Effect of Constant Glucose Feeding on the Production of Exopolysaccharides by <Emphasis Type="Italic">Tremella fuciformis</Emphasis> Spores

A fed-batch culture system with constant feeding (glucose 80 g L⁻¹, 0.25 ml min⁻¹) was used to study the influence of glucose on cell dry weight and exopolysaccharides production from submerged Tremella fuciformis spores in a 5-L stirred-tank bioreactor. The results showed that high levels of cell mass (9.80 g L⁻¹) and exopolysaccharides production (3.12 g L⁻¹) in fed-batch fermentation were obtained after 1 h of feeding, where the specific growth rate (μ) and exopolysaccharides yield on substrate consumed (YP/S) were 0.267 d⁻¹ and 0.14 g g⁻¹. Unlike batch fermentation, maximal cell mass and exopolysaccharides production merely reached 7.11 and 2.08 g L⁻¹; the specific growth rate (μ) and exopolysaccharides yield on substrate consumed (YP/S) were 0.194 d⁻¹ and 0.093 g g⁻¹, respectively. It is concluded that the synthesis of exopolysaccharides can be promoted effectively when feeding glucose at a late exponential phase.     

Redox Behavior of Neptunium(V) in Tributyl Phosphate and <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-Dihexyl Octanamide Extractants Dissolved in <Emphasis Type="Italic">n</Emphasis>-Dodecane

Spectrophotometric investigations have been carried out on the disproportionation of Np(V) to form Np(IV) and Np(VI) in 1.1 mol⋅L⁻¹ solutions of tributyl phosphate (TBP) and in N,N-dihexyl octanamide (DHOA) in n-dodecane medium. The Np(V) was found to coordinate with Np(IV) in 1.1 mol⋅L⁻¹ TBP solution in n-dodecane to form a mixed valence “cation–cation” complex by bonding through an axial oxo group on Np(V). By contrast, this interaction was less prominent in the case of 1.1 mol⋅L⁻¹ DHOA solutions. The effect of 1-octanol, added as phase modifier, on the disproportionation behavior of Np(V) was also investigated. An attempt was made to calculate the disproportionation/reduction rate constants for Np(V) under the conditions of these studies. Absorbance measurements on the Np stripped from organic phases revealed the occurrence of Np(V) in the aqueous phase.     

Spectrophotometric determination of <Emphasis Type="Italic">L</Emphasis>-ascorbic acid in pharmaceuticals

A simple and sensitive spectrophotometric method for the determination of L-ascorbic acid with leuco crystal violet is proposed. The determination is based on the oxidation of analyte by potassium iodate. The colourless oxidation products were formed in the quantity equivalent to iodide ions. The iodide ions react with the excess of iodate ions in acidic medium, to form free iodine which oxidized leuco crystal violet (LCV) to the liberated crystal violet (CV ⁺) dye, showing maximum absorption at 588 nm. The absorbance was measured at pH of 4.1–4.2 in 1 cm cuvettes. Beer’s law was obeyed in the concentration range 0.5–4.0 μg/mL. The molar absorptivity of the coloured compound is 4.14 × 10⁴ L/mol cm for L-ascorbic acid. The analytical parameters were optimized and the method was successfully applied to the determination of L-ascorbic acid in pharmaceuticals. The results were compared with those obtained by methods proposed in Polish Standard.     

Extraction studies of uranium into a third-phase of thorium nitrate employing tributyl phosphate and <Emphasis Type="Italic">N,N</Emphasis>-dihexyl octanamide as extractants in different diluents

