Optimization of keratinase production and enzyme activity using response surface methodology with streptomyces sp7

A two-step response surface methodology (RSM) study was conducted for the optimization of keratinase production and enzyme activity from poultry feather byStreptomyces sp7. Initially different combinations of salts were screened for maximal production of keratinase at a constant pH of 6.5 and feather meal concentration of 5 g/L. A combination of K₂HPO₄, KH₂PO₄, and NaCl gave a maximum yield of keratinase (70.9 U/mL) production. In the first step of the RSM study, the selected five variables (feather meal, K₂HPO₄, KH₂PO₄, NaCl, and pH) were optimized by a 2⁵ full-factorial rotatable central composite design (CCD) that resulted in 95 U/mL of keratinase production. The results of analysis of variance and regression of a second-order model showed that the linear effects of feather meal concentration (p<0.005) and NaCl (p<0.029) and the interactive effects of all variables were more significant and that values of the quadratic effects of feather meal (p<1.72e-5), K₂HPO₄ (p<4.731e-6), KH₂PO₄ (p<1.01e-10), and pH (p 7.63e-7) were more significant than the linear and interactive effects of the process variables. In the second step, a 2³ rotatable full-factorial CCD and response surface analysis were used for the selection of optimal process parameters (pH, temperature, and rpm) for keratinase enzyme activity. These optima were pH 11.0, 45°C, and 300 rpm.     

Optimization of inulinase production by Kluyveromyces marxianus using factorial design

Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase by Kluyveromyces marxianus. Sucrose was used as the carbon source instead of inulin. Initially, a fractional factorial design (2^5–1) was used in order to determine the most relevant variables for enzyme production. Five parameters were studied (sucrose, peptone, yeast extract, pH, and K₂HPO₄), and all were shown to be significant. Sucrose concentration and pH had negative effects on inulinase production, whereas peptone, yeast extract, and K₂HPO₄ had positive ones. The pH was shown to be the most significant variable and should be preferentially maintained at 3.5. According to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized in a full factorial design. The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose, 10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K₂HPO₄. The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization.     

Production of bacterial cellulose by Gluconacetobacter sp. RKY5 isolated from persimmon vinegar

The optimum fermentation medium for the production of bacterial cellulose (BC) by a newly isolated Gluconacetobacter sp. RKY5 was investigated. The optimized medium composition for cellulose production was determined to be 15 g/L glycerol, 8 g/L yeast extract, 3 g/L K₂HPO₄, and 3 g/L acetic acid. Under these optimized culture medium, Gluconacetobacter sp. RKY5 produced 5.63 g/L of BC after 144 h of shaken culture, although 4.59 g/L of BC was produced after 144 h of static culture. The amount of BC produced by Gluconacetobacter sp. RKY5 was more than 2 times in the optimized medium found in this study than in a standard Hestrin and Shramm medium, which was generally used for the cultivation of BC-producing organisms.     

Optimization of medium by orthogonal matrix method for submerged mycelial culture and exopolysaccharide production in Collybia maculata

Optimization of submerged culture conditions for the production of mycelial growth and exopolysaccharides (EPSs) by Collybia maculata was investigated. The optimum temperature and the initial pH for EPS production in a shake-flask culture of C. maculata were found to be 20°C and 5.5, respectively. Among the various medium’s constituents examined, glucose, Martone A-1, K₂HPO₄, and CaCl₂ were the most suitable carbon, nitrogen, and mineral sources for EPS production, respectively. The optimum concentration of the medium’s ingredients determined using the orthogonal matrix method was as follows: 30 g/L of glucose, 20 g/L of Martone A-1, 1g/L of K₂HPO₄, and 1g/L of CaCl₂. Under the optimized culture conditions, the maximum concentration of EPSs in a 5-L stirred-tank reactor was 2.4 g/L, which was approximately five times higher than that in the basal medium. A comparative fermentation result showed that the EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor despite the lower mycelial growth rate. The specific productivities and the yield coefficients in the airlift reactor were higher than those in the stirred-tank reactor even though the volumetric productivities were higher in the stirred-tank reactor than in the airlift reactor.     

