Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.     

<Emphasis Type="SmallCaps">d</Emphasis>-Psicose production from <Emphasis Type="SmallCaps">d</Emphasis>-fructose using an isolated strain, <Emphasis Type="Italic">Sinorhizobium</Emphasis> sp.

Sinorhizobium sp., which can convert d-fructose into d-psicose, was isolated from soil. The optimal pH, temperature, and cell concentration for d-psicose production with the isolated strain were 8.5, 40°C, and 60 mg/ml, respectively. The toluene-treated cells showed 2.5- and 4.8-fold increases in the d-psicose concentration and productivity compared with untreated washed cells. Under the optimal conditions, the toluene-treated cells produced 37 g d-psicose/l from 70% (w/v) (3.9 M) d-fructose after 15 h.     

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.     

Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

Fermentation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose and <Emphasis Type="SmallCaps">d</Emphasis>-xylose mixtures by <Emphasis Type="Italic">Candida tropicalis</Emphasis> NBRC 0618 for xylitol production

The fermentation of d-glucose and d-xylose mixtures by the yeast Candida tropicalis NBRC 0618 has been studied under the most favourable operation conditions for the culture, determining the most adequate initial proportion in these sugars for xylitol production. In all the experiments a synthetic culture medium was used, with an initial total substrate concentration of 25 g L⁻¹, a constant pH of 5.0 and a temperature of 30 °C. From the experimental results, it was deduced that the highest values of specific rates of production and of overall yield in xylitol were achieved for the mixtures with the highest percentage of d-xylose, specifically in the culture with the initial d-glucose and d-xylose concentrations of 1 and 24 g L⁻¹, respectively, with an overall xylitol yield of 0.28 g g⁻¹. In addition, the specific rates of xylitol production declined over the time course of the culture and the formation of this bioproduct was favoured by the presence of small quantities of d-glucose. The sum of the overall yield values in xylitol and ethanol for all the experiments ranged from 0.26 to 0.56 g bioproduct/g total substrate.     

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.     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from sugarcane molasses,sugarcane juice and sugar beet juice by <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l⁻¹) and sugarcane juice (133 g total sugar l⁻¹) was 107 g l⁻¹ and 120 g l⁻¹, respectively. With 10% (w/v) sugar beet juice (105 g total sugar l⁻¹), 84 g lactic acid l⁻¹ was produced. The optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.     

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.     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.     

Continuous 2-keto-<Emphasis Type="SmallCaps">l</Emphasis>-gulonic acid fermentation from <Emphasis Type="SmallCaps">l</Emphasis>-sorbose by <Emphasis Type="Italic">Ketogulonigenium vulgare</Emphasis> DSM 4025

A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h⁻¹, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Substrate specificity of a glucose-6-phosphate isomerase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> for monosaccharides

We purified recombinant glucose-6-phosphate isomerase from Pyrococcus furiosus using heat treatment and Hi-Trap anion-exchange chromatography with a final specific activity of 0.39 U mg⁻¹. The activity of the glucose-6-phosphate isomerase for l-talose isomerization was optimal at pH 7.0, 95°C, and 1.5 mM Co²⁺. The half-lives of the enzyme at 65°C, 75°C, 85°C, and 95°C were 170, 41, 19, and 7.9 h, respectively. Glucose-6-phosphate isomerase catalyzed the interconversion between two different aldoses and ketose for all pentoses and hexoses via two isomerization reactions. This enzyme has a unique activity order as follows: aldose substrates with hydroxyl groups oriented in the same direction at C2, C3, and C4 > C2 and C4 > C2 and C3 > C3 and C4. l-Talose and d-ribulose exhibited the most preferred substrates among the aldoses and ketoses, respectively. l-Talose was converted to l-tagatose and l-galactose by glucose-6-phosphate isomerase with 80% and 5% conversion yields after about 420 min, respectively, whereas d-ribulose was converted to d-ribose and d-arabinose with 53% and 8% conversion yields after about 240 min, respectively.     

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.     

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.     

Isolation of a Novel <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-lactosamine Specific Lectin from <Emphasis Type="Italic">Alocasia cucullata</Emphasis> (Schott.)

An N-acetyl-d-lactosamine (LacNAc) specific lectin from tubers of Alocasia cucullata was purified by affinity chromatography on asialofetuin-linked amino activated silica. The pure lectin showed a single band in SDS-PAGE at pH 8.8 and was a homotetramer with a subunit molecular mass of 13.5 kDa and native molecular mass of 53 kDa. It was heat stable up to 55 °C for 15 min and showed optimum hemagglutination activity from pH 2 to 11. The lectin was affected by denaturing agents such as urea (2 m), thiourea (2 m) and guanidine–HCl (0.5 m) and did not require Ca²⁺ and Mn²⁺ for its activity. It was a potent mitogen at 10 μg/ml towards human peripheral blood mononuclear cells with 50% growth inhibitory potential towards SiHa (human cervix ) cancer cell line at 100 μg/ml.     

Cloning and expression of <Emphasis Type="SmallCaps">d</Emphasis> -lactonohydrolase cDNA from <Emphasis Type="Italic">Fusarium moniliforme</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A d -lactonohydrolase gene of about 1.1 kb was cloned from Fusarium moniliforme. The ORF sequence predicted a protein of 382 amino acids with a molecular mass of about 40 kDa. An expression plasmid carrying the gene under the control of the triose phosphate isomerase gene promotor was introduced into Saccharomyces cerevisiae, and the d -lactonohydrolase gene was successfully expressed in the recombinant strains.Revisions requested 10 September 2004; Revisions received 15 October 2004The nucleotide sequence data reported in this paper has been assigned accession number AY728018 in the GeneBank database.     

Improving the thermostability of <Emphasis Type="Italic">N</Emphasis>-carbamyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolase by error-prone PCR

To facilitate the easier production of d-amino acids using N-carbamyl-d-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 °C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production of d-amino acids.     

Biotransformation of glycerol to <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid by <Emphasis Type="Italic">Acetobacter tropicalis</Emphasis>

Bacterial strains capable of converting glycerol to glyceric acid (GA) were screened among the genera Acetobacter and Gluconacetobacter. Most of the tested Acetobacter and Gluconacetobacter strains could produce 1.8 to 9.3 g/l GA from 10% (v/v) glycerol when intact cells were used as the enzyme source. Acetobacter tropicalis NBRC16470 was the best GA producer and was therefore further investigated. Based on the results of high-performance liquid chromatography analysis and specific rotation, the enantiomeric composition of the produced GA was d-glyceric acid (d-GA). The productivity of d-GA was enhanced with the addition of both 15% (v/v) glycerol and 20 g/l yeast extract. Under these optimized conditions, A. tropicalis NBRC16470 produced 22.7 g/l d-GA from 200 g/l glycerol during 4 days of incubation in a jar fermentor.     

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.