Production of high fructose syrup from <Emphasis Type="Italic">Asparagus</Emphasis> inulin using immobilized exoinulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial purification by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a significant improvement in the thermal stability of the biocatalyst after immobilization. Apparent K _m values for inulin, raffinose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (E _a) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co²⁺ and Mn²⁺ enhanced the activity, whereas Hg²⁺ and Ag²⁺ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was effectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.     

Cellulase production from <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> sp. NO3 isolated from the sea squirt <Emphasis Type="Italic">Halocynthia rorentzi</Emphasis>

Pseudoalteromonas sp. NO3 was isolated from the hemolymph of diseased sea squirts (Halocynthia rorentzi) with symptoms of soft tunic syndrome. The strain was found to produce an extracellular cellulase (CelY) that consisted of a 1,476 bp open reading frame encoding 491 amino acid residues with an approximate molecular mass of 52 kDa. Homologies of the deduced amino acid sequence of celY with the products of the celA, celX, celG and cel5Z genes were 92.6, 93.3, 92.6, and 59.1%, respectively. Additionally, CelY had 50–80% remnant catalytic activity at temperatures of 10–20°C. Highest carboxymethyl cellulose (CMC) hydrolysis was observed at pH 8.0 and 40°C. CMC activity was determined by zymogram active staining and different degraded product profiles for CelY were obtained when cellotetraose, cellopentaose, and CMC were used as substrates. This study identified a transglycosylation activity in CelY that allows the enzyme to digest G4 to G2 and G3 without the production of G1.     

Effect of trace metals on ethanol production from synthesis gas by the ethanologenic acetogen, <Emphasis Type="Italic">Clostridium</Emphasis><Emphasis Type="Italic">ragsdalei</Emphasis>

The effect of trace metal ions (Co²⁺, Cu²⁺, Fe²⁺, Mn²⁺, Mo⁶⁺, Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻) on growth and ethanol production by an ethanologenic acetogen, Clostridium ragsdalei was investigated in CO:CO₂-grown cells. A standard acetogen medium (ATCC medium no. 1754) was manipulated by varying the concentrations of trace metals in the media. Increasing the individual concentrations of Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻ from 0.84, 6.96, 1.06, and 0.68 μM in the standard trace metals solution to 8.4, 34.8, 5.3, and 6.8 μM, respectively, increased ethanol production from 35.73 mM under standard metals concentration to 176.5, 187.8, 54.4, and 72.3 mM, respectively. Nickel was necessary for growth of C. ragsdalei. Growth rate (μ) of C. ragsdalei improved from 0.34 to 0.49 (day⁻¹), and carbon monoxide dehydrogenase (CODH) and hydrogenase (H₂ase)-specific activities improved from 38.45 and 0.35 to 48.5 and 1.66 U/mg protein, respectively, at optimum concentration of Ni²⁺. At optimum concentrations of WO₄ ⁻ and SeO₄ ⁻, formate dehydrogenase (FDH) activity improved from 32.3 to 42.6 and 45.4 U/mg protein, respectively. Ethanol production and the activity of FDH reduced from 35 mM and 32.3 U/mg protein to 1.14 mM and 8.79 U/mg protein, respectively, upon elimination of WO₄ ⁻ from the medium. Although increased concentration of Zn²⁺ enhanced growth and ethanol production, the activities of CODH, FDH, H₂ase and alcohol dehydrogenase (ADH) were not affected by varying the Zn²⁺ concentration. Omitting Fe²⁺ from the medium decreased ethanol production from 35.7 to 6.30 mM and decreased activities of CODH, FDH, H₂ase and ADH from 38.5, 32.3, 0.35, and 0.68 U/mg protein to 9.07, 7.01, 0.10, and 0.24 U/mg protein, respectively. Ethanol production improved from 35 to 54 mM when Cu²⁺ was removed from the medium. The optimization of trace metals concentration in the fermentation medium improved enzyme activities (CODH, FDH, and H₂ase), growth and ethanol production by C. ragsdalei.     

