从火棘果中提取精氨酸的研究

以天然野生植物火棘果为原料分离提取精氨酸,考察了树脂类型、吸附方式、温度、pH值、洗脱液、脱色及精制方法等因素对提取精氨酸的影响,并对产物进行了分析鉴定。结果表明,在20℃、pH值7-8的条件下,选用D001型大孔阳离子交换树脂静态吸附,40 min即达到饱和;以3 mol.L-1氨水为洗脱液,流速控制在40 mL.h-1,精氨酸的洗脱率达到90%以上;采用711型阴离子树脂进行脱色,D001型大孔阳离子交换树脂动态精制,精氨酸的提取率达到95.83%。     

Elution and Inactivation of Bacteriophages on Soil and Cation-Exchange Resin

A variety of elution schemes was tested to determine the most effective procedure for eluting Arthrobacter bacteriophages from soil. A buffer solution of pH 8.0 was found to be the most satisfactory eluent. Bacteriophages were adsorbed to cation-exchange sites on soils, clays, and Dowex-50 resin and eluted. Eluted bacteriophages were detected by passive hemagglutination and plaque assay. Although bacteriophage antigen was successfully eluted, most recovered bacteriophages were noninfective. Inactivation was greatest in bacteriophages of Bradley group B and was associated with the elution process. Inactivation is believed to be caused by physical damage to bacteriophage tails upon elution. The significance of this inactivation relative to bacteriophage models for vertebrate viruses is discussed.     

Simultaneous analysis of homocysteine and methionine in plasma

The new isocratic cation exchange method separates up to eight different amino thiols. The separated sample components are detected electrochemically using a gold electrode and the integrated pulsed amperometry. The eluent composition is, for example, 0.15 M sodium perchlorate, 0.02 M perchloric acid and 5% acetonitrile. The report describes the optimization of chromatographic parameters such as column diameter and eluent composition. Quantitative performance is discussed for eight different amino thiols using standards. Also presented is a long term quantitative study for homocysteine and methionine in plasma samples. The preparation of plasma samples is simpler than with the previously reported version of the method. Only a reduction step is required, and neither column switching nor derivatization are necessary.     

大孔树脂分离纯化辣椒叶总黄酮的工艺研究

目的:筛选适合分离纯化辣椒叶总黄酮的一种大孔树脂,同时用响应面法进行优化得到最佳纯化工艺。方法:采用热回流法提取辣椒叶总黄酮,以吸附率和解吸率为考察指标,考察6种不同型号的大孔树脂(HPD100、HPD450、HPD600、HPD826、D101、AB-8)对辣椒叶总黄酮的吸附能力与解吸能力,确定最佳树脂。通过动态吸附解吸实验考察此树脂对辣椒叶总黄酮的最佳分离纯化工艺。结果:通过对辣椒叶总黄酮吸附分离性能的分析显示HPD600为最佳树脂,最优工艺为:上样浓度为10 mg/mL,上样量为10 mL,洗脱体积为4 BV,洗脱液流速为4 mL/min,洗脱液pH为7,依次用水、10%、30%乙醇冲洗树脂柱,50%乙醇为洗脱液。纯化后的黄酮纯度435.4 mg/g。结论:该方法简便,操作简单,对辣椒叶总黄酮的纯化效果较好。     

大孔树脂纯化赶黄草黄酮工艺及体外抗氧化活性

本研究以赶黄草地上部分为材料,研究大孔树脂纯化赶黄草黄酮的工艺,并评价体外抗氧化活性。根据大孔树脂对赶黄草黄酮的吸附和解吸性能,从7种不同类型的大孔树脂中筛选出适宜的树脂,进一步优化其纯化工艺,并比较纯化前后黄酮的体外抗氧化活性。试验结果表明,DM130大孔树脂对赶黄草黄酮有较好的吸附和解吸效果,其最佳纯化工艺参数:上样液黄酮浓度为1.0 mg/mL、pH为5、上样速度为1.0 mL/min、上样量为110 mL、洗脱液为70%乙醇、洗脱速度为1.0 mL/min和洗脱体积为40 mL。该工艺条件下,黄酮的纯度由20.04%提高至43.93%,提高了23.89%,表明DM130树脂对赶黄草黄酮的纯化效果较好。另外,纯化后赶黄草黄酮的DPPH自由基清除能力和还原力均显著提高。     

High-performance anion-exchange chromatography of ultraviolet-absorbing constituents of human urine

A 100-μl urine sample was chromatographed on a column packed with a strongly basic macroreticular anion-exchange resin (Diaion CDR-10, 5– μm diameter with a nominal 35% cross linkage). The elution was performed with a linear acetate gradient from 0 to 6.0 M at an average flow-rate of 0.72 ml/min and at an average pressure of 104 kg/cm². The relative standard deviation of retention times and peak height was ± 4% or less. The properties of the macroreticular anion-exchange resin, the effect of the particle size, the pH of acetate buffers, and the effect of the flow-rate of the eluent on the separation were investigated. Thirty three components of urine were then resolved and named.     

