离子排斥色谱法同时测定2,3-丁二醇发酵液中的葡萄糖和木糖及各种代谢产物

针对2,3-丁二醇发酵体系,以高交联度磺化苯乙烯-二乙烯基苯共聚物多孔微球为固定相的Aminex HPX-87H离子色谱柱为分析柱,利用示差折光检测器对该体系中的6种代谢产物(2,3-丁二醇、3-羟基丁酮、乙醇、乙酸、甲酸和乳酸)以及2种残留底物葡萄糖和木糖进行了分离.在65 ℃柱温下,以5 mmol/L H2SO4作为流动相,发酵液样品中底物葡萄糖和木糖及2,3-丁二醇等各种代谢产物的线性相关系数均在0.9990～0.9999之间,回收率为98.8%～103.2%,精密度为0.3%～2.1%.在最佳色谱条件下,能够同时测定2,3-丁二醇发酵体系中的残留底物和各种代谢产物的含量,适合于实时定量监测2,3-丁二醇发酵过程.     

有机酸的非抑制型离子排斥色谱法测定

研究了非抑制型离子排斥色谱分离测定7种有机酸(柠檬酸、酒石酸、苹果酸、琥珀酸、乳酸、甲酸、乙酸)的方法.以Shim-pack SCR-102H柱为分离柱,选用对甲苯磺酸-乙腈为淋洗液,考察了淋洗液浓度、流速、pH、色谱柱温度及淋洗液中乙腈含量等条件对分离和测定的影响.通过实验确定最佳的色谱条件:2.0 mmol/LP-甲苯磺酸-乙腈(体积比91:9)为淋洗液,流速1.0 mL/min,柱温50℃,pH 2.8.该法对所测有机酸的检出限在0.06～1.05.mg/L之间,相对标准偏差在3.3%以下(n=5),加标回收率在96%-101%之间,整个分析过程在11 min内完成.     

高效液相色谱法测定发酵醪液中的衣康酸

采用AminexHPX－87H色谱柱和折光检测器,以4mmol／L硫酸为洗脱剂,分析土曲霉发酵醪液中的衣康酸获得满意的结果。     

苯并芘DNA加合物anti-BPDE-N^2-dG四种立体异构体的合成及色谱分离

体外合成了苯并芘DNA加合物-邻二醇环氧苯并芘-脱氧鸟苷加合物(anti-BPDE-N2-dG)四种立体异构体(两对手性异构体)。通过优化体外反应条件,anti-BPDE-N2-dG四种异构体的合成产量较现有合成方法提高了2倍多,为定量检测生物体中anti-BPDE-N2-dG提供了标准品。并首次将五氟苯基色谱柱应用于该立体异构体的色谱分离提纯,通过优化色谱条件,采用常规的五氟苯基色谱柱(250 mm×4.6 mm,5μm),以乙腈-0.1%甲酸水(22.5∶77.5)为流动相,流速1.2 mL/min条件下,45 min内即可分离提纯四种立体异构体。该方法与常规C18柱(250 mm×4.6 mm,5μm)需要160 min,苯基柱(250 mm×4.6 mm,5μm)需要85~100 min才能将四种立体异构体实现色谱分离相比,缩短了分离时间,提高了提纯效率。通过紫外吸收光谱、质谱、圆二色谱对四种立体异构体进行表征,确定出峰顺序为trans(-)、trans(+)、cis(+)、cis(-)-anti-BPDE-N2-dG。此外,利用高效液相色谱-串联质谱(HPLC-MS/MS)检测anti-BPDE-N2-dG四种立体异构体标准品时,使用常规的五氟苯基色谱柱可在30 min内完成分离检测,与相同规格的苯基柱需要60 min相比提高了检测效率。     

离子交换色谱法对8种有机酸含量的同时测定

建立了淋洗液自动发生梯度淋洗的离子交换色谱法同时测定果汁中乳酸、乙酸、苹果酸、丙二酸、马来酸、草酸、柠檬酸、异柠檬酸8种有机酸的分析方法.样品经处理后用lonPac AS11-HC分离柱和lonPac AG11-HC型保护柱分离,以EG40自动淋洗液发生器生成的0.8～30 mmol/L KOH为淋洗液梯度洗脱,抑制型...     

反相高效液相色谱法测定丙酸发酵液中的有机酸

对于生物法制备丙酸的微生物发酵体系,利用Waters Atlantis dC18反相色谱柱,以0.01mol/L KH2PO4缓冲液(pH2.8)-乙腈(95∶5,V/V)作流动相,在流速为1.0mL/min,柱温为25℃时,于紫外波长215nm处检测,9min内发酵液中的草酸、甲酸、乙酸、乳酸、琥珀酸和丙酸能得到良好分离。6种有机酸线性相关系数均在0.9992~1.0000之间;方法的回收率为99%~102%;RSD为0.56~1.66。本方法能够简便、快速测定丙酸发酵体系中主产物丙酸与其它有机酸含量,适于指导整个发酵过程条件的优化及发酵产物的监控。     