Extraction behavior of 1 × 10⁻²–0.1 M U(VI) from aqueous phases containing 0.86 M Th(IV) at 4 M HNO₃ in 1.1 M tributyl phosphate (TBP) and 1.1 M N,N-dihexyl octanamide (DHOA) solutions in different diluents viz. n-dodecane, 10% 1-octanol + n-dodecane, and decahydronaphthalene (decalin) was studied. Third-phase formation was observed in both the extractants using n-dodecane as diluent. There was a gradual decrease in Th(IV) concentration in the third-phase (heavy organic phase, HOP) with increased aqueous U(VI) concentration [0.71 M (no U(VI))–0.61 M (0.1 M U(VI)) for TBP; 0.27 M (no U(VI))–0.22 M (0.1 M U(VI)) for DHOA]. The HOP volume in case of DHOA was ~2.2 times of that of TBP. Uranium concentration in HOP increased with its initial concentration in the aqueous phase [from 1.8 × 10⁻² M (0.01 M U(VI))–0.162 M (0.1 M U(VI)) for TBP; from 1.4 × 10⁻² M (0.01 M U(VI))–0.14 M (0.1 M U(VI)) for DHOA] suggesting that Th(IV) was being replaced by U(VI). An empirical correlation was developed for predicting the concentrations of uranium and thorium in HOP for both the extractants. No third-phase appeared during the extraction of uranium and thorium from the aqueous phases employing 10% 1-octanol + n-dodecane, or decalin as diluents, and therefore, were better choices as diluent for alleviating the third-phase formation during the reprocessing of spent thorium based fuels, and for the recovery of thorium from high-level waste solutions.     

Biosorption of uranium(VI) by a mangrove endophytic fungus <Emphasis Type="Italic">Fusarium sp</Emphasis>. #ZZF51 from the South China Sea

The uranium(VI) accumulation was studied in detail by using the biomass of mangrove endophytic fungus Fusarium sp.#ZZF51 from the South China Sea. The uranium(VI) biosorption process onto the tested fungus powders was optimized at pH 4.0, adsorption time 60 min, and uranium(VI) initial concentration 50 mg L⁻¹ with 61.89% of removal efficiency. According to Fourier transform infrared spectra for the tested fungus before and after loaded with uranium(VI), the results showed that both of hydroxyl and carboxyl groups acted as the important roles in the adsorption process. In addition, the experimental data were analyzed by using parameter and kinetic models, and it was obtained that the Langmuir isotherm model and the pseudo-second-order kinetic model provided better correlation with the experimental data for adsorption of uranium(VI).     

Kinetic and Stoichiometric Parameters in the Production of Carotenoids by <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> (CBS 2636) in Synthetic and Agroindustrial Media

With the objective of determining the kinetic behavior (growth, substrate, pH, and carotenoid production) and obtain the stoichiometric parameters of the fermentative process by Sporidiobolus salmonicolor in synthetic and agroindustrial media, fermentations were carried out in shaken flasks at 25°C, 180 rpm, and initial pH of 4.0 for 120 h in the dark, sampling every 6 h. The maximum concentrations of total carotenoids in synthetic (913 μg/L) and agroindustrial (502 μg/L) media were attained approximately 100 h after the start of the fermentative process. Carotenoid bioproduction is associated with cell growth and the ratio between carotenoid production and cell growth (Y _P/X) is 176 and 163 μg/g in the synthetic and agroindustrial media, respectively. The pH of the agroindustrial fermentation medium varied from 4.2 to 8.5 during the fermentation. The specific growth rate (μ _X) for S. salmonicolor in synthetic and agroindustrial media was 0.07 and 0.04 h⁻¹, respectively. The synthetic medium allowed for greater productivity, obtaining maximum cell productivity (P _x) of 0.08 g L⁻¹ h⁻¹ and maximum total carotenoid productivity (P _car) of 14.2 μg L⁻¹ h⁻¹. Knowledge of the kinetics of a fermentative process is of extreme importance when transposing a laboratory experiment to an industrial scale, as well as making a quantitative comparison between different culture conditions.     

High Level Expression of an Acid-Stable Phytase from <Emphasis Type="Italic">Citrobacter freundii</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni²⁺–NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC) had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being incubated under various buffer (pH 1.5–8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg⁻¹, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg⁻¹ min⁻¹, respectively. The enzyme activity was significantly improved by 1 mM of K⁺, Ca²⁺, and Mg²⁺. These characteristics contribute to its potential application in feed industry.     