Separation of urinary thiols as tributyltinmercaptides and determination using capillary isotachophoresis

The essential steps in the assay included electrolytic reduction of disulphides, neutralization, extraction of thiols with 0.1 M tributyltin hydroxide in octane, stripping of the extract with 2% acetic acid, fixing the washed-out amino thiols to a cation exchanger, elution with 2 M hydrochloric acid, oxidation with bromine and evaporation. The remaining octane extract was decomposed by dodecanethiol, the mercapto acids were washed out, oxidized with bromine and evaporated. Both residues were dissolved in water and analysed using capillary isotachophoresis at pH 3.0. Cysteamine was extracted from reduced urine at ca. pH 10, decomposed by dodecanethiol and re-extracted to boric acid followed by determination as a cation. The presence of the following thiols in urine has been confirmed: mercaptoacetic acid, 3-mercaptolactic acid, 2-mercaptopropionic acid, acetylcysteine, mercaptoethanol, cysteine, homocysteine and an un-identified amino thiol. Cysteamine and 3-mercaptopropionic acid could not be detected. Captopril, homocysteine and cysteine were determined quantitatively.     

Increased sensitivity and discrimination in screening through an immobilized-resin microbiological assay method

Summary Problems with present bioactive microbial product screening techniques include low sensitivity and insufficient discrimination capabilities. These problems are addressed by our new immobilized-resin microbiological assay. This technique concentrates bioactive samples on macroporous polymeric resins that are immobilized in hydrogel beads. These beads are then subjected to elution in the wells of an agar diffusion microbiological assay medium. With a strong base anion exchanger, the sensitivity to ampicillin of the -lactam-supersensitiveEscherichia coli mutant ESS-22-31 was increased 10-fold. Similar increases in sensitivity were obtained in the detection of streptomycin using a weak acid cation exchanger withBacillus subtilis and for cycloheximide by a neutral resin andSaccharomyces cerevisiae NRRL-Y-139. A judicious choice of resin type and eluent permitted a selective sensitivity increase based on the charge or hydrophobic nature of the desired product. This selectivity imparts a discrimination capability to the technique.     

大孔树脂分离纯化有柄石韦总黄酮的工艺优化

以总黄酮解吸率和滤液中总黄酮含量为指标,通过正交实验设计优选大孔树脂分离纯化有柄石韦总黄酮的最佳工艺。结果表明,D-101型大孔树脂分离纯化总黄酮的优选工艺条件为：上样液质量浓度0.5 mg·mL^-1,pH为6,洗脱剂乙醇浓度为50%,洗脱速率2 mL·min^-1。该优化方法稳定可行,结果可靠,提取的滤液中总黄酮含量达76.40%。     

Formation of proline metabolites in chick embryo bone: Interference with the measurement of free hydroxyproline by ion-exchange chromatography

Metabolites of -[¹⁴C]proline were found in the trichloroacetic acid-soluble fraction of 16-day-old chick embryo frontal bones. In several ion-exchange procedures these metabolites interfered with the analysis of hydroxyproline derived from the metabolic breakdown of collagen. The major metabolite was identified as glutamic acid by its chromatographic and crystallization properties. It was eluted from AG50 cation-exchange resin with 1.0 HCL in the hydroxyproline region, but was separated from hydroxyproline on a DC-6A column in the amino acid analyzer. Another metabolite was identified as aspartic acid. It was not separated from hydroxyproline on either AG50 using 1 HCL for elution or on DC-6A using 0.1 sodium citrate, pH 3.25, for elution, but adequate separation was obtained by elution with 0.2 sodium citrate buffer at pH 2.91. Formation of these metabolites was not related either to protein synthesis or proline hydroxylation. Therefore, it is possible to analyze for hydroxyproline accurately by using a separate unhydroxylated sample to correct for the presence of the metabolites. The formation of glutamic acid suggested that proline oxidase activity might be present in bone tissue, but none was detected using a sensitive radioisotopic assay. Although the amount of radioactivity found in the metabolites was 36% of the amount of [¹⁴C]proline incorporated into protein, no radioactive glutamic or aspartic acid was present in protein hydrolyzates. This observation suggests that the metabolites did not enter the major amino acid pool used for protein synthesis.     