Supramolecular inclusion of a pillared double-layered host by an anion-directed second-sphere coordination

In this paper, we have illustrated the utilisation of a second-sphere coordination approach to construct supramolecular inclusion solids with varieties of guest molecules. A flexible molecule N,N,N′,N′-tetra-p-methylbenzyl-ethylenediamine (L1) bearing doubly protonated H-bond donors was designed, capable of forming N–H…Cl hydrogen bonds with a crystallographically unique chloride anion, to construct an anion-directed ligand. The pillared double-layered host framework was constructed by an anion-directed ligand and primary coordination sphere [CoCl₄]^2 ?  through weak C–H…Cl hydrogen-bonding interactions. A variety of guest molecules, such as p-anisaldehyde, 1,4-dimethoxy-2,5-bis(methoxymethyl)benzene, can be included, leading to the formation of novel supramolecular inclusion solids: [L1]·4[H]⁺·[CoCl₄]^2 ? ·2Cl^? ·1.5[C₈H₈O₂]·0.25[CH₃OH] (1) and [L1]·4[H]⁺·[CoCl₄]^2 ? ·2Cl^? ·1.5[C₁₂H₂₀O₄]·0.5[CH₃OH] (2).

We have presented herein the utilisation of a second-sphere coordination approach to construct supramolecular inclusion solids with a variety of guest molecules. A novel type of a pillared double-layered host framework was constructed by a second-sphere coordination between the anion-directed ligand (L1 = N,N,N′,N′-tetra-p-methylbenzyl-ethylenediamine) and [CoCl₄]^2 ?  through weak C–H…Cl hydrogen-bonding interaction, and a variety of guest molecules, such as p-anisaldehyde, 1,4-dimethoxy-2,5-bis(methoxymethyl)benzene, can be included, leading to the formation of supramolecular inclusion solids: [L1]·4[H]⁺·[CoCl₄]^2 ? ·2Cl^? ·1.5[C₈H₈O₂]·0.25[CH₃OH] (1) and [L1]·4[H]⁺·[CoCl₄]^2 ? ·2Cl^? ·1.5[C₁₂H₂₀O₄]·0.5[CH₃OH] (2)

     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

Potassium gallium hydrogenphosphate fluoride,K2Ga[H(HPO4)2]F2

Hydrothermally synthesized dipotassium gallium {hydrogen bis[hydrogenphosphate(V)]} difluoride, K₂Ga[H(HPO₄)₂]F₂, is isotypic with K₂Fe[H(HPO₄)₂]F₂. The main features of the structure are ([Ga{H(HPO₄)₂}F₂]²⁻)_n columns consisting of centrosymmetric Ga(F₂O₄) octahedra [average Ga—O = 1.966 (3) Å and Ga—F = 1.9076 (6) Å] stacked above two HPO₄ tetrahedra [average P—O = 1.54 (2) Å] sharing two O‐atom vertices. The charge‐balancing seven‐coordinate K⁺ cations [average K—O,F = 2.76 (2) Å] lie in the intercolumn space, stabilizing a three‐dimensional structure. Strong [O...O = 2.4184 (11) Å] and medium [O...F = 2.6151 (10) Å] hydrogen bonds further reinforce the connections between adjacent columns.     

Spektrophotometrische Untersuchungen über Chlorokomplexe von Co++, Ni++ und Cu++ in acetonitril und Trimethylphosphat

Zusammenfassung Mit steigendem Chloridionenangebot lassen sich auf spektrophotometrischem Wege in Lösungen von Acetonitril und Trimethylphosphat folgende Cl-Koordinationsformen nachweisen: CoCl₂, CoCl₃ ^–, CoCl₄ ^–;K ₁<K ₂>K ₃>K ₄; NiCl₂, NiCl₃ ^–, NiCl₄ ^–; in TrimethylphosphatK ₂K ₃>K ₄; in AcetonitrilK ₃>K ₄; CuCl⁺, CuCl₂, CuCl₃ ^–, CuCl₄ ^–;K ₁>K ₂K ₃>K ₄. Die Löslichkeit des NiCl₂ steigt mit der Basizität des Lösungsmittels.Mit 9 Abbildungen     

A study on the adsorption-exchange of UO2 2+,137Cs,169Yb and HPO4 2− on modified peat

The adsorption-exchange equilibrium time and the adsorption isotherms of UO₂ ²⁺,¹³⁷Cs,¹⁶⁹Yb and HPO₄ ²⁻ on modified peat have been investigated by batch experiments. The effect of pH on the adsorption-exchange percentage (E) and the distribution coefficients (K _d) was also examined. It was found that the adsorption-exchange of UO₂ ²⁺ and¹⁶⁹Yb on the modified peat was described well by Freundlich isotherm, whereas the adsorption-exchange of¹³⁷Cs and HPO₄ ²⁻ on modified peat corresponded to a Langmuir isotherm and the maximum adsorption capacities of the modified peat for¹³⁷Cs and HPO₄ ²⁻ ions were 4.4 and 4.1 μg/g respectively. The optimum pH for the adsorption-exchange of UO₂ ²⁺,¹³⁷Cs,¹⁶⁹Yb and HPO₄ ²⁻ on the modified peat was 7.0 at 25°C.     