PVA-Hydrogel Entrapped <Emphasis Type="Italic">Candida Guilliermondii</Emphasis> for Xylitol Production from Sugarcane Hemicellulose Hydrolysate

Viable cells of Candida guilliermondii were immobilized by inclusion into polyvinyl alcohol (PVA) hydrogel using the freezing–thawing method. Entrapment experiments were planned according to a 2³ full factorial design, using the PVA concentration (80, 100, and 120 g L⁻¹), the freezing temperature (−10, −15, and −20 °C), and the number of freezing-thawing cycles (one, three, and five) as the independent variables, integrated with three additional tests to estimate the errors. The effectiveness of the immobilization procedure was checked in Erlenmeyer flasks as the pellet capability to catalyze the xylose-to-xylitol bioconversion of a medium based on sugarcane bagasse hemicellulosic hydrolysate. To this purpose, the yield of xylitol on consumed xylose, xylitol volumetric productivity, and cell retention yield were selected as the response variables. Cell pellets were then used to perform the same bioconversion in a stirred tank reactor operated at 400 rpm, 30 °C, and 1.04 vvm air flowrate. At the end of fermentation, a maximum xylitol concentration of 28.7 g L⁻¹, a xylitol yield on consumed xylose of 0.49 g g⁻¹ and a xylitol volumetric productivity of 0.24 g L⁻¹ h⁻¹ were obtained.     

Compactin Production Studies Using <Emphasis Type="Italic">Penicillium brevicompactum</Emphasis> Under Solid-State Fermentation Conditions

In the present study, compactin production by Penicillium brevicompactum WA 2315 was optimized using solid-state fermentation. The initial one factor at a time approach resulted in improved compactin production of 905 μg gds⁻¹ compared to initial 450 μg gds⁻¹. Subsequently, nutritional, physiological, and biological parameters were screened using fractional factorial and Box–Behnken design. The fractional factorial design studied inoculum age, inoculum volume, pH, NaCl, NH₄NO₃, MgSO₄, and KH₂PO₄. All parameters were found to be significant except pH and KH₂PO₄. The Box–Behnken design studied inoculum volume, inoculum age, glycerol, and NH₄NO₃ at three different levels. Inoculum volume (p = 0.0013) and glycerol (p = 0.0001) were significant factors with greater effect on response. The interaction effects were not significant. The validation study using model-defined conditions resulted in an improved yield of 1,250 μg gds⁻¹ compactin. Further improvement in yield was obtained using fed batch mode of carbon supplementation. The feeding of glycerol (20% v/v) on day 3 resulted in further improved compactin yield of 1,406 μg gds⁻¹. The present study demonstrates that agro-industrial residues can be successfully used for compactin production, and statistical experiment designs provide an easy tool to improve the process conditions for secondary metabolite production.     

Enhanced Production of <Emphasis Type="SmallCaps">l</Emphasis>-Arginine by Expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> Hemoglobin Using a Novel Expression System in <Emphasis Type="Italic">Corynebacterium crenatum</Emphasis>

Corynebacterium crenatum SYPA 5-5 is an aerobic and industrial l-arginine producer. It was proved that the Corynebacterium glutamicum/Escherichia coli shuttle vector pJC1 could be extended in C. crenatum efficiently when using the chloramphenicol acetyltransferase gene (cat) as a reporter under the control of promoter tac. The expression system was applied to over-express the gene vgb coding Vitreoscilla hemoglobin (VHb) to further increase the dissolved oxygen in C. crenatum. As a result, the recombinant C. crenatum containing the pJC-tac-vgb plasmid expressed VHb at a level of 3.4 nmol g⁻¹, and the oxygen uptake rates reached 0.25 mg A₅₆₂⁻¹ h⁻¹ which enhanced 38.8% compared to the wild-type strain. Thus, the final l-arginine concentration of the batch fermentation reached a high level of 35.9 g L⁻¹, and the biomass was largely increased to 6.45 g L⁻¹, which were 17.3% and 10.5% higher than those obtained by the wild-type strain, respectively. To our knowledge, this is the first report that the efficient expression system was constructed to introduce vgb gene increasing the oxygen and energy supply for l-arginine production in C. crenatum, which supplies a good strategy for the improvement of amino acid products.     

Variable Volume Fed-Batch Fermentation for Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp<Emphasis Type="Italic">. lactis W28</Emphasis>

A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l⁻¹) with 100 ml sucrose solution (190 g l⁻¹) being evenly fed (9–10 ml h⁻¹) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions, the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively. When the sucrose concentration was controlled at 5–10 g l⁻¹ in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l⁻¹, the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be scaled up, since various parameters involved in industrial production were taken into account.     