Expanded bed adsorption as a unique unit operation for the isolation of bacteriocins from fermentation media

Expanded bed adsorption using a strong cation exchanger allowed the direct isolation of amylovorin L471, a bacteriocin from Lactobacillus amylovorusDCE 471, from the fermentation medium. The pH of the loading and elution buffer were optimised in a packed bed with cell-free culture supernatant. Bound bacteriocin was eluted with 1.0 M NaCl. The highest recovery (30%) was obtained at the lowest pH (3.6). At higher pH values the recovery was lower, namely 12%, 15% and 7% at pH 4.5, 6.5 and 8.0, respectively. In expanded bed mode, direct isolation of the bacteriocin from the fermentation medium at pH 3.6 (loading and elution) initially resulted in a recovery of 12%. After optimisation of the pH (loading and elution at pH 3.6 and 6.5, respectively), the recovery for amylovorin L471 increased up to 30% and higher. Recovery of enterocin A from Enterococcus faeciumCTC 492 fermentation medium averaged 15% (loading and elution at pH 3.6 and 6.0, respectively). With pediocin, produced by Pediococcus acidilactici ATCC 8042, 26% recovery was obtained at a pH of 6.5 during loading and elution. Low recoveries can be ascribed to non-optimal operation conditions (pH of loading and elution buffer), inactivation of the bacteriocin on a cationic resin, and the formation of more insoluble and less active, strongly hydrophobic bacteriocin aggregates upon further purification.     

Systematic interpolation method predicts protein chromatographic elution with salt gradients,pH gradients and combined salt/pH gradients

A methodology is presented to predict protein elution behavior from an ion exchange column using both individual or combined pH and salt gradients based on high‐throughput batch isotherm data. The buffer compositions are first optimized to generate linear pH gradients from pH 5.5 to 7 with defined concentrations of sodium chloride. Next, high‐throughput batch isotherm data are collected for a monoclonal antibody on the cation exchange resin POROS XS over a range of protein concentrations, salt concentrations, and solution pH. Finally, a previously developed empirical interpolation (EI) method is extended to describe protein binding as a function of the protein and salt concentration and solution pH without using an explicit isotherm model. The interpolated isotherm data are then used with a lumped kinetic model to predict the protein elution behavior. Experimental results obtained for laboratory scale columns show excellent agreement with the predicted elution curves for both individual or combined pH and salt gradients at protein loads up to 45 mg/mL of column. Numerical studies show that the model predictions are robust as long as the isotherm data cover the range of mobile phase compositions where the protein actually elutes from the column.     

Analysis, Production, and Isolation of an Extracellular Laccase from Polyporus anceps

Methods are described for the analysis, production, and isolation of laccase produced by a strain of Polyporus anceps. A simple quantitative colorimetric assay based on the oxidation of syringaldazine to syringaldazine quinone is described. Using a defined medium supplemented with the amino acids cysteine and histidine and with elevated phosphate, consistently high titers of laccase were obtained. The enzyme was isolated directly from fermentation medium by binding to diethylaminoethyl cellulose, and, once bound to the ion exchanger, it could be stored for 6 months at -70°C with minimal loss of activity. The enzyme was quantitatively recovered from the resin by elution with 0.2 M phosphate buffer (pH 5.0).     

Preparative-scale isolation of isotopically labeled amino acids

Over 10 g of individual ²H, ¹⁵N-labeled amino acids was resolved and recovered on a laboratory-scale ion-exchange system from a crude bacterial protein hydrolyzate derived from 20 g of lyophilized cells. The 17 amino acids (cystine was not isolated) were recovered containing less than 1.0% of other contaminating amino acids except for proline (4.0%). The aromatic and basic amino acids were isolated on a dual-column carrier displacement system (390-ml resin bed volume) while most of the neutral and acidic amino acids were separated on a pyrazolium chloride elution system (560-ml resin bed volume). The two remaining overlapping pairs were resolved on small carrier displacement columns. In addition, the overlapping fractions from adjacent peaks of the pyrazolium chloride elution system represent only 3.5% (0.37 g) of the total sample.     

桑椹中黄酮类物质的大孔树脂纯化工艺

以桑椹中黄酮类物质的吸附量和解吸率为指标,对比分析HZ-801、HZ-816、HZ-818等12种大孔吸附树脂对桑椹提取液的分离纯化效果,优选出最佳树脂HZ-801并通过对上样液pH、上样液质量浓度、上样量、吸附流速、洗脱剂质量浓度、洗脱剂用量、洗脱流速等影响因素的考察,确定最优工艺:吸附阶段上样液pH=4,上样液质量浓度0.45mg/mL,上样量420mL,吸附流速120mL/h,动态吸附量(干树脂)25.34mg/g,吸附率84.25%;洗脱阶段的洗脱剂体积分数为60%乙醇,洗脱剂用量270mL,洗脱流速120mL/h。此优化工艺条件下的洗脱率为85.78%,总黄酮纯度从23.64%提高到82.36%。     