Isopiestic Determination of the Osmotic and Activity Coefficients of K<Subscript>2</Subscript>HPO<Subscript>4</Subscript>(aq), Including Saturated and Supersaturated Solutions,at <Emphasis Type="Italic">T</Emphasis>=298.15 K

The osmotic coefficients of K₂HPO₄(aq) have been measured at T=298.15 K by the isopiestic vapor pressure method over the range of molalities from 1.3846 mol⋅kg⁻¹ to 13.939 mol⋅kg⁻¹ (oversaturation) with CaCl₂(aq) as the reference solution. The molalities and osmotic coefficients of saturated solutions in equilibrium with K₂HPO₄⋅xH₂O(cr) were measured simultaneously by the same method. Available literature osmotic coefficients of K₂HPO₄(aq) at T=298.15 K, and our new experimental data, were combined and modeled using an extended form of Pitzer’s equation and the Clegg-Pitzer-Brimblecombe equation based on the mole-fraction-composition scale. These equations were used to calculate the activity coefficients of K₂HPO₄(aq) at T=298.15 K.     

Investigation of early antibiotic use in pediatric patients with acute respiratory infections by high-performance liquid chromatography

In this study, we developed and validated two reliable high-performance liquid chromatography (HPLC) methods for the qualitative detection of six oral β-lactams, which are commonly used in pediatric patients with acute respiratory infections (ARIs). Two distinct reverse-phase chromatographic separations of six β-lactams were obtained. Four β-lactams (cefadroxil, cephalexin, cefaclor and cefixime) in urine were separated using a gradient program with a mobile phase consisting of K₂HPO₄ buffer (20 mm , pH 2.8) and acetonitrile on a LichroCART 250 × 4.6 mm, Purospher STAR C₁₈ end-capped (5 μm) column. Two remained β-lactams (amoxicillin and cefuroxime) were analyzed using a gradient elution with the mobile phase containing K₂HPO₄ buffer (20 mm , pH 3.0) and acetonitrile on a LichroCart^® Purospher Star C₈ end-capped column (5 μm, 125 × 4.6 mm). Good linearity within the range of 0.3–30 μg/ml for cefadroxil, cephalexin, cefaclor and cefixime, and 0.2–20 μg/ml for amoxicillin and cefuroxime, was attained. The precisions were <14%. The accuracies ranged from 85.87 to 102.8%. The two validated methods were then applied to determine these six antibiotics in 553 urine samples of pediatric patients with ARIs. As a result, 32.2% were positive with one or more of six tested β-lactams. Cefixime was the most commonly detected agent, accounting for 9.8% of enrolled patients.     

Production and Characteristics of the Whole-Cell Lipase from Organic Solvent Tolerant <Emphasis Type="BoldItalic">Burkholderia</Emphasis> sp. ZYB002

The thermostable and organic solvent tolerant whole-cell lipase (WCL) was produced by Burkholderia sp. ZYB002 with broad spectrum organic solvent tolerance. The production medium of the WCL was primarily optimized, which resulted in the maximum activity of 22.8 U/mL and the 5.1-fold increase of the WCL yield. The optimized culture medium was as follows (% w/v or v/v): soybean meal 2, soybean oil 0.5, manganese sulfate 0.1, K₂HPO₄ 0.1, olive oil 0.5, initial pH 6.0, inoculum density 2, liquid volume 35 mL in 250-mL Erlenmeyer flask, and incubation time 24 h. The biochemical characterization of the WCL from Burkholderia sp. ZYB002 was determined, and the results showed that the optimal pH and temperature for lipolytic activity of the WCL was 8.0 and 65°C, respectively. The WCL was stable at temperature up to 70°C for 1 h and retained 79.2% of its original activity. The WCL was highly stable in the pH range from 3.0 to 8.5 for 6 h. Ca²⁺, K⁺, Na⁺, NO₃⁻, etc. ions stimulated its lipolytic activity, whereas Zn²⁺ ion caused inhibition effect. The WCL was also relatively stable in n-butanol at a final concentration of 50% (v/v) for 24 h. However, the WCL was strongly inhibited in Triton X-100 at a final concentration of 10% (v/v). The WCL with thermal resistance and organic solvent tolerance showed its great potential in various green industrial chemical processes.     