Adsorption of uranium(VI) onto <Emphasis Type="Italic">Ulva sp.</Emphasis>-sepiolite composite

Ulva sp. and sepiolite were used to prepare composite adsorbent. The adsorption of uranium(VI) from aqueous solutions onto Ulva sp.-sepiolite has been studied by using a batch adsorber. The parameters that affect the uranium(VI) adsorption, such as solution pH, initial uranium(VI) concentration, and temperature, have been investigated and the optimum conditions determined. The adsorption patterns of uranium on the composite adsorbent followed the Freundlich and Dubinin-Radushkevich (D-R) isotherms. The Freundlich, Langmuir, and Dubinin-Radushkevich (D-R) models have been applied and the data correlate well with Freundlich model. The sorption is physical in nature (sorption energy, E = 4.01 kJ/mol). The thermodynamic parameters such as variation of enthalpy ΔH, variation of entropy ΔS and variation of Gibbs free energy ΔG were calculated from the slope and intercept of lnK _d vs. 1/T plots. Thermodynamic parameters (ΔH _ads = −22.17 kJ/mol, ΔS _ads = −17.47 J/mol·K, ΔG ^o _ads (298.15 K) = −16.96 kJ/mol) show the exothermic heat of adsorption and the feasibility of the process. The results suggested that the Ulva sp-sepiolite composite adsorbent is suitable as a sorbent material for recovery and biosorption/adsorption of uranium ions from aqueous solutions.     

Conversion of Isoeugenol to Vanillin by <Emphasis Type="Italic">Psychrobacter</Emphasis> sp. Strain CSW4

To screen strains of halotolerant or halophile bacteria which are able to convert isoeugenol to vanillin, 36 different strains of bacteria isolated from the salty environments in Iran were investigated. During growth on isoeugenol, a moderately halotolerant Gram-negative coccobacil showed capability of converting isoeugenol to vanillin. Based on morphological, physiological, and phylogenetic studies, strain CSW4 was classified as a bacterium belonging to the genus Psychrobacter. The bioconversion products were confirmed by thin-layer chromatography, high-performance liquid chromatography, and spectral data obtained from UV/Vis spectroscopy, FTIR, and mass-spectroscopy. Using growing cells, vanillin reached its maximum level of 88.18 mg L⁻¹ after 24 h of reaction time in the presence of 1 g L⁻¹ isoeugenol, resulting in a molar yield of 10.2%. The use of resting cells led to the optimal yield of vanillin (16.4%) which was obtained after 18-h reaction using 1 g L⁻¹ isoeugenol and 3.1 g of dry weight of cells per liter harvested at the end of the exponential growth phase. To improve vanillin yield, the effect of substrate concentration on vanillin production under resting cells conditions was also investigated. Using 10 g L⁻¹ isoeugenol, the maximal vanillin concentration (1.28 g L⁻¹) was achieved after a 48-h reaction, without further optimization. The present study brings the first evidence for biotransformation of isoeugenol to vanillin in the genus Psychrobacter.     

A new cobalt complex of 1-decyl-1<Emphasis Type="Italic">H</Emphasis>-benzo[<Emphasis Type="Italic">d</Emphasis>]imidazole: synthesis,characterization and electrocatalysis

The present work reports on the synthesis, characterization and performance of a new cobalt(II) complex, [Co(C₁₀H₂₁-bim)₂(SCN)₂] (bim = benzimidazole) as electrocatalyst for trichloroacetic acid and bromate reduction. Its structure was characterized by X-ray crystallography, IR spectroscopy and elemental analysis. The cobalt atom adopts a distorted tetrahedral geometry by coordinating to four nitrogen atoms from two thiocyanates and two 1-decyl-1H-benzo[d]imidazole ligands. The electrochemical behavior and electrocatalysis of the title complex bulk-modified carbon paste electrode (Co-CPE) have been studied by cyclic voltammetry. The Co-CPE shows good electrocatalytic activities toward the reduction of trichloroacetic acid and bromate. The detection limit and the sensitivity are 0.02 μM, 34.63 μA μM⁻¹ for trichloroacetic acid detection, and 0.03 μM, 78.92 μA μM⁻¹ for bromate detection, respectively. This modified electrode shows good reproducibility, high stability, low detection limit, technical simplicity and possibility of rapid preparation, which is important for practical application.     