AB-8大孔树脂吸附分离红花桑寄生总黄酮的影响因子研究

AB-8大孔吸附树脂对红花桑寄生总黄酮静态吸附和动态洗脱的效果,受提取液质量浓度、pH值及环境温度、振速以及洗脱剂乙醇浓度、流速等因素影响。试验表明,提取液质量浓度和pH值对AB-8树脂的吸附效果有显著影响,其吸附分离总黄酮的工艺条件为:浓度为1.2~2.0 mg/ml、pH 3.0~4.0的红花桑寄生提取液,置于摇床上,于室温条件下振荡(振速160 r/min)吸附2~3 h,然后用5倍于树脂体积(5BV)的50%乙醇以1.5 ml/min流速进行柱上动态解吸。AB-8树脂对红花桑寄生总黄酮的饱和吸附量可达29.0 mg/g,动态洗脱率达95.0%,获得产品中黄酮纯度为46.0%,得率为5.5%。     

Highly pure 1,3‐propanediol: Separation and purification from crude glycerol‐based fermentation

A new separation and purification process was developed for recovering 1,3‐propanediol (1,3‐PD) from crude glycerol‐based fermentation broth with high purity. The downstream process integrated chitosan flocculation, activated carbon decolorization, fixed bed cation exchange resin adsorption, and vacuum distillation. Breakthrough curves were measured considering the effect of sample concentration, flow rate, temperature, and resin stack height. Yoon–Nelson model was proposed to fit the fixed bed adsorption. The characteristic column parameters were calculated. Optimal condition for adsorption was 1,3‐PD, 30.0 g/L; flow rate, 1.00 mL/min; stacking height, 30.0 cm; and temperature, 298 K. Ethanol‐water (75%, 1 mL/min) was used as eluent to separate 1,3‐PD and glycerol with 95.3% 1,3‐PD elution rate. After vacuum distillation, the overall purity and yield of 1,3‐PD were 99.2% and 80.8% in the purification process, respectively. This is a simple and efficient downstream strategy for 1,3‐PD purification.     

Performance of nonrigid ion-exchange packings of small particle size in the separation of cytochrome c and derivatives by high-pressure liquid chromatography

Conditions for high-pressure liquid chromatography separations of cytochrome c and derivatives were investigated to provide an analytical and preparative tool for biochemical and medical research. Cation exchangers with a polystyrene-divinylbenzene or cellulose matrix were found to be not useful. Better results were obtained with hydroxylapatite and (meth)acrylic cation exchangers. The chromatographic selectivity with respect to cytochrome c derivatives is described at varying cation concentrations, pH of the eluent, and temperature. The column efficiency and its dependence on particle size for cytochrome c-like compounds is shown. A number of chromatograms is presented to prove the potential of this type of columns.     

Observations on the use of a cation exchange resin for the preparation of metal-depleted media for lymphocyte culture

A chelating resin specific for divalent cations (Chelex) was used to prepare metal-depleted media for lymphocyte culture. A batch procedure (resin in pH 7.4 phosphate buffer/specimen, 1:1) removed 70-80% of iron, 77-87% of copper and 88-98% of zinc, calcium and magnesium. At variance with other reports, when a resin/specimen ratio of 1:4 was used, iron chelation decreased to 40%, whereas other cation chelation remained unchanged. Best chelation for iron and calcium was obtained at pH 5-6.4; for copper, zinc and magnesium, at pH 7.4-8.0. During the procedure protein content decreased by 8-10%; arginine and lysine by 80%; asparagine, cystine, tyrosine and phenylalanine by 60%, other amino acids by 35%. These new data suggest that cation-depleted media prepared with Chelex may be used to study the effects of cations on lymphocytes in culture, provided that the most appropriate pH and resin/specimen ratio are selected and adequate amino acid replacement is performed. Results on normal human lymphocytes are reported.     

用超大孔树脂吸附和低乙醇浓度洗脱提纯异黄酮的研究

传统大孔吸附树脂吸附分离中草药有效成分通常需要高浓度的有机溶剂作为洗脱剂,在大规模生产中具有安全隐患和对环境的污染。本文以异黄酮为目标产物,考察了8种不同骨架、不同孔径的树脂对异黄酮的吸附分离性能,其中包括本实验室自制的孔径为1000nm的超大孔树脂。实验结果表明,与传统大孔树脂吸附层析法相比,超大孔树脂吸附层析不仅工艺简单、快速而且大幅度降低了有机溶剂的使用量。洗脱时,乙醇浓度由70％降低到20％。