Ethanol fermentation of mixed-sugars using a two-phase, fed-batch process: method to minimize D-glucose repression of Candida shehatae D-xylose fermentations

Candida shehatae cells pre-grown on D-xylose simultaneously consumed mixtures of D-xylose and D-glucose, under both non-growing (anoxic) and actively growing conditions (aerobic), to produce ethanol. The rate of D-glucose consumption was independent of the D-xylose concentration for cells induced on D-xylose. However, the D-xylose consumption rate was approximately three times lower than the D-glucose consumption rate at a 50% D-glucose: 50% D-xylose mixture. Repression was not observed (substrate utilization rates were approximately equal) when the percentage of D-glucose and D-xylose was changed to 22% and 78%, respectively. In fermentations with actively growing cells (50% glucose and D-xylose), ethanol yields from D-xylose increased, the % D-xylose utilized increased, and the xylitol yield was significantly reduced in the presence of D-glucose, compared to anoxic fermentations (Y_ETOH,xylose = 0.2–0.40 g g⁻¹, 75–100%, and Y_xylitol = 0–0.2 g g⁻¹ compared to Y_ETOH,xylose = 0.15 g g⁻¹, 56%, Y_xylitol = 0.51 g g⁻¹, respectively). To increase ethanol levels and reduce process time, fed-batch fermentations were performed in a single stage reactor employing two phases: (1) rapid aerobic growth on D-xylose (μ = 0.32 h⁻¹) to high cell densities; (2) D-glucose addition and anaerobic conditions to produce ethanol (Y_ETOH,xylose = 0.23 g g⁻¹). The process generated high cell densities, 2 × 10⁹ cells ml⁻¹, and produced 45–50 g L⁻¹ ethanol within 50 h from a mixture of D-glucose and D-xylose (compared to 30 g L⁻¹ in 80 h in the best batch process). The two-phase process minimized loss of cell viability, increased D-xylose utilization, reduced process time, and increased final ethanol levels compared to the batch process. Received 23 February 1998/ Accepted in revised form 15 July 1998     

Simultaneous utilization of glucose and D-xylose by Candida shehatae in a chemostat

The kinetics of biomass formation, D-xylose utilization, and mixed substrate utilization were determined in a chemostat using the yeast Candida shehatae. The maximum growth rate of C. shehatae grown aerobically on D-xylose was 0.42 h⁻¹ and the Monod constant, K _s, was 0.06 g L⁻¹. The biomass yield, Y _{X/S}, ranged from 0.40 to 0.50 g g⁻¹ over a dilution rate range of 0.2–0.3 h⁻¹, when C. shehatae was grown on pure D-xylose. Mixtures of D-xylose and glucose (∼1 : 1) were simultaneously utilized over a dilution rate from 0.15 to 0.35 h⁻¹ at pH 3.5 and 4.5, but pH 3.5 reduced μ_max and reduced the dilution rate range over which D-xylose was utilized in the presence of glucose. At pH 4.5, μ_max was not reduced with the mixed sugar feed and the overall or lumped K _s value was not significantly increased (0.058 g L⁻¹ vs 0.06 g L⁻¹), when compared to a pure D-xylose feed. Kinetic data indicate that C. shehatae is an excellent candidate for chemostat production of value added products from renewable carbon sources, since simultaneous mixed substrate utilization was observed over a wide range of growth rates on a 1 : 1 mixture of glucose and D-xylose. Received 21 August 1997/ Accepted in revised form 28 May 1998     

Production, purification and characterization of a constitutive intracellular α-galactosidase from the thermophilic fungus Humicola sp

The thermophilic fungus Humicola sp constitutively produces intracellular α-galactosidase (1.33 U mg⁻¹ protein) within 48 h at 45°C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation, DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an optimum temperature of 65°C and an optimum pH of 5.0. Humicola α-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The K _m S for p-nitrophenyl-α-D-galactopyranoside, O-nitrophenyl-α-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly inhibited by Ag⁺ and Hg²⁺. D-Galactose inhibited α-galactosidase competitively and the inhibition constant (K _i) for galactose was 11 mM. Received 28 January 1999/ Accepted in revised form 07 April 1999     

