离子交换法从发酵液中提取聚苹果酸

用离子交换法提取发酵液中的聚苹果酸,比较了不同型号的阴离子树脂对聚苹果酸的静态吸附量,其中以D296树脂对聚苹果酸的吸附量最大.通过静态和动态方法,考察了不同操作条件对固定床分离工艺的影响.结果表明,发酵液经预处理后,调pH至9.5,以lBV/h流速上柱吸附,上柱量为4BV;用0.5mol/L NaCl洗脱,速度为1BV/h.该工艺能够将发酵液中大部分杂质去除,PMLA样品的纯度达到 93.2%,提取收率为79.46%.     

离子交换法从发酵液中提取L-亮氨酸

用离子交换法提取发酵液中的L-亮氨酸,比较了不同型号的强酸性阳离子树脂对L-亮氨酸的静态吸附量和吸附动力学,其中以WA-2型树脂对L-亮氨酸吸附量最大、吸附速度快,适于L-亮氨酸的提取.测定了WA-2型树脂对L-亮氨酸的吸附等温线,并回归得到Freundlich方程.考察了固定床操作工艺条件,结果表明:发酵液经预处理后,以1BV/h流速上柱吸附,上柱量为2BV;再用0.3mol/L的氨水洗脱,速度为1BV/h,洗脱效果较好,L-亮氨酸回收率达到95.7%.提取过程中WA-2型树脂不会受到不可逆的污染,也没有机械损坏,其使用寿命不受影响.     

Isolation of ATP from a yeast fermentation broth using a cryogel column at high flow velocities

This communication presents an effective method for isolating adenosine triphosphate (ATP) from a yeast fermentation broth using an anion‐exchange supermacroporous cryogel column at high flow velocities. The breakthrough and elution behaviors of pure ATP in the cryogel bed were investigated at flow velocities of 2, 5, and 10 cm/min and the ATP binding capacities were determined. Then the ATP‐containing yeast fermentation broth was employed as the test feedstock and various chromatographic runs were conducted to isolate ATP by the cryogel at different high flow velocities. The ATP samples obtained were analyzed quantitatively by HPLC. The results showed that even at a flow velocity of 5 or 10 cm/min, a product purity of 97.4 or 98.0% can be achieved, illustrating the potential of the present method for separation of high‐purity ATP directly from fermentation feedstock at high flow velocities.     

Separation of D‐psicose and D‐fructose using simulated moving bed chromatography

Simulated moving bed (SMB) processes have been widely used in the sugar industries with ion‐exchange resin as a stationary phase. D ‐Psicose, a rare monosaccharide known as a valuable pharmaceutical substrate, was synthesized by the enzymatic conversion from D ‐fructose. The SMB process was adopted to separate D ‐psicose from D ‐fructose. Before the SMB experiment, the reaction mixture including D ‐psicose and D ‐fructose was treated by a deashing process to remove contaminants, such as buffers, proteins, and other organic materials. Four columns packed with Dowex 50WX4‐Ca²⁺ (200–400 mesh) ion‐exchange resins were used in the four‐zone SMB. Single‐step frontal analysis was performed to estimate the isotherm parameters of each monosaccharide. The operating conditions of the SMB process were determined based on the Equilibrium Theory. According to the simulation of the SMB process, the purity and yield of extract product (D ‐psicose) achieved were 99.04 and 97.46%, respectively and those of raffinate product (D ‐fructose) were 99.06 and 99.53%, respectively. Under the optimized operating condition, complete separation (extract purity = 99.36%, raffinate purity = 99.67%) was achieved experimentally.     

