Development of a sensitive bioassay method for quorum sensing inhibitor screening using a recombinantAgrobacterium tumefaciens

Acylhomoserine lactones (AHLs) are known to be the triggering molecules in the quorum sensing mechanism of many gram-negative bacteria. In order to detect AHL inhibitors that are potential biofilm inhibitors, a convenient and sensitive bioassay was developed based on the β-galactosidase activity (β-GAL) of a recombinantAgrobacterium tumefaciens strain. A series of commercially available AHLs were tested for inducing β-GAL at varying concentrations in agar-plate and liquid cultures of the reporter strain. All AHLs tested exhibited a concentration-dependent induction, and octanoyl homoserine lactone (OHL) showed the highest sensitivity with a detection limit of 0.1 nM in the liquid culture assay. When fimbrolide, a known quorum sensing inhibitor, was added, induction of β-GAL by OHL was repressed. The repression at a constant OHL concentration was dependent on the fimbrolide concentration with the detection limit below 1 ppm, indicating that this assay is a sensitive method for screening AHL inhibitors.     

Microbial transformation of ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">Acremonium strictum</Emphasis>

Preparative-scale fermentation of ginsenoside Rb₁ (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg₃ (4), ginsenoside F₂ (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb₁ in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.     

Variation of acharan sulfate and monosaccharide composition and analysis of neutral <Emphasis Type="Italic">N</Emphasis>-glycans in African giant snail (<Emphasis Type="Italic">Achatina fulica</Emphasis>)

Acharan sulfate content from African giant snail (Achatina fulica) was compared in eggs and snails of different ages. Acharan sulfate was not found in egg. Acharan sulfate disaccharide →4)-α-d-GlcNpAc (1→4)-α-l-IdoAp2S(1→, analyzed by SAX (strong-anion exchange)–HPLC was observed soon after hatching and increases as the snails grow. Monosaccharide compositional analysis showed that mole % of glucosamine, a major monosaccharide of acharan sulfate, increased with age while mole % of galactose decreased with age. These results suggest that galactans represent a major energy source during development, while acharan sulfate appearing immediately after hatching, is essential for the snail growth. The structures of neutral N-glycans released from eggs by peptide N-glycosidase F (PNGase F), were next elucidated using ESI-MS/MS, MALDI-MS/MS, enzyme digestion, and monosaccharide composition analysis. Three types of neutral N-glycan structures were observed, truncated (Hex_2–4-HexNAc₂), high mannose (Hex_5–9-HexNAc₂), and complex (Hex₃-HexNAc_2–10) types. None showed core fucosylation.     

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.     

Clonal propagation of mesquite tree (<Emphasis Type="Italic">Prosopis laevigata</Emphasis> Humb. &; Bonpl. ex Willd. M.C. Johnston). I. Via cotyledonary nodes

Plantlet regeneration in Prosopis laevigata (Humb. & Bonpl. ex Willd.) Johnston (Fabaceae), a multipurpose tree, has been achieved from cotyledonary nodes excised from in vitro grown seedlings. The explants were cultured on MS media containing different concentrations of N-6 benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-d) and a mixture of organic components. The highest number (3.37 + 0.51) of multiple shoots was observed in MS media containing 2,4-d (9.05 μM) + BA (6.62 μM). The regenerated shoots were then transferred onto half-strength MS medium containing a plant growth regulator that was either: indole-3-butyric acid, 1-naphthaleneacetic, indole-3-acetic acid, or 2,4-d as well as phytagel or vermiculite for adventitious root initiation. Best rooting efficiency of 44.0% was obtained when NAA (16.11 μM) and vermiculite were used. After rooting, the cloned plantlets were successfully hardened to ex vitro conditions. This work may help to reduce the devastation caused by the overexploitation of this species.     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.     

Effect of growth rate on the production of<Emphasis Type="SmallCaps">l</Emphasis>-proline in the fed-batch culture of<Emphasis Type="Italic">Corynebacterium acetoacidophilum</Emphasis>

Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06, 0.08, and 0.1 h⁻¹. The results show that the highest production ofl-proline was obtained at μ=0.04 h⁻¹. The specificl-proline production rate (Q_p) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at μ=0.08 h⁻¹. Thus, the highest productivity ofl-proline was 1.66 g L⁻¹ h⁻¹ at μ=0.08 h⁻¹. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics.     