Kinetic and mechanistic studies on the substitution of aqua ligands from <Emphasis Type="Italic">cis</Emphasis>-diaqua-<Emphasis Type="Italic">bis</Emphasis>-(bypyridyl)-ruthenium(II) ion by vic-dioximes

Kinetics of aqua ligand substitution from cis-[Ru(bpy)₂(H₂O)₂]²⁺ by three vicinal dioximes, namely dimethylglyoxime (L¹H), 1,2-cyclohexane dionedioxime (L²H) and α-furil dioxime (L³H) have been studied spectrophotometrically in the 45–60 °C temperature range. The rate constants increase with increasing dioxime concentration and approach a limiting condition. We propose the following rate law for the reaction in the 3.5–5.5 pH range: where k ₂ is the interchange rate constant from outer sphere to inner sphere complex and K _E is the outer sphere association equilibrium constant. Activation parameters were calculated from the Eyring plots for all three systems: ΔH ^≠ = 59.2 ± 8.8, 63.1 ± 6.8 and 69.7 ± 8.5 kJ mol⁻¹, ΔS ^≠ = −122 ± 27, −117 ± 21 and −99 ± 26 J K⁻¹ mol⁻¹ for L¹H, L²H and L³H, respectively. An associative interchange mechanism is proposed for the substitution process. Thermodynamic parameters calculated from the temperature dependence of the outer sphere association equilibrium constants give negative ΔG ⁰ values for all the systems studied at all the temperatures (ΔH ⁰ = 30.05 ± 2.5, 18.9 ± 1.1 and 11.8 ± 0.2 kJ mol⁻¹; ΔS ⁰ = 123 ± 8, 94 ± 3 and 74 ± 1 J K⁻¹ mol⁻¹ for L¹H, L²H and L³H, respectively), which also support our proposition.     

<Emphasis Type="Italic">Rhizopus stolonifer</Emphasis> LAU 07: a novel source of fructosyltransferase

Increasing awareness of the importance of fructooligosaccharides (FOS) as ingredients of functional foods has led to intensive search of new sources of fructosyltransferases (FTase), enzymes responsible for the conversion of sucrose to fructooligosaccharides. A local strain of Rhizopus stolonifer isolated from spoilt orange fruit with high fructosyltransferase activity (U _t) of 12.31–45.70 U mL⁻¹ during a fermentation period of 24–120 h is herein reported. It showed low hydrolytic activity (U _h) in the range of 0.86–1.78 U mL⁻¹ during the same period. FOS yield of 34 % (1-kestose, GF₂, nystose, GF₃) was produced by FTase obtained from a 72 h-old culture using 60 g of sucrose per 100 mL of the substrate. When the isolate was grown in a defined submerged medium, its pH dropped sharply from the intial value of 5.5 to 1.0 within 24 h, and this value was maintained throughout the fermentation. The biomass content ranged from 8.8 g L⁻¹ at 24 h of fermentation to reach the maximum of 10 g L⁻¹ at 72 h. It was reduced to 5.6 g L⁻¹ at the end of 120 h of fermentation. This report represents the first reference to a strain of Rhizopus as a source of FTase for the production of FOS. The high U _t/U _h ratio shown by this isolate indicates that it may be a good strain for the industrial and commercial production of FOS. However, there is a need of further optimization of the bioprocess to increase the conversion efficiency of sucrose to FOS by the enzyme.     

Production of Butyric Acid from Glucose and Xylose with Immobilized Cells of <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis> in a Fibrous-bed Bioreactor

Butyric acid has many applications in chemical, food, and pharmaceutical industries. In the present study, Clostridium tyrobutyricum ATCC 25755 was immobilized in a fibrous-bed bioreactor to evaluate the performance of butyrate production from glucose and xylose. The results showed that the final concentration and yield of butyric acid were 13.70 and 0.46 g g⁻¹, respectively, in batch fermentation when 30 g L⁻¹ glucose was introduced into the bioreactor. Furthermore, high concentration 10.10 g L⁻¹ and yield 0.40 g g⁻¹ of butyric acid were obtained with 25 g L⁻¹ xylose as the carbon source. The immobilized cells of C. tyrobutyricum ensured similar productivity and yield from repeated batch fermentation. In the fed-batch fermentation, the final concentration of butyric acid was further improved to 24.88 g L⁻¹ with one suitable glucose feeding in the fibrous-bed bioreactor. C. tyrobutyricum immobilized in the fibrous-bed bioreactor would provide an economically viable fermentation process to convert the reducing sugars derived from plant biomass into the final bulk chemical (butyric acid).     