Recovery of actinorhodin from fermentation broth

In the present work, a new method of purification for actinorhodin was developed using an expanded bed chromatography technique in which antibiotic capture, feedstock clarification, centrifugation, dialysis and concentration are done in one step. The cation-exchanger (P-11) resulted in 26% adsorption and 2% recovery whereas the anion-exchanger (DE-52) resulted in 99% adsorption and 56% recovery of adsorbed antibiotic using methanol buffer and 2 M NH4Cl as eluting agent. Streamline DEAE anion-exchanger, which is especially designed for EBA applications, yields 82% adsorption and 50% elution of actinorhodin fed into the chromatography column directly from the fermentation broth. Isocratic elution resulted in extremely efficient yield compared to linear gradient elution, i.e. 13.5-fold more recovery in the column with an aspect ratio (L:D) of 4. Expansion by 150% of settled bed resulted in the best recovery of actinorhodin among 100 and 200% expansions. A comparison of breakthrough profiles in packed and expanded bed adsorption showed that the performance of the expanded bed is better (by 33%) at allowing more volume of the fermentation broth to pass through the chromatography column.     

L(+)-乳酸提取纯化新工艺

发酵制备L(+)-乳酸过程中,发酵醪液分离提纯的时间和成本占到整个生产过程的一半以上,大大限制了乳酸生产工业的发展.针对此种情况,需探索出低成本高效率的分离提纯精制L(+)-乳酸的新工艺,以提高产品收率和纯度.本工艺过程:发酵醪液微波灭菌、高速离心、活性碳除色、膜过滤除蛋白质、柱分离、柱精制、真空浓缩得成品.生产过程不产生硫酸钙废弃物,减少对环境的污染,提取率由原来50%提高到71%以上.     

An efficient strategy based on liquid‐liquid extraction and pH‐zone‐refining counter‐current chromatography for selective enrichment,separation, and purification of alkaloids and organic Acids from natural products

This study presents an efficient strategy based on liquid‐liquid extraction and pH‐zone‐refining counter‐current chromatography for selective enrichment, separation, and purification of alkaloids and organic acids from natural products. First, an acid or base modified two‐phase solvent system with maximum or minimum partition coefficient was developed for the liquid‐liquid extraction of the crude extract. As a result, alkaloids or organic acids could be selectively enriched in the upper or lower phase. Then pH‐zone‐refining counter‐current chromatography was employed to separate and purify the selectively enriched alkaloids or organic acids efficiently. The selective enrichment and separation of five bufadienolide from toad venom of Bufo marinus was used as an example to show the advantage of this strategy. As a result, 759 mg of selectively enriched bufadienolide was obtained from 2 g of crude extract and the total content of five targets was increased from 14.64 to 83%. A total of 31 mg of marinobufagin‐3‐adipoyl‐l ‐arginine, 42 mg of telocinobufagin‐3‐pimeloyl‐l ‐arginine, 51 mg of telocinobufagin‐3‐suberoyl‐l ‐arginine, 132 mg of marinobufagin‐3‐suberoyl‐l ‐arginine, and 57 mg of bufalin‐3‐suberoyl‐l ‐arginine were all simultaneously separated from 500 mg of selectively enriched sample, with the purity of 92.4, 97.5, 90.3, 92.1, and 92.8%, respectively.     

Improved adsorptive purification and effective separation of acidic and lactonic sophorolipid biosurfactant

Crude Sophorolipid extract obtained from fermentation broth contains both acidic and lactonic forms of Sophorolipid with different degrees of acetylation and varying lengths of the fatty acid chains. Carboxylic end in the acidic form of the fatty acid is free, whereas in the lactonic form, it is internally esterified. Sophorolipid show different physicochemical properties with wide range of applications for each structural compound. This work represents the separation and purification strategies of acidic and lactonic Sophorolipid obtained from crude extract. Depending on structure of Sophorolipid molecule different resin were selected for screening based on % binding per ml of resin, % elution and % assay purity of the product obtained.Based on binding study, uptake kinetic, adsorption isotherm and Simchrome software results, macroporous adsorbent SEPABEADS SP 825L was selected as the choice of resin for preparative separation. Breakthrough capacity of the resin was calculated to be 37.8 and 2.801 mg/mL for lactonic and acidic SL respectively. Column study was performed to justify the previous results and 10 g of crude product was processed to obtain 4.2 and 3.9 g of acidic and lactonic SL having the assay purity of more than 90%.     