The impact of different chelating leaving groups on the substitution kinetics of mononuclear PtII(1,2- trans - R , R -diaminocyclohexane)(X–Y) complexes

A set of three oxaliplatin derivatives containing 1,2-trans-R,R-diaminocyclohexane (dach) as a spectator ligand and different chelating leaving groups X–Y, viz., [Pt(dach)(O,O-cyclobutane-1,1-dicarboxylate)], or Pt(dach)(CBDCA), [Pt(dach)(N,O-glycine)]⁺, or Pt(dach)(gly), and [Pt(dach)(N,S-methionine)]⁺, or Pt(dach)(l-Met), where l-Met is l-methionine, were synthesized and the crystal structure of Pt(dach)(gly) was determined by X-ray diffraction. The effect of the leaving group on the reactivity of the resulting Pt(II) complexes was studied for the nucleophiles thiourea, glutathione (GSH) and l-Met under pseudo-first-order conditions as a function of nucleophile concentration and temperature, using UV–vis spectrophotometric techniques. ¹H NMR spectroscopy was used to follow the substitution of the leaving group by guanosine 5′-monophosphate (5′-GMP²⁻) under second-order conditions. The rate constants indicate for all reactions a direct substitution of the X–Y chelate by the selected nucleophiles, thereby showing that the nature of the chelate, viz., O–O (CBDCA²⁻), N–O (glycine) or S–N (l-Met), respectively, plays an important role in the kinetic and mechanistic behavior of the Pt(II) complex. The k ₁ values for the reaction with thiourea, l-Met, GSH and 5′-GMP²⁻ were found to be as follows (10³ k ₁, 37.5 °C, M⁻¹ s⁻¹): Pt(dach)(CBDCA) 61 ± 2, 21.6 ± 0.1, 23 ± 1, 0.352 ± 0.002; Pt(dach)(gly) 82 ± 3, 6.2 ± 0.2, 37 ± 1, 1.77 ± 0.01; Pt(dach)(l-Met) (thiourea, GSH) 62 ± 2, 24 ± 1. The activation parameters for all reactions studied suggest an associative substitution mechanism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

Development of medium for enhanced production of glutaminase-free <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase from <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> MTCC 1428

Glutaminase-free l-asparaginase is known to be an excellent anticancer agent. In the present study, statistically based experimental designs were applied to maximize the production of glutaminase-free l-asparaginase from Pectobacterium carotovorum MTCC 1428. Nine components of the medium were examined for their significance on the production of l-asparaginase using the Plackett–Burman experimental design. The medium components, viz., glucose, l-asparagine, KH₂PO₄, and MgSO₄·7H₂O, were screened based on their high confidence levels (P < 0.04). The optimum levels of glucose, l-asparagine, KH₂PO₄, and MgSO₄·7H₂O were found to be 2.076, 5.202, 1.773, and 0.373 g L⁻¹, respectively, using the central composite experimental design. The maximum specific activity of l-asparaginase in the optimized medium was 27.88 U mg⁻¹ of protein, resulting in an overall 8.3-fold increase in the production compared to the unoptimized medium.     

Xylose reductase activity in <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> UFV-170 cultivated in semi-synthetic medium and cotton husk hemicellulose hydrolyzate

To develop a new enzymatic xylose-to-xylitol conversion, deeper knowledge on the regulation of xylose reductase (XR) is needed. To this purpose, a new strain of Debaryomyces hansenii (UFV-170), which proved a promising xylitol producer, was cultivated in semi-synthetic media containing different carbon sources, specifically three aldo-hexoses (d-glucose, d-galactose and d-mannose), a keto-hexose (d-fructose), a keto-pentose (d-xylose), three aldo-pentoses (d-arabinose, l-arabinose and d-ribose), three disaccharides (maltose, lactose and sucrose) and a pentitol (xylitol). The best substrate was lactose on which cell concentration reached about 20 g l⁻¹ dry weight (DW), while the highest specific growth rates (0.58–0.61 h⁻¹) were detected on lactose, d-mannose, d-glucose and d-galactose. The highest specific activity of XR (0.24 U mg⁻¹) was obtained in raw extracts of cells grown on d-xylose and harvested in the stationary growth phase. When grown on cotton husk hemicellulose hydrolyzates, cells exhibited XR activities five to seven times higher than on semi-synthetic media.     

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C, with a t ₅₀ of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase).     

Chromosome number and DNA ploidy level reports from Central Europe — 2

Second part of commented chromosome number and DNA ploidy level reports from Central Europe comprising the whole Carpatho-Pannonian region includes the data for following taxa: Tephroseris aurantiaca (2n = 96), T. capitata (2n = 64) and T. integrifolia (2n = 48) by J. Kochjarová from Poland and Slovakia (reports nos. 12–14); Urtica diocia and U. kioviensis (both 2n = 26) by M. Kolník & K. Goliašová from Slovakia (nos. 15–16); Viola hirta (2n = 20), V. odorata (2n = 20), V. reichenbachiana (2n = 20), V. riviniana (2n = 40, 2n ∼ 8x, based on x = 5), V. suavis (2n = 40) and V. × bavarica [V. reichenbachiana × v. riviniana] (2n ∼ 6x, based on x = 5) by P. Mereďa jun., I. Hodálová, P. Mártonfi & V. Kolarčík from Slovakia (nos. 17–22); Fallopia × bohemica [F. japonica × F. sachalinensis] (2n = 66), Thladiantha dubia (2n = 18) and Hieracium longifoliosum (2n = 36) by P. Mráz from Romania and Slovakia (nos. 23–25); Amsinckia calycina (2n = 34) by M. Perny & H. Šípošová from Slovakia (no. 26). For further details and arrangements of reports see the first part (Mráz, 2005).     

