^T exhibited the highest 16S rRNA gene sequence similarity values of 97.5 and 97.1 % to the type strains of Thalassobius maritimus and Thalassococcus halodurans, respectively. The neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain YCS-24^T clustered with the type strain of T. halodurans. The DNA G+C content of strain YCS-24^T was 58.0 mol% and its DNA–DNA relatedness values with T. halodurans JCM 13833^T and T. maritimus GSW-M6^T were 17 ± 6.2 and 23 ± 9.2 %, respectively. The predominant ubiquinone found in strain YCS-24^T was Q-10 and the predominant fatty acid of strain YCS-24^T was C_18:1 ω7c. The major polar lipids of strain YCS-24^T were phosphatidylcholine, phosphatidylglycerol, one unidentified aminolipid and one unidentified lipid. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, demonstrated that strain YCS-24^T is distinguished from T. halodurans. On the basis of the data presented, strain YCS-24^T (= KCTC 32084^T = CCUG 62791^T) represents a novel species of the genus Thalassococcus, for which the name Thalassococcus lentus sp. nov. is proposed.     

Synthesis of α-galactooligosaccharides with α-galactosidase from<Emphasis Type="Italic"> Lactobacillus reuteri</Emphasis> of canine origin

Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities. When raffinose was used as the carbon source, -galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase. A 64 kDa enzyme was purified by ultra- and gel filtration, and characterized for its hydrolytic and synthetic activity. Highest hydrolytic activity was found at pH 5.0 at 50 °C (K_M 0.55 mM, V_max 0.80 mol min^–1 mg^–1 of protein). The crude cell-free extract was further used in glycosyl transfer reactions to synthesize oligosaccharides from melibiose and raffinose. At a substrate concentration of 23% (w/v) oligosaccharide mixtures were formed with main products being a trisaccharide at 26% (w/w) yield from melibiose after 8 h and a tetrasaccharide at 18% (w/w) yield from raffinose after 7 h. Methylation analysis revealed the trisaccharide to be 6 -galactosyl melibiose and the tetrasaccharide to be stachyose. In both cases synthesis ceased when hydrolysis of the substrate reached 50%.     

Different ratios of sucrose/raffinose-induced membrane depolarizations in the mesophyll of species with symplasmic (Catharanthus roseus, Ocimum basilicum) or apoplasmic (Impatiens walleriana, Vicia faba) minor-vein configurations

In mesophyll cells of species with a symplasmic (Ocimum basilicum, Catharanthus roseus, Magnolia denudata) or an apoplasmic (Vicia faba, Impatiens walleriana, Bellis perennis) minor-vein configuration, membrane depolarizations in response to 20 or 200 mol·m^–3 raffinose and sucrose were measured. Ageing period and resting potential marginally affected the degree of depolarization. The symplasmic species showed similar depolarization responses to 20 and 200 mol·m^–3 sucrose or raffinose. In the apoplasmic species, depolarization increased statistically significantly from 20 to 200 mol·m^–3 sucrose, whereas the depolarization response to raffinose was equal at both concentrations. In the apoplasmic species, moreover, the depolarization response to raffinose was significantly weaker than to sucrose at all concentrations. A major difference between symplasmic and apoplasmic species seems to lie in the scantiness of raffinose carriers in the mesophyll plasma membrane of species with the apoplasmic mode of phloem loading.Abbreviations 20R(200R) 20(200) mol·m^–3 raffinose - 20S(200S) 20(200) mol·m^–3 sucrose     

Xylose utilisation by recombinant strains of Saccharomyces cerevisiae on different carbon sources

Autoselective xylose-utilising strains of Saccharomyces cerevisiae expressing the xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes of Pichia stipitis were constructed by replacing the chromosomal FUR1 gene with a disrupted fur1::LEU2 allele. Anaerobic fermentations with 80 g l⁻¹ d-xylose as substrate showed a twofold higher consumption of xylose in complex medium compared to defined medium. The xylose consumption rate increased a further threefold when 20 g l⁻¹ d-glucose or raffinose was used as co-substrate together with 50 g l⁻¹ d-xylose. Xylose consumption was higher with raffinose as co-substrate than with glucose (85% versus 71%, respectively) after 82 h fermentations. A high initial ethanol concentration and moderate levels of glycerol and acetic acid accompanied glucose as co-substrate, whereas the ethanol concentration gradually increased with raffinose as co-substrate with no glycerol and much less acetic acid formation. Received: 12 March 1999 / Received revision: 31 June 1999 / Accepted: 5 July 1999