Separation of salvianic acid A from the fermentation broth of engineered Escherichia coli using macroporous resins

Salvianic acid A (also known as danshensu) is a plant‐derived polyphenolic acid, and has a variety of physiological and pharmacological activities. Our laboratory previously constructed an unprecedented artificial biosynthetic pathway in Escherichia coli and established the fermentation process to produce salvianic acid A. Here, we developed an efficient method for separating salvianic acid A from the fermentation broth of engineered Escherichia coli by macroporous resins. Among ten tested macroporous resins, the static and dynamic adsorption/desorption experiments demonstrated that X5 resin was the best to separate salvianic acid A from fermentation broth. Other parameters during static and dynamic procedures were also investigated. Under the optimum separation conditions, the average adsorption capacity of SAA were 10.66±0.54 mg/g dry resin and the desorption ratio was 85.6±4.1%. The purity and recovery yield of salvianic acid A in the final dry product were 90.2±1.5 and 81.5±2.3%, respectively. The results show that adsorption separation with macroporous resin X5 was an efficient method to prepare salvianic acid A from fermentation broth. This work will benefit the development and application of plant‐derived salvianic acid A and its derivatives.     

大孔吸附树脂提取麦拓莱霉素的工艺研究

研究了用大孔吸附树脂从发酵液中分离提取新型大环内酯类抗生素-麦拓莱霉素的吸附工艺过程，对吸附条件进行了优化，得到了适宜的操作条件．结果表明：NKA树脂为最佳吸附剂：吸附最佳pH值为10．32；吸附流速选择为1ml／min；NaCl的加入对吸附不利，选择NaCl浓度为0．6％左右，本实验条件下，发酵液中的麦拓莱霉素浓度为0．18mg／ml；NKA的吸附量可达33．7mg／g树脂。     

PURIFICATION OF FLAVONES OF GINKGO LEAVES BY ADSORPTION HZ-841 RESIN

The purification process of total flavones of ginkgo leaves by resin HZ-841 from ethanol extract was studied. First, the total flavone was extracted from the clef aired powder of ginkgo Lbiloba leaves. Effects of solvents and operation conditions were examined to get a relative high yield and purity in this step. The crude extract was further purified by resin HZ-841. Both adsorption and elution process were studied to obtain an optimized conditions, i.e., pH, flow rate, concentration. A yellow powder was obtained, of which 37.3% was flavones, obviously higher than the basic international standard of 24%.     

PURIFICATION OF FLAVONES OF GINKGO LEAVES BY ADSORPTION HZ-841 RESIN

The extraction of dry ginkgo biloba L. leaves is a mixture of some effective agents like flavones and lactones, which have therapeutic activities of peripheral vasoregulation, PAF (platelet aggregation factor) antagonism and prevention of membrane damage caused by free radicals, etc. Thus broad application prospect in the field of sanitarian product and cosmetics is expected [1]. The total flavonoid can be extracted in distilled water or in organic solvents, usually methanol, ethanol, or ace…     

Hydrophobic charge‐induction resin with 5‐aminobenzimidazol as the functional ligand: preparation,protein adsorption and immunoglobulin G purification

A new hydrophobic charge‐induction chromatography resin was prepared with 5‐aminobenzimidazol as functional ligand and polyacrylic ester beads as matrix. Adsorption isotherms and adsorption in columns were investigated using human immunoglobulin G and bovine serum albumin as model proteins, and the influence of pH and NaCl concentration was discussed. Results showed that the ligand density was 195 μmol/mL gel, and protein selectivity can be improved by controlling pH and salt addition. An optimized purification process (sample loading at pH 8.0 with 0.2 M NaCl and elution at pH 5.0) was performed to purify human immunoglobulin G from bovine serum albumin containing feedstock, which resulted in human immunoglobulin G purity of 99.7% and recovery of 94.6%. A similar process was applied for the purification of monoclonal antibody from cell culture supernatant, which showed antibody purity of 94.9% and recovery of 92.5%. The results indicated that the new resin developed had comparable performance as Protein A chromatography and would be suitable for antibody purification from complex feedstock.     