The dipeptidyl carboxypeptidase of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue: overproduction and molecular characterization of the recombinant enzyme

To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His₆-tagged EcDCP (His₆-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni²⁺-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His₆-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His₆-EcDCP was stimulated by 1.5 fold. The K _M and k _cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s⁻¹, respectively. His₆-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His₆-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.     

A novel GH43 α-l-arabinofuranosidase from Humicola insolens: mode of action and synergy with GH51 α-l-arabinofuranosidases on wheat arabinoxylan

A novel α-l-arabinofuranosidase (α-AraF) belonging to glycoside hydrolase (GH) family 43 was cloned from Humicola insolens and expressed in Aspergillus oryzae. ¹H-NMR analysis revealed that the novel GH43 enzyme selectively hydrolysed (1→3)-α-l-arabinofuranosyl residues of doubly substituted xylopyranosyl residues in arabinoxylan and in arabinoxylan-derived oligosaccharides. The optimal activity of the cloned enzyme was at pH 6.7 and 53 °C. Two other novel α-l-arabinofuranosidases (α-AraFs), both belonging to GH family 51, were cloned from H. insolens and from the white-rot basidiomycete Meripilus giganteus. Both GH51 enzymes catalysed removal of (1→2) and (1→3)-α-l-arabinofuranosyl residues from singly substituted xylopyranosyls in arabinoxylan; the highest arabinose yields were obtained with the M. giganteus enzyme. Combinations (50:50) of the GH43 α-AraF from H. insolens and the GH51 α-AraFs from either M. giganteus or H. insolens resulted in a synergistic increase in arabinose release from water-soluble wheat arabinoxylan in extended reactions at pH 6 and 40 °C. This synergistic interaction between GH43 and GH51 α-AraFs was also evident when a GH43 α-AraF from a Bifidobacterium sp. was supplemented in combination with either of the GH51 enzymes. The synergistic effect is presumed to be a result of the GH51 α-AraFs being able to catalyse the removal of single-sitting (1→2)–α-l-arabinofuranosyls that resulted after the GH43 enzyme had catalysed the removal of (1→3)–α-l-arabinofuranosyl residues on doubly substituted xylopyranosyls in the wheat arabinoxylan.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning,sequencing, overexpression and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose isomerase from <Emphasis Type="Italic">Bacillus pallidus</Emphasis> Y25 for rare sugar production

The l-rhamnose isomerase gene (L -rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L -rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60°C for 60 min. The apparent affinity (K _m) and catalytic efficiency (k _cat/K _m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 10⁵ M⁻¹ min⁻¹, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.     

Synthesis of p-nitrophenyl sulfated disaccharides with beta-D-(6-sulfo)-GlcNAc units using beta-N-acetylhexosaminidase from Aspergillus oryzae in a transglycosylation reaction

When α-d-GlcNAc-OC₆H₄NO₂ -p and β-d-(6-sulfo)-GlcNAc-OC₆H₄NO₂-p (2) were used as substrates, β-N-acetylhexosaminidase from Aspergillus oryzae transferred the β-d-(6-sulfo)-GlcNAc(unit from 2 to α-d-GlcNAc-OC₆H₄NO₂ -p to afford β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-GlcNAc-OC₆H₄NO₂-p (3) in a yield of 94% based on the amount of donor, 2, added. β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-Glc-OC₆H₄NO₂-p (4) was obtained with α-d-Glc-OC₆H₄NO₂ -p as acceptor in a similar manner. With a reaction mixture of 2 and β-d-GlcNAc-OC₆H₄NO₂-p (1) in a molar ratio of 6:1, the enzyme mediated the transfer of β-d-GlcNAc from 1 to 2, affording disaccharide β-d-GlcNAc-(1→4)-β-(6-sulfo)-d-GlcNAc-OC₆H₄NO₂-p (5) in a yield of 13% based on the amount of 1 added.     

Enhancement of xylitol productivity and yield using a xylitol dehydrogenase gene-disrupted mutant of Candida tropicalis under fully aerobic conditions

The effects of glycerol and the oxygen transfer rate on the xylitol production rate by a xylitol dehydrogenase gene (XYL2)-disrupted mutant of Candida tropicalis were investigated. The mutant produced xylitol near the almost yield of 100% from d-xylose using glycerol as a co-substrate for cell growth and NADPH regeneration: 50 g d-xylose l⁻¹ was completely converted into xylitol when at least 20 g glycerol l⁻¹ was used as a co-substrate. The xylitol production rate increased with the O₂ transfer rate until saturation and it was not necessary to control the dissolved O₂ tension precisely. Under the optimum conditions, the volumetric productivity and xylitol yield were 3.2 g l⁻¹ h⁻¹ and 97% (w/w), respectively.