Purification and Biochemical Characterization of Phytase Enzyme from <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> (<Emphasis Type="Italic">MH121153</Emphasis>)

Phytase (myo-inositol hexaphosphate phosphohydrolase) belongs to phosphatases. It catalyzes the hydrolysis of phytate to less-phosphorylated inorganic phosphates and phytate. Phytase is used primarily for the feeding of simple hermit animals in order to increase the usability of amino acids, minerals, phosphorus and energy. In the present study, phytase isolation from the Lactobacillus coryniformis strain, isolated from Lor cheese sources, phytase purification and characterization were studied. The phytase was purified in simple three steps. The enzyme was obtained with 2.60% recovery and a specific activity of 202.25 (EU/mg protein). The molecular mass of the enzyme was determined to be 43.25 kDa with the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The optimum temperature and pH for the enzyme were found as 60 °C and 5.0 and respectively. To defined the substrate specificity of the phytase, the hydrolysis of several phosphorylated compounds by the purified enzyme was studied and sodium phytate showed high specificity. Furthermore, the effects of Ca²⁺, Ag⁺, Mg²⁺, Cu²⁺, Co²⁺, Pb²⁺, Zn²⁺ and Ni²⁺ metal ions on the enzyme were studied.     

Molecular cloning,sequence analysis,expression and characterization of the endochitinase gene from <Emphasis Type="Italic">Trichoderma</Emphasis> sp. in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively. There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions: the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation by 1 mM Mg²⁺ and 5 mM Fe²⁺ and inhibition by 5 mM Co²⁺ and 5 mM Hg²⁺ were observed. The kinetic constants K_m, V_max and K_cat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min⁻¹ and 6.23 min⁻¹, respectively.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Purification and characterization of a <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase produced by <Emphasis Type="Italic">Talaromyces emersonii</Emphasis> during growth on algal fucoidan

A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (K_m value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH 5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg²⁺, Co²⁺ and Fe³⁺.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Purification and Characterization of Extracellular Phytase from a Marine Yeast <Emphasis Type="Italic">Kodamaea ohmeri</Emphasis> BG3

The extracellular phytase in the supernatant of cell culture of the marine yeast Kodamaea ohmeri BG3 was purified to homogeneity with a 7.2-fold increase in specific phytase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex™ G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow Anion-Exchange). According to the data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 98.2 kDa while the molecular mass of the purified enzyme was estimated to be 92.9 kDa and the enzyme was shown to be a monomer according to the results of gel filtration chromatography. The optimal pH and temperature of the purified enzyme were 5.0 and 65°C, respectively. The enzyme was stimulated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Ba²⁺, Mg²⁺ and Co²⁺ (at a concentrations of 5.0 mM), but it was inhibited by Cu²⁺, Hg²⁺, Fe²⁺, Fe³⁺, Ag⁺, and Zn²⁺ (at a concentration of 5.0 mM). The enzyme was also inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid (at a concentration of 1.0 mM), and phenylgloxal hydrate (at a concentration of 5.0 mM), and not inhibited by EDTA and 1,10-phenanthroline (at concentrations of 1.0 mM and 5.0 mM). The K _m, V _max, and K _cat values of the purified enzyme for phytate were 1.45 mM, 0.083 μmol/ml · min, and 0.93 s^-1, respectively.     

Granulosain I,a Cysteine Protease Isolated from Ripe Fruits of <Emphasis Type="Italic">Solanum granuloso</Emphasis><Emphasis Type="Italic">-leprosum</Emphasis> (Solanaceae)

A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.     

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.     

Malate Dehydrogenase from the Thermophilic Bacterium <Emphasis Type="Italic">Vulcanithermus medioatlanticus</Emphasis>

Thermostable dimeric malate dehydrogenase (MDH) was isolated from the microorganism of hydrothermal vents Vulcanithermus medioatlanticus. The enzyme was electrophoretically homogeneous and possessed the specific activity of 6.9 U/mg. The large molecular weight of the subunits (55 kD) is likely to provide the rigidity of the enzyme structure (the activation energy of the enzymatic reaction is 32.6 kJ/mol). The thermophilic MDH differs little from the mesophilic enzyme in terms of kinetic and regulatory characteristics.     