大孔吸附树脂分离纯化异甘草素的研究

研究大孔吸附树脂分离纯化异甘草素的工艺条件及参数。通过研究HPD-600、D4020、D101、AB-8、NKA-II、AL-2和NKA-9树脂对异甘草素的吸附和解吸附能力,筛选最佳树脂为AB-8,并研究了其对异甘草素的吸附和解吸附性能,确定了最佳的吸附与解吸附工艺参数,吸附:pH=5,室温,流速1.5BV/h,溶液处理量为5BV;脱附:洗脱剂为70%的乙醇溶液,流速1BV/h,洗脱剂用量4.5BV。异甘草素样品溶液经AB-8树脂吸附与脱附后回收率为76.7%,纯度由2.02%提高到29.1%,提高了14.4倍。实验结果表明,AB-8树脂对异甘草素的吸附量大,脱附容易,可以应用于异甘草素的分离纯化。     

Optimization of an α‐(Amino acid)‐N‐carboxyanhydride polymerization using the high vacuum technique: Examining the effects of monomer concentration,polymerization kinetics,polymer molecular weight,and monomer purity

l ‐Ornithine‐based poly(peptides) have been widely utilized in the field of drug delivery, however few studies have been conducted examining the details of polymerization. In this article, the effects of monomer concentration, polymerization kinetics, polymer molecular weight and monomer purity were investigated using l ‐carboxybenzyl (Cbz)‐ornithine as a model monomer. The mechanism of polymerization herein follows the normal amine mechanism to produce poly(peptides) having controlled molecular weights, known chain ends and a narrow polydispersity index (PDI). A preferred monomer concentration range was determined, which required minimal polymerization times and allowed for predictable and reproducible molecular weights with narrow PDIs. The impact of monomer purity on the polymerization was established and monomer purification conditions are reported, which produce high‐purity monomer after a single recrystallization. Additionally, the optimized polymerization conditions and monomer purification protocol were combined with a sequential monomer addition technique to produce high molecular weight poly(ornithine) with a narrow PDI and known chain ends. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 1385–1391     

Purification and Preparation of Moenomycin A from Fermentation Broth by Multidimensional Chromatography

Based on various adsorption characteristics of resins, a novel method for purifying and preparing moenomycin A from fermentation broth was established by combining different chromatographic modes into a three-step preparative chromatography process. Fermentation broth of moenomycins was firstly prepurified by macroporous adsorbent XAD7HP to remove most strong polar impurities, then further purified by anion exchange resin FPA98Cl, and finally refined moenomycin A was obtained by use of semi-preparative reversed-phase chromatographic column packed with Chromtorex C8 silica gel. As the main indicators, purity and yield of moenomycin A were examined in order to optimise the chromatographic process for each step. Under optimized chromatographic conditions, the purity and total yield of moenomycin A were 95.0 and 22.2 %, and the biological potency of moenomycin A was 2232 U mg⁻¹, significantly higher than 1395 U mg⁻¹, which is the potency of the standard from Agriculture Ministry of China. Three-step preparative chromatographic mode could gradually and effectively remove impurities. The present method is practical, easy to be operated with less solvent consumption, and provides a new idea for the preparation of moenomycin A with high purity.