Effect of counterion condensation on the acid-base equilibrium of a pH-sensitive merocyanine dye covalently attached to polyelectrolytes

Effects of monovalent and divalent counterions on the acid-base equilibrium of a pH-sensitive merocyanine dye covalently attached to copolymers of acrylic acid and acrylamide with varying charge densities (0.28 < ξ < 2.8) were investigated. Added chloride salts of Li⁺, Na⁺, K⁺, and NH⁺₄ (< 0.2 mM) had essentially no effect on pK observed (pK^obs) for the equilibrium. By contrast, the salts of Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺ caused a significant decrease in pK^obs for the copolymers with larger ξ. With smaller ξ, most likely when ξ < 0.5, no decrease in pK^obs was observed upon addition of the salts of divalent cations. A competitive effect of Ca²⁺ and Na²⁺ ions on pK^obs in the presence of an excess of Na⁺ ions implied that Ca²⁺ ions at very low concentrations were preferentially, and therefore exhaustively, condensed on the polyanions with sufficiently large ξ probably until effective charge density was lowered to 0.5. The observed difference in the influence of the monovalent and divalent cations on pK^obs was discussed in terms of the difference in the microscopic behavior of the condensed monovalent and divalent cations. © 1993 John Wiley & Sons, Inc.     

Coupled diffusion produced by hydrolysis of aqueous potassium phosphate

Conductimetric and diaphragm cell techniques have been used to measure diffusion of aqueous potassium phosphate solutions at 25°C from 0.01 to 0.10 mol-dm^–3 (M). A significant portion of the aqueous K₃PO₄ component diffuses as equimolar amounts of potassium hydrogen phosphate and potassium hydroxide produced by hydrolysis: K₃PO₄+H₂O=K₂HPO₄+KOH. Because OH^– diffuses more rapidly than HPO ₄ ^2– , the total flow of KOH exceeds the flow of K₂HPO₄. The extra flow of KOH constitutes coupled transport of a second solute component. Ternary diffusion coefficients that describe interacting flows of K₃PO₄ and KOH components are reported. At low concentrations where phosphate is strongly hydrolyzed, the molar flux of the KOH component produced by diffusion of K₃PO₄ is six times larger than the flux of the K₃PO₄ component. Binary diffusion coefficients for aqueous K₂HPO₄ solutions are also reported. It is shown that ternary transport coefficients for K₃PO₄ solutions can be estimated from the properties of binary solutions of K₂HPO₄ and KOH.     

MZn2HPO4PO4 (M=Na+, K+)的合成、表征和标准摩尔生成焓

采用低温固相法和水热法制备MZn2HPO4PO4 (M=Na+, K+) 并用XRD, FT-IR, TG and SEM对其进行表征,用等温量热计测定热化学性质。按照Hess’s定律,设计一新的热化学循环。结果表明,所合成的物质是等结构三斜晶系的目标产物,具有片层结构,分解温度分别为： 415 ℃和430 ℃。从测定的溶解焓和其他的标准热化学数据,计算出MZn2HPO4PO4 (M=Na+, K+) 的标准摩尔生成焓分别为：ΔfHm [NaZn2HPO4PO4, s]=-3042.38±0.31 kJ·mol-1; ΔfHm [KZn2HPO4PO4,s]=-3093.46 ±0.27 kJ·mol-1。     

Effects of Nonionic, Cationic, and Mixed Nonionic-Cationic Surfactants on the Acid-Base Behavior of Phenyl Salicylate

The effects of Brij 35 micelles, CTABr micelles, and mixed Brij 35–CTABr micelles on the acid–base behavior of phenyl salicylate (PST) have been studied in aqueous solution containing 2% v/v acetonitrile. The apparent pK_b (pK^app_b) of PST is decreased by 1.5 pK units with the increase in [Brij 35] from 0 to 0.02 M which is attributed to micellar medium effect. The values of pK^app_b remain almost independent of [CTABr] within its range 0.01–0.03 M. The increase in [CTABr] from 0 to 0.03 M in aqueous solution containing 0.02 M Brij 35 has not resulted in a change in pK^app_b. This shows that the characeristic structural features of nonionic Brij 35 micelles remain essentially unchanged on addition of CTABr under the present experimental conditions.     