An Oxidized Ergosterol from <Emphasis Type="Italic">Pleurotus cystidiosus</Emphasis> Active Against Anthracnose Causing <Emphasis Type="Italic">Colletotrichum</Emphasis><Emphasis Type="Italic">gloeosporioides</Emphasis>

This study was undertaken to study the antifungal activity of Pleurotus cystidiosus against Colletotrichum gloeosporioides. This was achieved by fractionating the mushroom, P. cystidiosus initially to acetone (A), dichloromethane (D), and hexane (H) and studying the antifungal activity using the standard poisoned food technique. All the test solutions used were in the concentration of 20,000 ppm. The percentage inhibition of extracts A, D, and H was 12, 7, and 0.4%, respectively. Antifungal assay guided fractionation of the most active extract A resulted in four fractions; A1, A2, A3, and A4 having 12, 22, 0, and 17% percentage inhibitions, respectively. Fractions A2 and A4 were selected for further purifications. Normal phase column chromatography of A2 gave A2-1, A2-2, A2-3, and A2-4, with percentage inhibitions 7, 5, 26, and 13%, respectively. The fraction with the highest inhibitory activity (A2-3) was further separated using the Chromatotron and a single compound (A2-3-13) with 41% inhibition was isolated. Structure elucidation of this compound using 1D and 2D NMR spectroscopy proved this compound to be 3beta, 5alpha, 6beta-trihydroxyergosta-7,22-diene.     

Reassessment of hydrogen tolerance in <Emphasis Type="Italic">Caldicellulosiruptor saccharolyticus</Emphasis>

Background

Caldicellulosiruptor saccharolyticus has the ability to produce hydrogen (H₂) at high yields from a wide spectrum of carbon sources, and has therefore gained industrial interest. For a cost-effective biohydrogen process, the ability of an organism to tolerate high partial pressures of H₂ (P_H2) is a critical aspect to eliminate the need for continuous stripping of the produced H₂ from the bioreactor.

Results

Herein, we demonstrate that, under given conditions, growth and H₂ production in C. saccharolyticus can be sustained at P_H2 up to 67 kPa in a chemostat. At this P_H2, 38% and 16% of the pyruvate flux was redirected to lactate and ethanol, respectively, to maintain a relatively low cytosolic NADH/NAD ratio (0.12 mol/mol). To investigate the effect of the redox ratio on the glycolytic flux, a kinetic model describing the activity of the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was developed. Indeed, at NADH/NAD ratios of 0.12 mol/mol (K i of NADH = 0.03 ± 0.01 mM) GAPDH activity was inhibited by only 50% allowing still a high glycolytic flux (3.2 ± 0.4 mM/h). Even at high NADH/NAD ratios up to 1 mol/mol the enzyme was not completely inhibited. During batch cultivations, hydrogen tolerance of C. saccharolyticus was dependent on the growth phase of the organism as well as the carbon and energy source used. The obtained results were analyzed, based on thermodynamic and enzyme kinetic considerations, to gain insight in the mechanism underlying the unique ability of C. saccharolyticus to grow and produce H₂ under relatively high P_H2.

Conclusion

C. saccharolyticus is able to grow and produce hydrogen at high P_H2, hence eliminating the need of gas sparging in its cultures. Under this condition, it has a unique ability to fine tune its metabolism by maintaining the glycolytic flux through regulating GAPDH activity and redistribution of pyruvate flux. Concerning the later, xylose-rich feedstock should be preferred over the sucrose-rich one for better H₂ yield.

     

Quantitative analysis and functional evaluation of zinc ion in the <Emphasis Type="SmallCaps">d</Emphasis>-hydantoinase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> YZ-26

d-Hydantoinase (HDT) is a metal-dependent enzyme that is widely used in industrial bioconversion to d-amino acids as valuable intermediates in the fields of food, pharmaceutical industry and agriculture. In this report, we prepared apo-HDT (metal-removed HDT) and Zn²⁺-HDT (Zn²⁺-added HDT) in vitro from a recombinant HDT (re-HDT) expressed in E. coli. The Zn²⁺-HDT and re-HDT contain 2.17 and 0.95 mol Zn²⁺ per mol subunit, respectively, and they have comparable enzymatic activities. In contrast, the apo-HDT only retains 0.04 mol Zn²⁺ per mol subunit with less than 10% activity, compared with the re-HDT. When the apo-HDT was reconstituted with ZnCl₂, the enzymatic activity recovery was about 75%. Moreover, the fluorescence intensity, circular dichroism spectra and thermo-stability of the apo-HDT and Zn²⁺-HDT are quite different from those of the re-HDT. These data suggest that the re-HDT may have two Zn²⁺-binding sites, one is an intrinsic or tight-binding site (zinc-α) essential for its activity and the other is a vacant or loose-binding site (zinc-β) possibly non-essential for the activity.     