Production of d-hydantoinase by halophilic Pseudomonas sp. NCIM 5109

About 1000 bacterial colonies isolated from sea water were screened for their ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine as a criterion for the determination of hydantoinase activity. The strain M-1, out of 11 hydantoinase-producing strains, exhibited the maximum ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine. The strain M-1 appeared to be a halophilic Pseudomonas sp. according to morphological and physiological characteristics. Optimization of the growth parameters revealed that nutrient broth with 2% NaCl was the preferred medium for both biomass and enzyme production. d-Hydantoinase of strain M-1 was not found to be inducible by the addition of uracil, dihydrouracil, β-alanine etc. The optimum temperature for enzyme production was about 25 °C and the organism showed a broad pH optimum (pH 6.5–9.0) for both biomass and hydantoinase production. The organism seems to have a strict requirement of NaCl for both growth and enzyme production. The optimum pH and temperature of enzyme activity were 9–9.5 and 30 °C respectively. The biotransformation under the alkaline conditions allowed the conversion of 80 g l⁻¹ dl-5-phenylhydantoin to 82 g l⁻¹ d(−)N-carbamoylphenylglycine within 24 h with a molar yield of 93%. Received: 15 September 1997 / Received revision: 5 January 1998 / Accepted: 6 January 1998     

Continuous production of l(+)-lactic acid by Lactobacillus casei in two-stage systems

A two-stage two-stream chemostat system and a two-stage two-stream immobilized upflow packed-bed reactor system were used for the study of lactic acid production by Lactobacillus casei subsp casei. A mixing ratio of D ₁₂/D ₂= 0.5 (D = dilution rate) resulted in optimum production, making it possible to generate continuously a broth with high lactic acid concentration (48 g l⁻¹) and with a lowered overall content of initial yeast extract (5  g l⁻¹), half the concentration supplied in the one-step process. In the two-stage chemostat system, with the first stage at pH 5.5 and 37 °C and a second stage at pH 6.0, a temperature change from 40 °C to 45 °C in the second stage resulted in a 100% substrate consumption at an overall dilution rate of 0.05 h⁻¹. To increase the cell mass in the system, an adhesive strain of L. casei was used to inoculate two packed-bed reactors, which operated with two mixed feedstock streams at the optimal conditions found above. Lactic acid fermentation started after a lag period of cell growth over foam glass particles. No significant amount of free cells, compared with those adhering to the glass foam, was observed during continuous lactic acid production. The extreme values, 57.5 g l⁻¹ for lactic acid concentration and 9.72 g l⁻¹ h⁻¹ for the volumetric productivity, in upflow packed-bed reactors were higher than those obtained for free cells (48 g l⁻¹  and 2.42 g l⁻¹ h⁻¹) respectively and the highest overall l(+)-lactic acid purity (96.8%) was obtained in the two-chemostat system as compared with the immobilized-cell reactors (93%). Received: 4 December 1997 / Received revision: 23 February 1998 / Accepted: 14 March 1998     

Physiology and kinetics of trimethylamine conversion by two methylotrophic strains in continuous cultivation systems

The change of dilution rate (D) on both Methylophilus methylotrophus NCIMB11348 and Methylobacterium sp. RXM CCMI908 growing in trimethylamine (TMA) chemostat cultures was studied in order to assess their ability to remove odours in fish processing plants. M. methylotrophus NCIMB11348 was grown at dilution rates of 0.012–0.084 h⁻¹ and the biomass level slightly increased up to values of D around 0.07 h⁻¹. The maximum cell production rate was obtained at 0.07 h⁻¹ corresponding to a maximum conversion of carbon into cell mass (35%). The highest rate of TMA consumption was 3.04 mM h⁻¹ occurring at D=0.076 h⁻¹. Methylobacterium sp. RXM CCMI908 was grown under similar conditions. The biomass increased in a more steep manner up to values of D around 0.06 h⁻¹. The maximum cell production rate (0.058 g l⁻¹h⁻¹) was obtained in the region close to 0.06 h⁻¹ where a maximum conversion of the carbon into cell mass (40%) was observed. The maximum TMA consumption was 2.33 mM h⁻¹ at D=0.075 h⁻¹. The flux of carbon from TMA towards cell synthesis and carbon dioxide in both strains indicates that the cell is not excreting products but directing most of the carbon source to growth. Carbon recovery levels of approximately 100% show that the cultures are carbon-limited. Values for theoretical maximum yields and maintenance coefficients are presented along with a kinetic assessment based on the determination of the substrate saturation constant and maximum growth rate for each organism. Received: 25 February 1999 / Received revision: 14 May 1999 / Accepted: 17 May 1999     

