Use of expanded bed adsorption to purify flavonoids from Ginkgo biloba L.

Three techniques (liquid–liquid extraction, packed bed adsorption and expanded bed adsorption) have been compared for the purification of flavonoids from the leaves of Ginkgo biloba L. A crude Ginkgo extract was obtained by refluxing with ethanol for 3 h. The yield of flavonoids achieved by this crude extraction was about 19% (w/w) and the purity of flavonoids in the concentrated extract was between 1.9 and 2.3% (w/w). The crude extract was then dissolved in deionized water and centrifuged where necessary to prepare clarified feedstock for further purification. For the method using liquid–liquid extraction with ethyl acetate, the purity, concentration ratio and yield of flavonoids were 25.4–31.0%, 16–18 and >98%, respectively. For the method using packed bed adsorption, Amberlite XAD7HP was selected as the adsorbent and clarified extract was used as the feedstock. The dynamic adsorption breakthrough curves and elution profiles were measured. For a feedstock containing flavonoids at a concentration of 0.25 mg/mL, the appropriate loading volume to reach a 5% breakthrough point during the adsorption stage was estimated to be 550–600 mL for a packed bed of volume 53 mL and a flow rate of 183 cm/h. The results from the elution stage indicated that the majority of impurities were eluted by ethanol concentrations of 40% (v/v) or below and efficient separation of flavonoids from the impurities could be achieved by elution of the flavonoids with 50–80% ethanol reaching an average purity of ∼25%. The recovery yield of flavonoids using the packed bed purification method was about 60% of the flavonoids present in the clarified feedstock (corresponding to around 30% for the total flavonoids in the unclarified crude extract). For the method using expanded bed adsorption also conducted with Amberlite XAD7HP as the adsorbent, the optimal operation conditions scouted during the packed bed experiments were used but unclarified crude extract could be loaded directly into the column. For an expanded bed with a settled bed height of 30 cm, the loss of flavonoids in the column flow-through was about 30%. The two-step elution protocol again proved to be effective in separating the adsorbed impurities and flavonoids. More than 96% of the bound impurities were completely removed by 40% ethanol in the first elution stage and less than 4% remained in the final product eluted by 90% ethanol in the second elution stage. Also, ∼74% of the adsorbed flavonoids on column (corresponding to 51% of the total flavonoids in the unclarified feedstock) were recovered in the product. In addition to higher recovery yield, the average process time to obtain the same amount of product was decreased in the expanded bed adsorption (EBA) process. The results suggest that the adoption of EBA procedures can greatly simplify the process flow sheet and in addition reduce the cost and time to purify flavonoids from Ginkgo biloba. These results clearly demonstrate the potential for the use of EBA to purify pharmaceuticals from plant sources.     

Purification, cloning and expression of an Aspergillus niger lipase for degradation of poly(lactic acid) and poly(ε-caprolactone)

A lipase from Aspergillus niger MTCC 2594 was purified 53.8-fold to homogeneity by hydrophobic interaction chromatography using octyl sepharose and the enzyme showed two protein bands with apparent molecular mass of 35 and 37 kDa respectively. The lipase exhibited maximum activity at pH 7.0 and 37 °C and was stable between pH 4.0 and 10.0 and temperatures up to 50 °C. The values of K_m and V_max were 3.83 mM and 32.21 μmol/min/mg respectively, using olive oil as substrate. Lipase encoding gene, lipA, coded for 297 amino acid residues with conserved pentapeptide sequence, G-H-S-L-G, was cloned and expressed in Pichia pastoris. Although lipA showed high homology with the known Aspergillus lipases, it exhibited differences in putative lid domain. Both native and recombinant lipases have potential for degradation of poly(lactic acid) and poly(ε-caprolactone), and the present study will serve as a baseline of initial studies for its exploitation in polymer degradation.     

Study of kinetic desorption rate constant in fish muscle and agarose gel model using solid phase microextraction coupled with liquid chromatography with tandem mass spectrometry

This study aims to use solid phase microextraction (SPME), a simple tool to investigate diffusion rate (time) constant of selected pharmaceuticals in gel and fish muscle by comparing desorption rate of diffusion of the drugs in both agarose gel prepared with phosphate-buffered saline (PBS; pH 7.4) and fish muscle. The gel concentration (agarose gel model) that could be used to simulate tissue matrix (fish muscle) for free diffusion of drugs under in vitro and in vivo conditions was determined to model mass transfer phenomena between fibre polymer coating and environmental matrix such that partition coefficients and desorption time constant (diffusion coefficient) can be determined. SPME procedure involves preloading the extraction phase (fibre) with the standards from spiked PBS for 1 h via direct extraction. Subsequently, the preloaded fibre is introduced to the sample such fish or agarose gel for specified time ranging from 0.5 to 60 h. Then, fibre is removed at specified time and desorbed in 100 μL of desorption solution (acetonitrile: water 1:1) for 90 min under agitation speed of 1000 rpm. The samples extract were immediately injected to the instrument and analysed using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The limit of detection of the method in gel and fish muscle was 0.01–0.07 ng mL⁻¹ and 0.07–0.34 ng g⁻¹, respectively, while the limit quantification was 0.10–0.20 ng mL⁻¹ in gel samples and 0.40–0.97 ng g⁻¹ in fish sample. The reproducibility of the method was good (5–15% RSD). The results suggest that kinetics of desorption of the compounds in fish tissue and different viscosity of gel can be determined using desorption time constant. In this study, desorption time constant which is directly related to desorption rate (diffusion kinetics) of selected drugs from the fibre to the gel matrix is faster as the viscosity of the gel matrix reduces from 2% (w/v) to 0.8% (w/v). As the concentration of gel reduces, viscosity of the gel will be reduced therefore allowing faster diffusion which invariably affect desorption time constant. Also, desorption time constant of model drugs in the fish muscle and 0.8–0.9% (w/v) gel model are similar based on free diffusion of studied compounds. In addition, in vitro and in vivo desorption time constant comparison shows that desorption time constant in an in vivo system (live fish muscle) is generally higher than an in vitro system (dead fish muscle) except for sertraline and nordiazepam. This study demonstrates SPME as a simple investigative tool to understand kinetics of desorption in an in vivo system with a goal to measure desorption rate of pharmaceuticals in fish.     

Hollow fiber based affinity selection combined with high performance liquid chromatography–mass spectroscopy for rapid screening lipase inhibitors from lotus leaf

A novel kind of immobilized enzyme affinity selection strategy based on hollow fibers has been developed for screening inhibitors from extracts of medicinal plants. Lipases from porcine pancreas were adsorbed onto the surface of polypropylene hollow fibers to form a stable matrix for ligand fishing, which was called hollow fibers based affinity selection (HF-AS). A variety of factors related to binding capability, including enzyme concentration, incubation time, temperature, buffer pH and ion strength, were optimized using a known lipase inhibitor hesperidin. The proposed approach was applied in screening potential lipase bound ligands from extracts of lotus leaf, followed by rapid characterization of active compounds using high performance liquid chromatography–mass spectrometry. Three flavonoids including quercetin-3-O-β-d-arabinopyranosyl-(1 → 2)-β-d-galactopyranoside, quercetin-3-O-β-d-glucuronide and kaempferol-3-O-β-d-glucuronide were identified as lipase inhibitors by the proposed HF-AS approach. Our findings suggested that the hollow fiber-based affinity selection could be a rapid and convenient approach for drug discovery from natural products resources.     

FT-IR measurement of tagitinin C after solvent extraction from Tithonia diversifolia

Tagitinin C, an antiplasmodial compound, identified as one major compound of the subtropical medicinal plant, Tithonia diversifolia, was determined by FT-IR spectroscopy method. The crude ether extracts from aerial parts of the plant were evaporated to dryness and re-dissolved in tetrachloroethylene (C₂Cl₄) before analysis.The magnitude of the absorbance of the very specific CO stretching vibration (ν_CO) at 1664.8 cm⁻¹ was exploited in order to quantify tagitinin C. The determination coefficient (r²) of the calibration scale was 0.9994, the detection limit was lower than 3 μg ml⁻¹ and the quantification limit was lower than 10 μg ml⁻¹.Recovery values from 100.5 to 101.7% were found for spiked concentration levels from 19.91 to 89.95 μg ml⁻¹. The main characteristics of the curves obtained from the calibration standards and from the standard addition technique were not statistically different (Student t-test) suggesting that matrix effects were negligible.The results obtained for the determination of tagitinin C in the crude ether extract from aerial parts of T. diversifolia by LC and FT-IR spectroscopic method agreed well: 0.76±0.02 and 0.773±0.009, of tagitinin C in dried plant respectively.     

Americium and plutonium separation by extraction chromatography for determination by accelerator mass spectrometry

A simple method was developed to separate Pu and Am using single column extraction chromatography employing N,N,N′,N′-tetra-n-octyldiglycolamide (DGA) resin. Isotope dilution measurements of Am and Pu were performed using accelerator mass spectrometry (AMS) and alpha spectrometry. For maximum adsorption Pu was stabilized in the tetra valent oxidation state in 8 M HNO₃ with 0.05 M NaNO₂ before loading the sample onto the resin. Am(III) was adsorbed also onto the resin from concentrated HNO₃, and desorbed with 0.1 M HCl while keeping the Pu adsorbed. The on-column reduction of Pu(IV) to Pu(III) with 0.02 M TiCl₃ facilitated the complete desorption of Pu. Interferences (e.g. Ca²⁺, Fe³⁺) were washed off from the resin bed with excess HNO₃. Using NdF_3, micro-precipitates of the separated isotopes were prepared for analysis by both AMS and alpha spectrometry. The recovery was 97.7 ± 5.3% and 95.5 ± 4.6% for ²⁴¹Am and ²⁴²Pu respectively in reagents without a matrix. The recoveries of the same isotopes were 99.1 ± 6.0 and 96.8 ± 5.3% respectively in garden soil. The robustness of the method was validated using certified reference materials (IAEA 384 and IAEA 385). The measurements agree with the certified values over a range of about 1–100 Bq kg⁻¹. The single column separation of Pu and Am saves reagents, separation time, and cost.     

Characterization and evaluation of a macroporous adsorbent for possible use in the expanded bed adsorption of flavonoids from Ginkgo biloba L.

The suitability of the use of macroporous adsorbent Amberlite XAD7HP in expanded bed adsorption processes for the isolation of flavonoids from crude extracts of Ginkgo biloba L. has been assessed. The expansion and hydrodynamic properties of expanded beds were investigated and analyzed. The bed expansion as a function of operational fluid velocity was measured and correlated with the Richardson–Zaki equation. Theoretical predictions of the correlation parameters (the terminal settling velocity u_t and exponent n) were improved by modifying equations in the literature. Residence time distributions (RTDs) were studied using acetone as a tracer. Three measures of liquid phase dispersion (the height equivalent of theoretical plate, Bodenstein number and axial distribution coefficient) were investigated and compared to values previously obtained with commercial EBA adsorbents developed for protein purification. A suitable bed expansion ratio was found to be 1.25 times the settled bed height, which occurred at a corresponding flow velocity of 183 cm/h. For an initial settled bed height of 42 cm, the mean residence time of liquid in the expanded bed was around 28 min. Under these flow conditions, the axial mixing coefficient D_ax was 7.54 × 10⁻⁶ m²/s and the Bodenstein number was 28; the number of theoretical plates (N) was 19 and the height equivalent of a theoretical plate (HETP) was 2.77 cm. Rutin trihydrate was used as a model flavonoid for the characterization of the adsorption properties of Amberlite XAD7HP. Adsorption was observed to reach equilibrium within 3 h with 70% of the adsorption capacity being achieved within 30 min. The estimated maximum equilibrium adsorption capacity for rutin was estimated to be 43.0 mg/(g resin) when the results were fitted to Langmuir isotherms. The adsorption performance was not seriously impaired by the physical presence of G. biloba leaf powders. Assessment of the kinetics of the adsorption of rutin revealed that the rate constant for adsorption was only reduced by 15% in the presence of leaf powders at a concentration of 50 mg/mL. The results demonstrated that Amberlite XAD7HP should be suitable for expanded bed adsorption of flavonoids from crude extracts of G. biloba L.     

Contribution of blotting techniques to the study of rapeseeds (Brassica napus L.) lipases

A recent advance in the study of plant lipases involving immunological techniques is presented. In an attempt to characterize lipases of cotyledons from germinating rapeseed seedlings and to investigate an eventual cross-reactivity with animal lipases, we have prepared anti-porcine pancreatic lipase antibodies raised in rabbit. It is shown by enzyme-linked immunosorbent assay and dot-blotting that these antibodies react with lipases in the rapeseed crude extract and in the different cellular fractions obtained by differential centrifugation. Preincubation of the antiserum with the rapeseed crude extract affects the amount of antibodies binding to the porcine pancreatic lipase. We demonstrate immunochemical cross-reactivity between rapeseed and porcine pancreatic lipase. Using the immunoblotting procedure, it is found that antibodies bind specifically to a single polypeptide with a molecular mass of about 55 kDa. Rapeseed lipase activity decreased after immunoprecipitation suggesting that antibodies were bound to some catalytic site residues. We conclude from the data obtained in this study that the two different lipase species present close similarities in amino acid sequence and antigen characteristics.     

Development of a validated liquid chromatographic method for the quality control of Prunellae Spica: Determination of triterpenic acids

A simple and rapid reversed-phase HPLC-UV method was developed for the determination of triterpenic acids in the crude extract of Prunellae Spica. Five triterpenic acids were extracted and isolated from P. Spica as marker compounds for use in the quality control of herbal medicines. Various solvent extraction techniques were evaluated, and the greatest efficiency was observed with sonication in 100% ethanol. Elemental compositions of the five marker compounds were determined by high-resolution mass spectroscopy. The dynamic range of the HPLC-UV method depended on the specific analyte, and acceptable quantitation was obtained between 10 and 250 μg mL⁻¹ for oleanolic acid, between 10 and 300 μg mL⁻¹ for ursolic acid, between 3 and 75 μg mL⁻¹ for 2α,3α,24-trihydroxyolean-12en-28oic acid, between 5 and 100 μg mL⁻¹ for euscaphic acid, and between 5 and 100 μg mL⁻¹ for 2α,3α-dihydroxyurs-12en-28oic acid. The method was deemed satisfactory by inter- and intra-day validation and exhibited both high accuracy and precision (relative standard deviation <9.4%). Overall limits of quantitation and detection were approximately 0.5-2.5 μg mL⁻¹ at a signal-to-noise ratio (S/N) of 3 and were about 3.0-10.0 μg mL⁻¹ at a S/N of 10. In addition, principal component analysis (PCA) was performed on the analytical data of 15 different P. Spica samples in order to classify samples collected from different regions.     

Development of a temperature-responsive agarose-based ion-exchange chromatographic resin

A temperature-responsive ion-exchange resin (ItBA) has been prepared by grafting poly(N-isopropylacrylamide-co-acrylic acid-co-tert-butylacrylamide; ItBA) onto cross-linked agarose. A carboxymethylated ion exchanger (CM) of similar charge density was also prepared. Maximum adsorption capacities (B_max) for lactoferrin at 20 °C and 50 °C were determined for both resins by batch adsorption procedures. Dynamic adsorption and desorption characteristics of the CM and ItBA with lactoferrin were established, as well as the ability of ItBA to selectively adsorb and desorb lactoferrin in the presence of other proteins. With the CM-agarose resin there was no significant difference between the B_max values obtained at 20 °C and 50 °C. However, for the agarose-based ItBA resin the B_max value at 50 °C was almost three times higher than the B_max value at 20 °C. Dynamically, lactoferrin adsorbed to the ItBA packed column at 50 °C with a significant proportion of the adsorbed lactoferrin desorbed by reducing the temperature to 20 °C. In addition, anionic proteins did not adsorb to the ItBA packed column, and did not interfere with the dynamic adsorption/desorption behaviour of lactoferrin. These results indicate that this new temperature-responsive agarose-based ItBA resin has potential for the fractionation of whey proteins, with good selectivity for cationic proteins.     

In situ simultaneous determination the photolysis of multi-component PAHs adsorbed on the leaf surfaces of living Kandelia candel seedlings

A fiber-optic fluorimetry for in situ simultaneous determination of fluorine (Flu), phenanthrene (Phe) and fluoranthene (Fla) adsorbed on the leaf surfaces of Kandelia candel (Kc) seedlings was developed. Experimental results showed that the linear ranges for determination of Flu, Phe and Fla adsorbed on Kc leaves were 35-700, 5-900 and 2-450 ng/spot, respectively. The detection limits for Flu, Phe and Fla were 9.11, 1.65 and 0.90 ng/spot and with the relative standard deviations less than 10.32%, 7.56% and 4.29% (n = 9), respectively. The recovery results for Flu, Phe and Fla adsorbed on Kc leaves were 83.0-91.2, 78.5-88.5 and 91.5-107.3%, respectively. Under the laboratory experimental conditions, the photolysis processes of Flu, Phe and Fla individual and in mixtures adsorbed on the leaf surfaces of living Kc seedlings were studied. Results showed that the photolysis of Flu, Phe and Fla individual and in mixtures adsorbed on the leaf surfaces of Kc seedlings followed first-order kinetics with photolysis rates in the order of Flu > Fla > Phe on the Kc leaves. An antagonistic effect was found when the three polycyclic aromatic hydrocarbons (PAHs) were co-adsorbed on living Kc seedlings. The experimental results also indicated that photolysis was the main transformation pathway for Flu, Phe and Fla both individual and in mixtures adsorbed on Kc leaves, whereas disappearance of the adsorbed Flu, Phe and Fla as a result of volatilization and leaf absorption could be negligible during the experimental period.     

Effect of fermentation conditions in the enzymatic activity and stereoselectivity of crude lipase from Candida rugosa

Different fed-batch cultures of Candida rugosa were carried out using oleic acid as the only carbon source. The crude lipases obtained under several operational conditions and downstream processes showed different catalytic activity and isoenzymes ratio. This fact implied that the performance of the lipase produced could be modulated by using different operational fermentation conditions. These powders were compared with commercial lipase from Sigma (St. Louis, MO) in hydrolysis and synthesis reactions. Especially interesting was the fact that the enantioselectivity of a crude lipase was higher than that observed with commercial lipase in the resolution of recemic Ketoprofen. In addition, response of both lipases in the presence of water was different.     

On-line coupling of counter-current chromatography and macroporous resin chromatography for continuous isolation of arctiin from the fruit of Arctium lappa L.

In this work, we have developed a novel hybrid two-dimensional counter-current chromatography and liquid chromatography (2D CCC × LC) system for the continuous purification of arctiin from crude extract of Arctium lappa. The first dimensional CCC column has been designed to fractionalize crude complex extract into pure arctiin effluent using a one-component organic/salt-containing system, and the second dimensional LC column has been packed with macroporous resin for on-line adsorption, desalination and desorption of arctiin which was effluent purified from the first CCC dimension. Thus, the crude arctiin mixture has been purified efficiently and conveniently by on-line CCC × LC in spite of the use of a salt-containing solvent system in CCC separation. As a result, high purity (more than 97%) of arctiin has been isolated by repeated injections both using the ethyl acetate–8% sodium chloride aqueous solution and butanol–1% sodium chloride aqueous solution. By contrast with the traditional CCC processes using multi-component organic/aqueous solvent systems, the present on-line CCC × LC process only used a one-component organic solvent and thus the solvent is easier to recover and regenerate. All of used solvents such as ethyl acetate, n-butanol and NaCl aqueous solution are low toxicity and environment-friendly. Moreover, the lower phase of salt-containing aqueous solution used as mobile phase, only contained minor organic solvent, which will save much organic solvent in continuous separation. In summary, our results indicated that the on-line hybrid 2D CCC × LC system using one-component organic/salt-containing aqueous solution is very promising and powerful tool for high-throughput purification of arctiin from fruits of A. lappa.     

Direct recovery of recombinant nucleocapsid protein of Nipah virus from unclarified Escherichia coli homogenate using hydrophobic interaction expanded bed adsorption chromatography

A direct recovery of recombinant nucleocapsid protein of Nipah virus (NCp-NiV) from crude Escherichia coli (E. coli) homogenate was developed successfully using a hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC). The nucleic acids co-released with the recombinant protein have increased the viscosity of the E. coli homogenate, thus affected the axial mixing in the EBAC column. Hence, DNase was added to reduce the viscosity of feedstock prior to its loading into the EBAC column packed with the hydrophobic interaction chromatography (HIC) adsorbent. The addition of glycerol to the washing buffer has reduced the volume of washing buffer applied, and thus reduced the loss of the NCp-NiV during the washing stage. The influences of flow velocity, degree of bed expansion and viscosity of mobile phase on the adsorption efficiency of HI-EBAC were studied. The dynamic binding capacity at 10% breakthrough of 3.2 mg/g adsorbent was achieved at a linear flow velocity of 178 cm/h, bed expansion of two and feedstock viscosity of 3.4 mPa s. The adsorbed NCp-NiV was eluted with the buffer containing a step gradient of salt concentration. The purification of hydrophobic NCp-NiV using the HI-EBAC column has recovered 80% of NCp-NiV from unclarified E. coli homogenate with a purification factor of 12.5.     

Purification and partial characterization of CMP-Neu5Ac synthetase from rat brain

N-Acetyl-neuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase), which catalyzes the formation of cytidine-5′-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac) from cytidine-5′-triphosphate (CTP) and N-acetyl-neuraminic acid (Neu5Ac), was purified from rat brains aged 8-9 days, which presented the highest specific activity, and partially characterized. Partial protein fractionation in the crude extract was achieved by using 40-60% ammonium sulphate. Subsequently, CMP-Neu5Ac synthetase was purified by column chromatography on Sephacryl S-200 (gel filtration), Yellow-86-Agarose (affinity) and Phenyl-Sepharose (hydrophobic affinity). The pure enzyme had a specific activity of 3.6555 U/mg of protein and was purified 1662-fold, with an 18% yield. The purified CMP-Neu5Ac synthetase had a molecular weight of about 46 ± 1 kDa. Its purity was confirmed by sodium dodecyl sulphate and polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). The active enzyme chromatographed on a gel filtration column at 190 kDa, suggesting it exists in its native form as a tetramer. The greatest activity of enzyme was observed a temperature of 40 °C for a period of 45 min of incubation, revealing a certain thermal stability. The enzyme was found to remain stable in the pH range 8.5-9.5 at 40 °C, specifically at pH 9.0 for a 45 min incubation period. The enzyme was blocked by thiol-modifying reagents and such heavy metal cations as Mn²⁺, Cu²⁺, Sn²⁺, Co²⁺, Zn²⁺ and Hg²⁺, but was not inhibited by thiol-containing reagents like reduced glutathione (GSH), mercaptoethanol and cysteine. Finally, in the presence of 0.01 M of dithiothreitol (DTT) or 0.06 M of NaF, the enzyme showed activity losses of approximately 20 and 17%, respectively.     

Isolation of a polysaccharide with anticancer activity from Auricularia polytricha using high-speed countercurrent chromatography with an aqueous two-phase system

Polysaccharides from a crude extract of Auricularia polytricha were separated by high-speed countercurrent chromatography (HSCCC). The separation was performed with an aqueous two-phase system of PEG1000–K₂HPO₄–KH₂PO₄–H₂O (0.5:1.25:1.25:7.0, w/w). The crude sample (2.0 g) was successfully separated into three polysaccharide components of AAPS-1 (192 mg), AAPS-2 (137 mg), and AAPS-3 (98 mg) with molecular weights of 162, 259, and 483 kDa, respectively. These compounds were tested for growth inhibition of transplanted S180 sarcoma in mice. AAPS-2 had an inhibition rate of 40.4%. The structure of AAPS-2 was elucidated from partial hydrolysis, periodate oxidation, acetylation, methylation analysis, and NMR spectroscopy (¹H, ¹³C). These results showed AAPS-2 is a polysaccharide with a backbone of (1 → 3)-linked-β-d-glucopyranosyl and (1 → 3, 6)-linked-β-d-glucopyranosyl residues in a 2:1 ratio, and has one terminal (1→)-β-d-glucopyranosyl at the O-6 position of (1→3, 6)-linked-β-d-glucopyranosyl of the main chain.     

Development and validation of a capillary zone electrophoresis method for the determination of propranolol and N-desisopropylpropranolol in human urine

A simple, rapid and sensitive procedure using solid phase extraction and capillary zone electrophoresis for the determination of propranolol (a beta-blocker) and one of its metabolites, N-desisopropylpropranolol, has been developed and validated. The optimum separation of both analytes was obtained in a 37 cm × 75 μm fused silica capillary using 20 mmol/L phosphate buffer (pH 2.2) as electrolyte, at 25 kV and 30 °C, and hydrodynamic injection for 5 s. Prior to the electrophoretic separation, the samples were cleaned up and concentrated using a C₁₈ cartridge and then, eluted with methanol, allowing a concentration factor of 30.Good results were obtained in terms of precision, accuracy and linearity. The limits of detection were 28 and 30 μg/L for N-desisopropylpropranolol and propranolol, respectively. Additionally, a robustness test of the method was carried out using the Plackett-Burman fractional factorial model with a matrix of 15 experiments.The presented method has been applied to the determination of both compounds in human urine.     

In Silico and Experimental Characterization of Chimeric Bacillus thermocatenulatus Lipase with the Complete Conserved Pentapeptide of Candida rugosa Lipase

Lipases are one of the highest value commercial enzymes as they have broad applications in detergent, food, pharmaceutical, and dairy industries. To provide chimeric Bacillus thermocatenulatus lipase (BTL2), the completely conserved pentapeptide (¹¹²Ala-His-Ser-Gln-Gly¹¹⁶) was replaced with similar sequences (²⁰⁷Gly-Glu-Ser-Ala-Gly²¹¹) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. For this purpose, three mutations including A112G, H113E, and Q115A were inserted in the conserved pentapeptide sequence of btl2 gene. Based on the crystal structures of 2W22, the best structure of opened form of the chimeric lipases were garnered using the MODELLER v9.10 software. The native and chimeric lipases were docked to a set of ligands, and a trial version of Molegro Virtual Docker (MVD) software was used to obtain the energy values. Docking results confirmed chimeric lipase to be better than the native lipase. Following the in silico study, cloning experiments were conducted and expression of native and chimeric btl2 gene in Pichia pastoris was performed. The native and chimeric lipases were purified, and the effect of these mutations on characteristics of chimeric lipase studied and then compared with those of native lipase. Chimeric lipase exhibited 1.6-fold higher activity than the native lipase at 55 °C. The highest percentage of both lipases activity was observed at 60 °C and pH of 8.0. The ion Ca²⁺ slightly inhibited the activity of both lipases, whereas the organic solvent enhanced the lipase stability of chimeric lipase as compared with the native lipase. According to the results, the presence of two glycine residues at the conserved pentapeptide region of this chimeric lipase (¹¹² Gly-Glu-Ser-Ala-Gly ¹¹⁶) may increase the flexibility of the nucleophilic elbow region and affect the enzyme activity level.     

Comparative study of biosurfactant produced by microorganisms isolated from formation water of petroleum reservoir

Biosurfactant produced by Pseudomonas aeruginosa, Bacillus subtilis and Rhodococcus erythropolis that isolated from the formation water of Chinese petroleum reservoir has been compared in surface abilities and oil recovery. Maximum biosurfactant production reached to about 2.66 g/l and the surface tension of liquid decreased from 71.2 to 22.56 mN/m using P. aeruginosa. Three strains exhibited a good ability to emulsify the crude oil, and biosurfactant of P. aeruginosa attained an emulsion index of 80% for crude oil which was greater than other strains. Stability studies were carried out under the extreme environmental conditions, such as high temperature, pH, salinity and metal ions. Results showed an excellent resistance of all biosurfactants to retain their surface-active properties at extreme conditions. It was found that the biosurfactants from three isolated bacteria showed a good stability above pH of 5, but at lower pH (from 1 to 5) they will harmfully be affected. They were able to support the condition up to 20 g/l salinity. P. aeruginosa biosurfactant was even stable at the higher salinity. Regarding temperature, all produced biosurfactants demonstrated a good stability in the temperature up to 120 °C. But stability of three biosurfactants was affected by monovalent and trivalent ions. Oil recovery experiments in physical simulation showed 7.2-14.3% recovery of residual oil after water flooding when the biosurfactant of three strains was added. These results suggest that biosurfactants of these indigenous isolated strains are appropriate candidates for enhanced oil recovery with a preference to biosurfactant of P. aeruginosa.     

Development of electrospun poly(vinyl alcohol) fibers immobilizing lipase highly activated by alkyl-silicate for flow-through reactors

Electrospun fibrous membranes composed of poly(vinyl alcohol) (PVA) fibers of approximately 1 μm in diameter, and immobilizing highly activated lipase entrapped in silicate cages with smaller dimensions than the fibers, were developed; and their feasibility as a component of flow-through reactors was studied. The electrospun PVA fibers were prepared from a mixture of PVA solution and a sol obtained from silicon alkoxide(s)—either tetramethoxysilane (TMOS) or dimethyldimethoxysilane (DMDMOS), or both, containing lipase. The fastest initial transesterification rate converting (s)-glycidol to glycidyl n-butyrate with vinyl n-butyrate in batchwise reactions was accomplished by treatment of lipase using the sol obtained from DMDMOS and TMOS together. The values were 4.5-, 21.8-, and 1.8-fold faster than those of systems using lipases that were either non-modified or modified using TMOS alone or DMDMOS alone, respectively. The higher activity of the lipase modified using both DMDMOS and TMOS and immobilized in PVA fibers resulted in a flow-through reactor having a higher degree of conversion at the same retention time compared with that using immobilized non-modified lipase. These results show the feasibility of flow-through reactors composed of electrospun PVA fibers immobilizing lipase highly activated by alkyl-silicate.