Cloning and characterization of a heat-stable CMP-<Emphasis Type="Italic">N</Emphasis>-acylneuraminic acid synthetase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

In this study, we report the cloning, recombinant expression, and biochemical characterization of a heat-stable CMP-N-acylneuraminic acid (NeuAc) synthetase from Clostridium thermocellum ATCC 27405. A high throughput electrospray ionization mass spectrometry (ESI-MS)-based assay demonstrates that the enzyme has an absolute requirement for a divalent cation for activity and reaches maximum activity in the presence of 10 mM Mn²⁺. The enzyme is active at pH 8–13 in Tris–HCl buffer and at 37–60 °C, and maximum activity is observed at pH 9.5 and 50 °C in the presence of 0.2 mM dithiothreitol. In addition to NeuAc, the enzyme also accepts the analog N-glycolylneuraminic acid (NeuGc) as a substrate. The apparent Michaelis constants for cytidine triphosphate and NeuAc or NeuGc are 240 ± 20, 130 ± 10, and 160 ± 10 μM, respectively, with corresponding turnover numbers of 3.33, 2.25, and 1.66 s⁻¹, respectively. An initial velocity study of the enzymatic reaction indicates an ordered bi–bi catalytic mechanism. In addition to demonstration of a thermostable and substrate-tolerant enzyme, confirmation of the biochemical function of a gene for CMP-NeuAc synthetase in C. thermocellum also opens the question of the biological function of CMP-NeuAc in such nonpathogenic microorganisms.     

L-<Emphasis Type="Italic">myo</Emphasis>-inositol-1-phosphate synthase: partial purification and characterisation from <Emphasis Type="Italic">Gleichenia glauca</Emphasis>

A screening for the enzyme L-myo-inositol-1-phosphate synthase [EC 5.5.1.4] has been made first time in both vegetative and reproductive parts of the representative members of pteridophytes: Lycopodium, Selaginella, Equisetum, Polypodium, Dryopteris, and Gleichenia. The enzyme has been partially purified following low-speed centrifugation, streptomycin sulphate precipitation, ammonium sulphate fractionation, chromatography on DEAE-cellulose and gel-filtration through Sephadex G-200, and characterised from the reproductive pinnules of Gleichenia glauca Smith. The enzyme has a pH optimum at 7.5. The K_m for glucose-6-P and NAD⁺ were 0.922 × 10^–3 M and 0.9 × 10^–4 M, respectively. A basal activity of the enzyme has been recorded in absence of exogenous NAD⁺. The enzyme activity was augmented with NH₄Cl, but heavy metals like Hg²⁺, Cu²⁺ and Zn²⁺ inactivated it.     

Enzymatic properties of cellobiose 2-epimerase from <Emphasis Type="Italic">Ruminococcus albus</Emphasis> and the synthesis of rare oligosaccharides by the enzyme

The gene for cellobiose 2-epimerase (CE) from Ruminococcus albus NE1 was overexpressed in Escherichia coli cells. The recombinant CE was purified to homogeneity by a simple purification procedure with a high yield of 88%, and the molecular mass was 43.1 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and 44.0 kDa on gel chromatography. It exhibited optimal activity around at 30 degrees C and pH 7.5, and the enzyme activity was inhibited by Al3+, Fe3+, Co2+, Cu2+, Zn2+, Pb2+, Ag+, N-bromosuccinimide, iodoacetate, and 4-chloromercuribenzoate. In addition to cello-oligosaccharides, the enzyme was found to effectively 2-epimerize lactose to yield 4-O-beta-D-galactopyranosyl-D-mannose (epilactose), which occurs in cow milk as a rare oligosaccharide. The Km and kcat/Km values toward lactose were 33 mM and 1.6 s(-1) mM(-1), and those toward cellobiose were 13.8 mM and 4.6 s(-1) mM(-1), respectively. N-Acetyl-D-glucosamine, uridine 5'-diphosphate-glucose, D-glucose 6-phosphate, maltose, sophorose, laminaribiose, and gentiobiose were inert as substrates for the recombinant CE. We demonstrated that epilactose was resistant to rat intestinal enzymes, utilized by human adult bifidobacteria, and stimulated the tight junction permeability in Caco-2 cells. These results strongly suggest that this rare disaccharide is promising for use as a prebiotic.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.