Production of β-galactosidase from thermophilic fungus Rhizomucor sp

A thermostable β-galactosidase was produced extracellularly by a thermophilic Rhizomucor sp, with maximum enzyme activity (0.21 U mg⁻¹) after 4 days under submerged fermentation condition (SmF). Solid state fermentation (SSF) resulted in a nine-fold increase in enzyme activity (2.04 U mg⁻¹). The temperature range for production of the enzyme was 38–55°C with maximum activity at 45°C. The optimum pH and temperature for the partially purified enzyme was 4.5 and 60°C, respectively. The enzyme retained its original activity on incubation at 60°C up to 1 h. Divalent cations like Co²⁺, Mn²⁺, Fe²⁺ and Zn²⁺ had strong inhibitory effects on the enzyme activity. The K _m and V _max for p-nitrophenyl-β- _D-galactopyranoside and o-nitrophenyl-β - _D-galactopyranoside were 0.39 mM, 0.785 mM and 232.1 mmol min⁻¹ mg⁻¹ respectively. The K _m and V _max for the natural substrate lactose were 66.66 μM and 0.20 μ mol min⁻¹ mg⁻¹. Received 10 March 1997/ Accepted in revised form 17 July 1997     

Formation of α-D-glucose-1-phosphate by thermophilic α-1,4-D-glucan phosphorylase

For the production of α-D-glucose-1-phosphate (G-1-P), α-1,4-D-glucan phosphorylase from Thermus caldophilus GK24 was partially purified to a specific activity of 13 U mg⁻¹ and an enzyme recovery of 15%. The amount of G-1-P reached maximum (18%) when soluble starch was used as substrate, and the smallest substrate for G-1-P formation was maltotriose. The structure of purified G-1-P was confirmed by comparison to ¹³C-NMR data for an authentic sample. In addition to G-1-P, glucose-6-phosphate (12%) was simultaneously produced when 10 mM maltoheptaose was used as substrate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 89–93. Received 12 May 1999/ Accepted in revised form 29 August 1999     

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.     

A new route to l-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity d-threonine aldolase-catalyzed stereospecific resolution

A new enzymatic resolution process was established for the production of l-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using the recombinant low-specificity d-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized dl-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichiacoli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l⁻¹) l-threo-MTPS was obtained from 200 mM (45.5 g l⁻¹) dl-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess. Received: 2 September 1998 / Received revision: 27 October 1998 / Accepted: 29 November 1998     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Expression of glf Z.m. increases D-mannitol formation in whole cell biotransformation with resting cells of Corynebacterium glutamicum

A recombinant oxidation/reduction cycle for the conversion of D-fructose to D-mannitol was established in resting cells of Corynebacterium glutamicum. Whole cells were used as biocatalysts, supplied with 250 mM sodium formate and 500 mM D-fructose at pH 6.5. The mannitol dehydrogenase gene (mdh) from Leuconostoc pseudomesenteroides was overexpressed in strain C. glutamicum ATCC 13032. To ensure sufficient cofactor [nicotinamide adenine dinucleotide (reduced form, NADH)] supply, the fdh gene encoding formate dehydrogenase from Mycobacterium vaccae N10 was coexpressed. The recombinant C. glutamicum cells produced D-mannitol at a constant production rate of 0.22 g (g cdw)⁻¹ h⁻¹. Expression of the glucose/fructose facilitator gene glf from Zymomonas mobilis in C. glutamicum led to a 5.5-fold increased productivity of 1.25 g (g cdw)⁻¹ h⁻¹, yielding 87 g l⁻¹ D-mannitol from 93.7 g l⁻¹ D-fructose. Determination of intracellular NAD(H) concentration during biotransformation showed a constant NAD(H) pool size and a NADH/NAD⁺ ratio of approximately 1. In repetitive fed-batch biotransformation, 285 g l⁻¹ D-mannitol over a time period of 96 h with an average productivity of 1.0 g (g cdw)⁻¹ h⁻¹ was formed. These results show that C. glutamicum is a favorable biocatalyst for long-term biotransformation with resting cells. Dedicated to Prof. Hermann Sahm on the occasion of his 65th birthday.     