Basicity of some aliphatic amines in mixed H2O?CH3CN solvents

The values of pK_a^ms (K_a^ms represents ionization constant of conjugate acid of amine base in mixed water–acetonitrile solvent) for all amines, except for charged amine bases, show a mild decrease (ca. 0.1–0.4 pK units) with the increase in CH₃CN content from 2 to ∼60% v/v. However, the pK_a^ms values at 70% v/v CH₃CN become nearly equal or slightly larger (by ≤0.7 pK units) than the corresponding pK_a^ms at 2% v/v CH₃CN for all neutral and charged amines. The values of pK_a^ms for phenol increase from 10.17 to 13.38 with the increase in the content of CH₃CN from 2 to 70% v/v in mixed aqueous solvent. Taft reaction constants, ρ*, obtained from the plots of pK_a^ms against ∑σ* for primary and secondary amines decrease by ca. 0.8 ρ* units with the increase in the CH₃CN content from 2 to 70% v/v. The values of pK_a^ms show an empirical linear relationship with the corresponding values of pK_a^w (where pK_a^w represents the pK_a obtained in aqueous solvent containing 2% v/v CH₃CN), which allows the estimation of a pK_a in mixed H₂O CH₃CN solvents from that in water. © 2000 John Wiley & Sons, Inc. Int J Chem Kinet 32: 146–152, 2000     

Production of <Superscript>122</Superscript>Sb for the study of environmental pollution

This article presents, ¹²²Sb (T _1/2 = 2.723 days, I _β- = 97.59%) was produced via the ^natSn(p,xn) nuclear process at the AMIRS (Cyclone-30, IBA, Belgium). The electrodeposition experiments were carried out by potassium stannate trihydrate (K₂Sn(OH)₆) and potassium hydroxide. The optimum conditions of the electrodeposition of tin were as follows: 40 g/L ^natSn, 20 g/L KOH, 115 g/L K₂Sn(OH)₆, DC current density of 5 A/dm² with a bath temperature of 75 °C. The electroplated Tin-target was irradiated with 26.5 MeV protons at current of 180 μA for 20 min. Solvent extraction of no-carrier-added ¹²²Sb from irradiated Tin-natural target hydrochloric solution was investigated using di-n-butyl ether (C₈H₁₈O). Yields of about 3.61 MBq/μAh were experimentally obtained.     

Dissociation constants of the ampholyte glycine in 50 mass % methanol-water from 5 to 55°C,with related thermodynamic quantities

The two thermodynamic dissociation constants of glycine at 11 temperatures from 5 to 55°C in 50 mass % methanol-water mixed solvent have been determined from precise emf measurements with hydrogen-silver bromide electrodes in cells without liquid junction. The first acidic dissociation constant (K ₁)for the process HG⁺H⁺+G^± is expressed as a function ofT(^oK) by the equation pK ₁ = 2043.5/T – 9.6504 + 0.019308T. At 25°C, pK ₁is 2.961 in the mixed solvent, as compared with 2.350 in water, with H°=1497 cal-mole^–1, G°=4038 cal-mole^–1, S°=–8.52 cal-°K^–1-mole^–1, and C _p ^o =–53 cal-°K^–1-mole^–1. The second acidic dissociation constant (K ₂)for the process G^±H⁺+G^– over the temperature range studied is given by the equation pK ₂ = 3627.1/T – 7.2371 + 0.015587T. At 25°C, pK ₂is 9.578 in MeOH–H₂O as compared with 9.780 in water, whereas H° is 10,257 cal-mole^–1, G° is 13,063 cal-mole^–1, S° is –9.41 cal-°K^–1-mole^–1, and C _p ^o is –43 cal-°K^–1-mole^–1. The protonated glycine becomes weaker in 50 mass % methanol-water, whereas the second dissociation process becomes stronger despite the lower dielectric constant of the mixed solvent (=56.3 at 25°C).     

Benchmarking and validating algorithms that estimate p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> values of drugs based on their molecular structures

The REGDIA regression diagnostics algorithm in S-Plus is introduced in order to examine the accuracy of pK _a predictions made with four updated programs: PALLAS, MARVIN, ACD/pKa and SPARC. This report reviews the current status of computational tools for predicting the pK _a values of organic drug-like compounds. Outlier predicted pK _a values correspond to molecules that are poorly characterized by the pK _a prediction program concerned. The statistical detection of outliers can fail because of masking and swamping effects. The Williams graph was selected to give the most reliable detection of outliers. Six statistical characteristics (F _exp, R ², , MEP, AIC, and s(e) in pK _a units) of the results obtained when four selected pK _a prediction algorithms were applied to three datasets were examined. The highest values of F _exp, R ², , the lowest values of MEP and s(e), and the most negative AIC were found using the ACD/pK _a algorithm for pK _a prediction, so this algorithm achieves the best predictive power and the most accurate results. The proposed accuracy test performed by the REGDIA program can also be applied to test the accuracy of other predicted values, such as log P, log D, aqueous solubility or certain physicochemical properties of drug molecules.