     

Resolution and quantification of arginine,monomethylarginine, asymmetric dimethylarginine,and symmetric dimethylarginine in plasma using HPLC with internal calibration

N^G,N^G‐dimethyl‐l ‐arginine (asymmetric dimethylarginine, ADMA),N^G‐monomethyl‐l ‐arginine (l ‐NMMA) and N^G,N^G’‐dimethyl‐l ‐arginine (symmetric dimethylarginine, SDMA) are released during hydrolysis of proteins containing methylated arginine residues. ADMA and l ‐NMMA inhibit nitric oxide synthase by competing with l ‐arginine substrate. All three methylarginine derivatives also inhibit arginine transport. To enable investigation of methylarginines in diseases involving impaired nitric oxide synthesis, we developed a high‐performance liquid chromatography (HPLC) assay to simultaneously quantify arginine, ADMA, l ‐NMMA and SDMA. Our assay requires 12 μL of plasma and is ideal for applications where sample availability is limited. We extracted arginine and methylarginines with mixed‐mode cation‐exchange columns, using synthetic monoethyl‐l ‐arginine as an internal standard. Metabolites were derivatized with ortho‐phthaldialdeyhde and 3‐mercaptopropionic acid, separated by reverse‐phase HPLC and quantified with fluorescence detection. Standard curve linearity was ≥0.9995 for all metabolites. Inter‐day coefficient of variation (CV) values were ≤5% for arginine, ADMA and SDMA in human plasma and for arginine and ADMA in mouse plasma. The CV value for l ‐NMMA was higher in human (10.4%) and mouse (15.8%) plasma because concentrations were substantially lower than ADMA and SDMA. This assay provides unique advantages of small sample volume requirements, excellent separation of target metabolites from contaminants and validation for both human and mouse plasma samples. © 2015 The Authors Biomedical Chromatography published by John Wiley & Sons, Ltd.     

Separation of isorhamnetin 3‐sulphate and astragalin from Flaveria bidentis (L.) Kuntze using macroporous resin and followed by high‐speed countercurrent chromatography

D4020 resin offered the best dynamic adsorption and desorption capacity for total flavonoids based on the research results from ten kinds of macroporous resin. A column packed with D4020 resin was used to optimize the separation of total flavonoids from Flaveria bidentis (L.) Kuntze extracts. The content of flavonoids in the product was increased from 4.3 to 30.1% with a recovery yield of 90%. After the treatment with gradient elution on D4020 resin, the contents of isorhamnetin 3‐sulfate and astragalin were increased from 0.49 to 8.70% with a recovery yield of 74.1% and 1.16 to 30.8%, with a recovery yield of 92.2%, respectively. Further purification was carried out by one‐run high‐speed countercurrent chromatography yielding 4.5 mg of isorhamnetin 3‐sulfate at a high purity of 96.48% and yielding 24.4 mg of astragalin at a high purity of over 98.46%.     

Model-based design of a pilot-scale simulated moving bed for purification of citric acid from fermentation broth

One of the conventional processes used for the recovery of citric acid from its fermentation broth is environmentally harmful and cost intensive. In this work an innovative benign process, which comprises simulated moving bed (SMB) technology and use of a tailor-made tertiary poly(4-vinylpyridine) (PVP) resin as a stationary phase is proposed. This paper focuses on a model-based design of the operation conditions for an existing pilot-scale SMB plant. The SMB unit is modeled on the basis of experimentally determined hydrodynamics, thermodynamics and mass transfer characteristics in a single chromatographic column. Three mathematical models are applied and validated for the prediction of the experimentally attained breakthrough and elution profiles of citric acid and the main impurity component (glucose). The transport dispersive model was selected for the SMB simulation and design studies, since it gives a satisfactory prediction of the elution profiles within acceptable computational time. The equivalent true moving bed (TMB) and SMB models give a good prediction of the experimentally attained SMB separation performances, obtained with a real clarified and concentrated fermentation broth as a feed mixture. The SMB separation requirements are set to at least 99.8% citric acid purity and 90% citric acid recovery in the extract stream. The complete regeneration in sections 1 and 4 is unnecessary. Therefore the net flow rates in all four SMB sections have been considered in the unit design. The influences of the operating conditions (the flow rate in each section, switching time and unit configuration) on the SMB performances were investigated systematically. The resulting SMB design provides 99.8% citric acid purity and 97.2% citric acid recovery in the extract. In addition the citric acid concentration in the extract is a half of its concentration in the pretreated fermentation broth (feed).