Biosynthetic origin and longevity in vivo of α-d-mannopyranosyl-(1 → 4)-α-d-glucuronopyranosyl-(1 → 2)-myo-inositol, an unusual extracellular oligosaccharide produced by cultured rose cells

A non-reducing trisaccharide, α-D-mannopyranosyl-(1 → 4)-α-D-glucuronopyranosyl-(1 → 2)-myo-inositol (MGI) accumulated in the spent medium of cell-suspension cultures of `Paul's Scarlet' rose (Rosa sp.) predominantly during the period of rapid cell growth. This trisaccharide was also produced by cultures of sycamore (Acer pseudoplatanus L.) but not by those of the graminaceous monocots maize (Zea mays L.) and tall fescue grass (Festuca arundinacea Schreb.). When added to cultured Rosa cells, [¹⁴C]MGI was neither taken up by the cells nor bound to the cell surface and was not metabolised extracellularly. When D-[6-¹⁴C]glucuronic acid was fed to cultured Rosa cells, extracellular [¹⁴C]MGI started to appear only after a 5-h lag period, compared with a 0.5-h lag period for labelling of extracelluar polysaccharides. Furthermore, [¹⁴C]MGI continued to accumulate in the medium for at least 20 h after the accumulation of ¹⁴C-polymers had ceased. These observations indicate that extracellular MGI was produced from a slowly turning-over pool of a pre-formed intermediate. Structural considerations indicate that the intermediate could be a glucuronomannan or a phytoglycolipid (glycophosphosphingolipid). No Rosa polysaccharides could be found that generated MGI in the presence of living Rosa cells. We therefore favour phytoglycolipids as the probable biosynthetic origin of MGI. Received: 29 April 1999 / Accepted: 13 June 1999     

Analysis of the gene for β-fructosidase (invertase, inulinase) of the hyperthermophilic bacterium Thermotoga maritima, and characterisation of the enzyme expressed in Escherichia coli

This is the first report describing the gene structure and the enzymatic properties of a β-fructosidase of a hyperthermophilic organism. The bfrA gene of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue, polypeptide of about 50 kDa, with significant sequence similarity to other β-fructosidases. On the basis of its primary structure, BfrA can be assigned to glycosyl hydrolase family 32. The bfrA gene was expressed in Escherichia coli and the recombinant enzyme was purified and characterised. BfrA was specific for the fructose moiety and the β-anomeric configuration of the glycosidic linkages of its substrates. The enzyme released fructose from sucrose and raffinose, and the fructose polymer inulin was hydrolysed quantitatively in an exo-type fashion. BfrA displayed similar catalytic efficiencies for the hydrolysis of sucrose and inulin with k _cat/K _m values (at 75 °C, pH 5.5) of about 4.1 × 10⁴M⁻¹s⁻¹ and 3.1 × 10⁴M⁻¹s⁻¹ respectively. BfrA had an optimum temperature of 90–95 °C (10-min assay) and was extremely insensitive to thermo-inactivation. During 5 h at temperatures up to 80 °C at pH 7, the enzyme retained at least 85% of its initial activity. Thus, BfrA is the most thermostable β-fructosidase and also the most thermostable inulinase described to date. In conclusion, the T. maritima enzyme can be classified as an exo-β-d-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase activity. Its catalytic properties along with the extreme thermostability recommend it for use in biotechnology. Received: 28 August 1997 / Received revision: 19 January 1998 / Accepted: 24 January 1998     

The production of xylitol from d-xylose by fermentation with Hansenula polymorpha

We have analysed the influence of the initial pH of the medium and the quantity of aeration provided during the batch fermentation of solutions of d-xylose by the yeast Hansenula polymorpha (34438 ATCC). The initial pH was altered between 3.5 and 6.5 whilst aeration varied between 0.0 and 0.3 vvm. The temperature was kept at 30 °C during all the experiments. Hansenula polymorpha is known to produce high quantities of xylitol and low quantities of ethanol. The most favourable conditions for the growth of xylitol turned out to be: an initial pH of between 4.5 and 5.5 and the aeration provided by the stirring vortex alone. Thus, at an initial pH of 5.5, the maximum specific production rate (μ_m) was 0.41 h⁻¹, the overall biomass yield (Y _x/s ^G) was 0.12 g g⁻¹, the specific d-xylose-consumption rate (q _s ) was 0.075 g g⁻¹ h⁻¹ (for t = 75 h), the specific xylitol-production rate (q _Xy ) was 0.31 g g⁻¹ h⁻¹ (for t = 30 h) and the overall yields of ethanol (Y _E/s ^G) and xylitol (Y _Xy/s ^G) were 0.017 and 0.61 g g⁻¹ respectively. Both q _s and q _Xy decreased during the course of the experiments once the exponential growth phase had finished. Received: 26 March 1998 / Received revision: 30 June 1998 / Accepted: 2 July 1998     