1,3,5,7-Tetramethyl-8-(<Emphasis Type="Italic">N</Emphasis>-hydroxysuccinimidyl butyric ester)difluoroboradiaza-<Emphasis Type="Italic">s</Emphasis>-indacene as a new fluorescent labeling reagent for HPLC determination of amino acid neurotransmitters in the cerebral cortex of mice

1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene (TMBB-Su), a new BODIPY-based fluorescent probe, was designed and synthesized for the labeling of amino compounds. It was used as a pre-column derivatizing reagent for determination of amino acid neurotransmitters by high-performance liquid chromatography (HPLC). The fluorescence quantum yield in acetonitrile increased from 0.84 to 0.95 when it reacted with amino acid neurotransmitters. Derivatization of TMBB-Su with seven amino acid neurotransmitters was completed within 30 min at 25 °C in 24.0 mmol L⁻¹ pH 7.8 boric acid buffer. The separation was performed on a C₁₈ column with methanol–water–buffer 55:35:10 (v/v) as mobile phase (buffer: 0.10 mol L⁻¹ H₃Cit–0.10 mol L⁻¹ NaOH). Interference from the other concomitant amino acids was eliminated successfully by means of pH gradient elution. With fluorescence detection at 494 and 504 nm for excitation and emission, respectively, the limits of detection (signal-to-noise ratio = 3) were from 2.1 to 12.0 nmol L⁻¹. The proposed method has been used to determine amino acid neurotransmitters in the cerebral cortex of mice with cerebral ischemia at the convalescence stage with satisfactory recoveries varying from 94.9 to 105.2%.     

Mechanistic aspects of ligand substitution on <Emphasis Type="Italic">cis</Emphasis>-diaqua(<Emphasis Type="Italic">cis</Emphasis>-1,2-diaminocyclohexane)platinum(II) by Glycine-<Emphasis Type="SmallCaps">l</Emphasis>-Leucine

The kinetics of the interaction of glycine-l-leucine (Glyleu) with cis-[Pt(cis-dach)(OH₂)₂]²⁺ (dach = 1,2-diaminocyclohexane) has been studied spectrophotometrically as a function of [cis-[Pt(cis-dach)(OH₂)₂]²⁺], [Glyleu] and temperature at pH 4.0, where the complex exists predominantly as the diaqua species and Glyleu as a zwitterion. The substitution reaction shows two consecutive steps: the first is the ligand-assisted anation and the second is the chelation step. The activation parameters for both the steps were evaluated using Eyring’s equation. The low ∆H₁^≠ (51.9 ± 2.8 kJmol⁻¹) and large negative value of ∆S₁^≠ (−152 ± 8 JK⁻¹mol⁻¹) as well as ∆H₂^≠ (54.4 ± 1.7 kJmol⁻¹) and ∆S₂^≠ (−162 ± 5 JK⁻¹mol⁻¹) indicate an associative mode of activation for both the aqua ligand substitution processes.     

Cloning of a Heat-Stable Chitin Deacetylase Gene from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> and its Functional Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A gene encoding chitin deacetylase was cloned by polymerase chain reaction from Aspergillus nidulans. Sequencing result showed 40% homology to the corresponding gene from Colletotrichum lindemuthianum. The complete gene contains an open reading frame of 747 nucleotides encoding a sequence of 249 amino acid residues. The chitin deacetylase gene was subcloned into a pET28a expression vector and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a His-bind column. The purified chitin deacetylase demonstrated an activity of 0.77 U ml⁻¹ for the glycol chitin substrates, and its specific activity was 4.17 U mg⁻¹ for it. The optimal temperature and pH of the purified enzyme were 50 °C and 8.0, respectively. When glycol chitin was used as the substrate, K _m was 4.92 mg ml⁻¹, and K _cat showed 6.25 s⁻¹, thus the ratio of K _cat and K _m was 1.27 ml s⁻¹ mg⁻¹. The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid.