Application of response surface methodology to optimization of the fixation of ribonuclease a on glutaraldehyde activated amine silica

Summary We have used response surface methodology to study the interactions between various parameters (pH, temperature, enzyme concentration) controlling the immobilization of ribonuclease A on to glutaraldehyde activated-amine Spherosil beads. The optimum activity of immobilized ribonuclease A was observed at pH=9.1. The specific activity of the immobilized enzyme was affected by the pH and by the enzyme concentration.     

Production of L-lysine by immobilized trypsin. Study of DL-lysine methyl ester resolution

The possibility of producing L-lysine from chemically synthesized DL-lysine has been investigated. Optical resolution of racemic DK-lysine may be achieved by using the stereospecific esterasic activity of trypsin on DL-lysine methyl ester, which gives L-lysine and unchanged D-lysine methyl ester. SL-lysine methyl ester spontaneous hydrolysis may be neglected when operating at pH 5.5 and 30 degrees C. Effect of pH and substrate concentration on hydrolysis rate has been investigated when using as a catalyst either soluble or immobilized trypsin. For this purpose, trypsin was coupled onto an amine porous silica, Spherosil, activated with glutaraldehyde. The optimal pH is 5.8 for soluble trypsin and 6.0 for immobilized trypsin. It was yet possible to lower the parent optimal pH of immobilized trypsin, and thus increase its activity at 5.5, by co-grafting onto Spherosil an aminosilane, for enzyme coupling via glutaraldehyde activation and a positively charged diethyl amino ethyl (DEAE) silane, for decreasing the pH of trypsin microenvironment.     

High-performance liquid chromatographic determination of pipotiazine in human plasma and urine

A high-performance liquid chromatographic method has been developed for the determination of pipotiazine in human plasma and urine. After selective extraction, pipotiazine and the internal standard (7-methoxypipotiazine) are chromatographed on a column packed with Spherosil XOA 600 (5 μm) using a 7:3 (v/v) mixture of diisopropyl ether—isooctane (1:1, v/v) + 0.2% triethylamine and diisopropyl ether—methanol (1:1, v/v) + 0.2% triethylamine + 2.6% water. The eluted compounds are measured by fluorescence detection. The sensitivity of the method was established at 0.25 ng/ml pipotiazine in plasma and 2 ng/ml pipotiazine in urine (C.V. < 5%). The method has been successfully applied to a pharmacokinetic study following a single oral administration of 10 mg of pipotiazine.     

Selective adsorption of flavonoid compounds from the leaf extract of Ginkgo bilobaL.

A simple purification method has been developed in which flavonoid compounds are selectively adsorbed onto a polycarboxyl ester resin (XAD-7) from methanol extract of ginkgo leaves. When 1.0 g dried gingko leaves was extracted with 100 ml of methanol, about 98% of total flavonoid compounds was recovered by selective adsorption. The pH did not affect the adsorption of flavonoids but at high pH the chemical structure of flavonoids was changed and the adsorption decreased to almost zero. As the solution polarity played a key role in the selective adsorption, the amount of water added to the methanol extract was critical for the recovery and loading of flavonoids. Using standard flavonoids, kaempferol and quercetin, the optimal water content was 80% and the recovery was 80% with 10 g of resin dosage/l.     

Application of response surface methodology to optimization of glutaraldehyde activation of a support for enzyme immobilization

Summary We have studied the influence of several parameters (glutaraldehyde concentration, pH and contact time) on the activation of an amine silica with glutaraldehyde. In order to take interactions between parameters into consideration, we have used the Response Surface Methodology which allows us to obtain an equation (not a single estimation). The optimal conditions for glutaraldehyde activation of amine Spherosil beads were as follow: the porous silica was activated for 1 h at 25°C with an 8% (V/V) solution of glutaraldehyde at pH 6.4 these conditions were the same with RNA or CMP used as substrates.     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.     

Influence of the conditions of trypsin immobilization onto Spherosil on coupling efficiency

Summary The effect of several parameters (pH, time of reaction, temperature, enzyme concentration) on trypsin immobilization onto glutaraldehyde-activated amine-Spherosil was investigated. This activated support could be stored over long periods of time without any important loss of capacity for trypsin coupling. When increasing the amount of trypsin bound to the carrier, enzymatic activity shows an optimal value, beyond which an augmentation of Spherosil enzyme content results in a lowered activity. The influence of the number of available reactive aldehyde groups on silica was investigated by coupling L-lysine to activated support either prior to or simulataneously with trypsin immobilization. In both cases, the activity of trypsin derivatives is decreased when L-lysine concentration is increased, yet the activity of trypsin derivatives is never equal to zero, even in presence of a large excess of L-lysine. This suggests the presence of two types of reactive groups on the activated support.     

Preparation of a monoclonal antibody to common amino acid sequence of LHRH and its application

In order to prepare an antibody directed at the common amino acid sequence of mammalian, avian, and fish luteinizing hormone-releasing hormones (LHRHs), C-terminal free LHRH was conjugated with bovine thyroglobulin, and was used as the antigen. A monoclonal antibody (LRH13) was obtained as an ascitic fluid by fusing the spleen cells of a BALB/c donor mouse immunized with the antigen to X63.Ag8.653 mouse myeloma cells followed by limiting dilution cloning and transplanting a positive clone to BALB/c mice. This monoclonal antibody seems to belong to IgG2b as it was eluted from protein A-Sepharose CL-4B with citrate buffer pH 3.5. competitive binding experiment using fragment peptides of LHRH indicated the binding site of LRH13 was a region around serine and tyrosine, and modification of mammalian LHRH by radioiodination caused a marked decrease in the binding activity. LRH13 has an affinity constant of 0.134 X 10(9) M-1 to native mammalian LHRH, and binds C-terminal free LHRH with a similar affinity (1.6X), however, it binds with higher affinities to N- and C-terminal free LHRH (12.9X), N-terminal free LHRH (10.4X), salmon LHRH (8.3X) and chicken LHRH-I (6.0X). Chicken LHRH-II, where tyrosine is replaced for histidine, has a lower affinity (0.3X) than that of mammalian LHRH. From its high affinity to N-, C-terminal free LHRH, LRH13 is also expected to bind possible precursor peptides of LHRH. Immunohistochemical staining of the brain sections obtained from rats, mice, chickens, Japanese quail, and rainbow trout successfully visualized cell bodies and fibers distributed from the olfactory bulb to the median eminence, indicating high LHRH specificity and wide crossreactivity in animal classes of this monoclonal antibody. With this antibody, LHRH-like immunoreactive substance in the pineal gland was also stained with fixation at neutral pH.     

Rapid determination of zearalenone in edible oils by HPLC with fluorescence detection

A fast, cost-efficient, sensitive and accurate assay method for zearalenone in edible oils is described, as an alternative to gel permeation chromatography (GPC). Oil samples were extracted with an alkaline mixture of methanol and water (methanol +10 g/l aqueous ammonium carbaminate solution, pH 9; 9 + 1, v+v). The pH of the extract was neutralized with hydrochloric acid and then concentrated to dryness. The residue was dissolved with HPLC solvent, and zearalenone was determined by high-performance liquid chromatography with fluorometric detection (HPLC-FLD). The method was successfully validated for two matrices, maize oil and rapeseed oil. The recovery rate was 87%, and the coefficient of variation was 2.8% in a rapeseed oil sample contaminated with 27 μg zearalenone/kg. At a signal-to-noise ratio of 3:1, the method detection limit was 10 μg/kg, which was considered to be adequate in view of the present European Union maximum level of 400 μg/kg.     

Short term kinetics of luteinizing hormone secretion studied in dissociated pituitary cells attached to manipulable substrates

With the use of poly-L-lysine, a method has been developed which induces acutely dissociated rat anterior pituitary cells to attach to glass and polyacrylamide surfaces. In these attached cells the recovery of the secretory response, which is impaired in acutely dissociated cells, has been followed, and it has been established that, in terms of their ability to secrete luteinizing hormone (LH) in response to the specific secretogogue luteinizing-hormone-releasing hormone (LHRH), the cells become maximally responsive after 48 h. The attached cells also allow the short-term kinetics of LH secretion to be followed with great facility; and, when cells allowed to recover for 48 h are used, it is shown that in response to LHRH the pattern of LH release is biphasic.     

Alterations in hypothalamic content of luteinizing hormone-releasing hormone associated with pineal-mediated testicular regression in the golden hamster

The adult male golden hamster will undergo testicular regression when exposed to a short photoperiod, blinding, or late afternoon injections of melatonin. The present study was conducted to compare the effects of all three treatments on serum gonadotropin levels and testicular weights, and to evaluate the effects of these treatments on hypothalamic content of both immunoreactive and bioactive luteinizing hormone-releasing hormone (LHRH) levels. Hamsters were blinded (BL), exposed to a short photoperiod (SP), or received daily injections of melatonin (MEL) for 15 wk. Each treatment (BL, SP, MEL) induced a temporally similar decline in serum luteinizing hormone (LH), serum follicle-stimulating hormone (FSH), and testicular weight. Spontaneous recrudescence occurred earliest in the MEL group, with serum gonadotropins and testicular weight returning to normal by 15 wk. The SP group exhibited recovery of serum gonadotropins but not testicular weight by 15 wk. The BL group demonstrated partial recovery of serum FSH levels by 15 wk, with no recovery in either serum LH or testicular weight. Each treatment group demonstrated increased hypothalamic content of immunoreactive LHRH which was temporally correlated with the decreases of serum gonadotropins. Additionally, the MEL and SP groups demonstrated decreased immunoreactive LHRH levels during spontaneous recrudescence. Extracts of hypothalami from all treatment groups were bioactive on control hamster pituitary cells. These results indicate that there are temporal differences among the three common treatments and that these differences are manifested in serum gonadotropins, testicular weight and hypothalamic LHRH. Hypothalamic LHRH levels determined by radioimmunoassay and bioassay show periods of increase and decrease which coincide with periods of altered serum gonadotropin levels in all groups.     

Isocratic liquid chromatographic assay for monitoring the degradation of luteinizing hormone releasing hormone by extracts from the gastrointestinal tract of possums

A simple HPLC method to separate human luteinizing hormone releasing hormone (LHRH) from its metabolites using an isocratic elution is described. Intact LHRH and five metabolites were separated in 11.4 min. The calibration curve (peak area versus concentration) was linear over the concentration range 1.25-35 microg/ml (r(2)=0.99) with the intercept not significantly different from zero (P>0.05). Intra-day and inter-day variability of the assay was less than 5% for repeat injections of 5, 14.5 and 29 microg/ml. The method was applied to evaluate the susceptibility of LHRH to enzymes present in the lumen and mucosal extracts of the gastrointestinal tract of possums. The major degradation products of LHRH were identified by HPLC separation, amino acid analysis and mass spectrometry as LHRH (1-5), LHRH (1-4), LHRH (1-3) and LHRH (3-4).     

Receptor-specific large-scale purification of cholera toxin on silica beads derivatized with lysoGM1 ganglioside

1. A receptor-specific affinity chromatographic method for large-scale purification of cholera toxin is described. The receptor ganglioside for cholera toxin, GM1, is hydrolysed to lysoGM1 which is then covalently coupled, via stabilized Schiff's bases, to porous silica beads (Spherosil) onto which a layer of DEAE-dextran has been adsorbed and cross-linked before coupling. Columns of these Spherosil-DEAE-dextran-lysoGM1 beads, in contrast to particles derivatized with lysoGA1, bound the cholera toxin of Vibrio cholerae culture filtrates, after which the toxin could be eluted with the aid of an acid citrate buffer (pH 2.8). 2. The toxin-binding capacity was directly proportional to the amount of lysoGM1 in the column: 2.3 mg/mu mol lysoGM1. The yield of purified toxin after acid elution and pH neutralization was essentially quantitative (83-107%). 3. The affinity-purified toxin contained less than 5% impurities, but consisted of a mixture of predominantly intact holotoxin and B subunit protomer which could readily be separated by gel filtration on Sephadex G-100. 4. Scaling up of the technique was possible: a 1 kg column enabled us to treat 1000-1 cultures of V. cholerae and thus to isolate 20 g of cholera toxin per cycle.     

Quantitative high-performance liquid chromatographic determinations of aminopyrine and its metabolites in man

A quantitative high-performance liquid chromatographic method, using a polystyrene—divinyl benzene (Hitachi No. 3010 gel) column and aqueous methanol as the mobile phase, was employed for the determination of aminopyrine and its related compounds, 4-acetylaminoantipyrine, 4-aminoantipyrine and 4-monomethylaminoantipyrine. Baseline separation could be achieved within 25 min. The method was applied to the recovery of these materials from control urine and human urine. Before separation human urine was adjusted to pH 9 and extracted with ethyl acetate, chloroform and diethyl ether.     

Highly active immobilized hydrogenase from Desulfovibrio gigas

The hydrogenase from the sulfate reducer $\text{Desulfovibrio gigas}$ has been immobilized by covalent coupling onto a porous silica support. Two methods have been used: glutaraldehyde activation of aliphatic amino Spherosil and diazotation of aromatic amino Spherosil. The effect of cytochrome C₃ and CC₃ addition during coupling has been investigated. The highest enzymatic activity (4440 U/g support) and immobilization yield (29 %) was obtained when coupling hydrogenase in the presence of cytochrome C₃ or CC₃ with diazotized aromatic amino silica. This immobilized hydrogenase preparation which shows a very good resistance to oxygen inactivation seems suitable for hydrogen photoproduction by coupling with illuminated chloroplasts.     

利用反相高效液相色谱测定香椿叶中槲皮素含量

用乙醇对香椿叶粉末进行提取,树脂柱浓缩,真空冷冻干燥,甲醇溶解,制备香椿黄酮。用7.6 mol.L-1盐酸85℃水浴回流2 h进行水解,以反相ODS柱甲醇-水(体积比为50:50,磷酸调pH至2.53)为流动相,在波长368 nm处对香椿叶中的槲皮素进行分离、测定。结果表明:香椿叶中含槲皮素平均质量分数为1.28%,平均加样回收率为98.9%,RSD为1.75%,反相高效液相色谱法测定香椿叶中槲皮素含量操作简便易行、结果准确可靠。     

Gonadotropin-releasing hormone release from the mediobasal hypothalamus of fetal baboons

Pulsatile luteinizing-hormone releasing hormone (LHRH) secretion was measured from the mediobasal-suprachiasmatic-preoptic (MBH-SCN-POA) region of the hypothalamus from fetal baboons (Papio anubis) at midgestation (day 100; term = day 184). The entire MBH-SCN-POA (48 ± 5 mg) was obtained between 1100 and 1200 hours and was immediately placed in icecold phosphate buffer (pH 7.4). The MBH-SCN-POA units were halved at the midline and superfused in parallel at 37.5°C for 5 hours. Then 500 μl of superfusate was collected at 10-minute intervals, and LHRH concentration was measured by radioimmunoassay using the Chen-Ramirez antibody. In fetuses of untreated baboons (N = 3), LHRH pulse amplitude (mean ± SE) was 16.0 ± 4.2 pg, with a period of 30 ± 1 minute; the average 10-minute output of LHRH was 9.4 ± 2.0 pg. In fetal baboons in which the hormonal milieu in the mother was modulated by androstenedione treatment of midpregnancy (N = 3), average LHRH pulse amplitude was 1.7 ± 0.3 pg, with a period of 33.5 ± 4.9 minutes; the average 10-minute output of LHRH was 1.2 ± 0.2 pg. Collectively, these data suggest that as early as midgestation, fetal baboons secrete LHRH in vitro in a pulsatile fashion and with a periodicity of 30–35 minutes. In addition, the decrease in LHRH pulse amplitude and the average 10-minute LHRH output (P < .01, P < .05) in tissue from fetal baboons of mothers in which the normal pattern of steroidogenesis is altered suggest that the output of LHRH systems in the fetus is sensitive to changes in the maternal hormonal milieu.     

Rapid assay for in vitro degradation of luteinizing hormone releasing hormone

A radiochemical method for measuring luteinizing hormone releasing hormone (LHRH) degrading enzymatic activity in vitro was developed using LHRH labeled at the N-terminal 5-pyrrolidone-2-carboxylic acid (<Glu) residue. The intact labeled peptide is separated from the labeled fragments formed by cleavage by a cation-exchange batchwise procedure. The assay reflects the degradation of LHRH specifically in terms of inactivation of hormonal activity, is more rapid than a radioimmunoassay, is independent of LHRH concentration, and is not influenced by high protein concentrations. It can be used for studying the degradation of LHRH by subcellular fractions and enzymes. With this assay a highly active enzymatic degradation system was detected in the rat ovary, a recently discovered target organ for LHRH.     

Characteristics of immobilized thermostable amylases from two Bacillus licheniformis strains

Covalent immobilization of thermostable α-amylases from catabolite resistant and sensitive Bacillus licheniformis strains on controlled pore glass (CPG) and porous silica (Spherosil) beads and ionic binding on DEAE-cellulose, Amberlite and Dowex were investigated. Preparations with satisfactory operational stabilities and activities up to 1,600 U/g of support (ionic binding) and 800 U/g carrier (covalent coupling) were obtained. Immobilization led to a narrowing of the pH interval of maximum activity. The fixed amylases were stable in limited pH regions around the optimum pH level. An enhancement of the enzyme thermostability was observed. Apparent shifts of the optimum temperatures were not found. The apparent V_max decreased up to 80 times. The K_m′ remained unchanged (for amylopectin as the substrate) or increased up to 10 times (soluble starch). Maltose, maltotriose and maltopentaose were the main products of the hydrolysis. A significant increase in maltopentaose content was observed.     

Supercritical fluid disruption of Ralstonia eutropha for poly(beta-hydroxybutyrate) recovery

A new method for disruption of Gram-negative bacterium Ralstonia eutropha by supercritical CO(2) for poly(beta-hydroxybutyrate) (PHB) recovery is proposed. The effects of different parameters such as exposure time, pressure, temperature, volume of methanol as a modifier, and culture history on cell disruption efficiency were investigated using Taguchi's statistical approach to determine optimum conditions. The optimum conditions for cell disruption and PHB recovery were as follows: exposure time, 100 min; pressure, 200 atm; temperature, 40 degrees C; volume of methanol, 0.2 mL. The cell culture time was less significant. At optimum conditions, the maximum efficiency of PHB recovery was found to be 89%. The proposed method is comparable with other recovery methods in terms of the percentage of PHB recovery, while it is environmentally more benign.