Fagopyritol B1, O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol, a galactosyl cyclitol in maturing buckwheat seeds associated with desiccation tolerance

O-α-D-Galactopyranosyl-(1→2)-D-chiro-inositol, herein named fagopyritol B1, was identified as a major soluble carbohydrate (40% of total) in buckwheat (Fagopyrum esculentum Moench, Polygonaceae) embryos. Analysis of hydrolysis products of purified compounds and of the crude extract led to the conclusion that buckwheat embryos have five α-galactosyl D-chiro-inositols: fagopyritol A1 and fagopyritol B1 (mono-galactosyl D-chiro-inositol isomers), fagopyritol A2 and fagopyritol B2 (di-galactosyl D-chiro-inositol isomers), and fagopyritol B3 (tri-galactosyl D-chiro-inositol). Other soluble carbohydrates analyzed by high-resolution gas chromatography included sucrose (42% of total), D-chiro-inositol, myo-inositol, galactinol, raffinose and stachyose (1% of total), but no reducing sugars. All fagopyritols were readily hydrolyzed by α-galactosidase (EC 3.2.1.22) from green coffee bean, demonstrating α-galactosyl linkage. Retention time of fagopyritol B1 was identical to the retention time of O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol from soybean (Glycine max (L.) Merrill, Leguminosae), suggesting that the α-ga-lactosyl linkage is to the 2-position of D-chiro-inositol. Accumulation of fagopyritol B1 was associated with acquisition of desiccation tolerance during seed development and maturation in planta, and loss of fagopyritol B1 correlated with loss of desiccation tolerance during germination. Embryos of seeds grown at 18 °C, a condition that favors enhanced seed vigor and storability, had a sucrose-to-fagopyritol B1 ratio of 0.8 compared to a ratio of 2.46 for seeds grown at 25 °C. We propose that fagopyritol B1 facilitates desiccation tolerance and storability of buckwheat seeds. Received: 21 May 1997 / Accepted: 5 June 1997     

Effect of growth rate on α-amylase production by Streptomyces sp. IMD 2679

The α-amylase of Streptomyces sp. IMD 2679 was subject to catabolite repression. Four different growth rates were achieved when the organism was grown at 40 °C and 55 °C in the presence and absence of cobalt, with an inverse relationship between α-amylase production and growth rate. Highest α-amylase yields (520 units/ml) were obtained at the lowest growth rate (0.062 h⁻¹), at 40 °C in the absence of cobalt, while at the highest growth rate (0.35 h⁻¹), at 55 °C in the presence of cobalt, α-amylase production was decreased to 150 units/ml. As growth rate increased, the rate of specific utilisation of the carbon source maltose also increased, from 46 to 123 μg maltose (mg biomass)⁻¹ h⁻¹. The pattern and levels of α-glucosidase (the enzyme degrading maltose) detected intracellularly in each case, indicate that growth rate effectively controls the rate of feeding of glucose to the cell, and thus catabolite repression. Received: 17 February 1997 / Received revision: 29 April 1997 / Accepted: 11 May 1997     

Reduction of Cr(VI) by a Bacillus sp.

A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside the cell. When Cr(VI) was 10 and 40 μg ml⁻¹, >95% of the total Cr(VI) was reduced in 24 and 72 h of growth, respectively, whereas at 80 μg Cr(VI) ml⁻¹ only 50% of Cr(VI) was reduced. However growth was not affected; the cell mass was 0.7–0.8 mg ml⁻¹ in all cases. The cell-free extract showed Cr(VI) reducing enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was optimal at pH 6.0 and 30 °C. The enzyme was stable up to 30 °C and from pH 5.5 to 8, but from pH 4 to 5 the enzyme was severely destabilized. Its K_m and V_max were 14 μm and 3.8 nmol min⁻¹ mg⁻¹ respectively. The enzyme activity was enhanced by Cu²⁺ and Ni²⁺ and inhibited by Hg²⁺. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Identification and purification of O -acetyl-l-serine sulphhydrylase in Penicillium chrysogenum

We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K _m value for O-acetyl-l-serine and V _max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein⁻¹ h⁻¹ respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4. Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.