高效液相色谱法测定味精厂发酵渣中的氨基酸

建立了37种标准氨基酸混合液的分离方法,并以此分离条件测定发酵渣和大米中各种氨基酸含量,测定结果表明发酵渣中氨基酸含量是大米的六倍以上,可提纯作为营养品。 本法系离子交换色谱法,使氨基酸混合液通过锂柱,控制不同pH值的流动相进行梯度淋洗,使各种氨基酸分离,柱后依序加入次氯酸钠     

高效液相色谱法测定克鲁维酵母菊芋发酵液中的乙醇,糖和有机酸类代谢成分

建立了高效液相色谱法同时分析菊芋发酵液中的乙醇和有机酸的方法.采用HPLC有机酸分析柱, 流动相为0.01 mol/L H2SO4,流速为0.5 mL/min, 以紫外和示差折光检测器作为双通道检测手段,同时对克鲁维酵母菊芋发酵液中的柠檬酸、α-酮戊二酸、葡萄糖、丙酮酸、果糖、琥珀酸、乳酸、甘油、乙酸、乙醇进行了定量分析,本方法的回收率为95.8%～109.6%;RSD为0.33%～4.0%,结果表明,本方法简单、快速、准确,适用于监控克鲁维酵母发酵产物并指导整个发酵过程条件的优化.     

HPLC-MS联用测定发酵液中的紫杉醇

用HPLC-MS测定发酵液中的紫杉醇,分别考察了实验条件下(+)ESI和(-)ESI方式的MS检测灵敏度,苯基柱和C18柱的分离效果,研究表明以(-)ESI方式的MS检测灵敏度较(+)ESI方式优,苯基柱的分离效果优于C18柱,本文报道的实验方法用于测定发酵液中的紫杉醇效果快速、准确、可靠.     

聚酰胺-胺树枝状高分子的柱色谱提纯研究

采用发散法合成了0.5～4.0代(G)聚酰胺-胺(PAMAM)树枝状高分子,通过薄层色谱分析产物组成,并进一步选用柱色谱法对半代产物进行了提纯与精制.用红外,核磁,高效液相色谱(HPLC)和热重分析等方法对提纯后产物进行了表征.结果表明,提纯得到的各PAMAM产物具有较完整的分子结构,小分子含量很少;且0.5GPAMAM产物HPLC测试纯度达到97.81%.     

离子色谱法定量检测酒曲发酵液中的河豚毒素

建立了一种离子色谱定量检测酒曲发酵液中河豚毒素的分析方法。样品经乙腈(含0.1%磷酸)溶液提取和阳离子交换柱净化后,采用离子交换色谱柱分离和紫外检测。在优化的条件下,酒曲样品中的河豚毒素在10～100 mg/L内呈良好的线性关系(r2=0.997),加标回收率为90%～103%,相对标准偏差小于4.9%,检出限(信噪比为3)为1.0 mg/L。结果表明,该方法能达到定量检测的目的。将该方法应用于实际样品的检测,验证了方法的可靠性。河豚毒素初步降解实验发现,随着时间的推移,酒曲中河豚毒素的含量逐渐减少,表明酒曲发酵液对河豚毒素的降解效果显著。     

Reactive Extraction of Lactic Acid by Using Tri‐n‐octylamine: Structure of the Ionic Phase

Lactic acid is a promising biogenic platform chemical which can be produced by fermentation of cellulose and hemicellulose. However, separating lactic acid from the fermentation broth is extremely costly and technically complex. We therefore investigated whether liquid/liquid extraction of lactic acid with tri‐n‐octylamine is a cost‐effective alternative to the existing downstream processing method. In order to find an answer to this question, the structure of the middle phase of the occurring three‐phase region, which is enriched with up to 20 wt. % lactic acid, was explored. The results of our IR, small‐angle X‐ray scattering and NMR measurements show that this phase is ionic and has a bicontinuous structure. Due to the analogy with bicontinuous microemulsions, it should be possible to further enrich the lactic acid, which could lead to a rethink regarding the design of extraction processes.     

Esterification Synthesis of Ethyl Oleate in Solvent-Free System Catalyzed by Lipase Membrane from Fermentation Broth

In this study, the immobilized lipase was prepared by fabric membrane adsorption in fermentation broth. The lipase immobilization method in fermentation broth was optimized on broth activity units and pH adjustments. The viscose fermentation broth can be used with a certain percentage of dilution based on the original broth activity units. The fermentation broth can be processed directly without pH adjustment. In addition, the oleic acid ethyl ester production in solvent-free system catalyzed by the immobilized lipase was optimized. The molar ratio of ethanol to oil acid, the enzyme amount, the molecular amount, and the temperature were 1:1, 12% (w/w), 9% (w/w)(based the total amount of reaction mixture), and 30 °C, respectively. Finally, the optimal condition afforded at least 19 reuse numbers with esterification rate above 80% under stepwise addition of ethanol. Due to simple lipase immobilization preparation, acceptable esterification result during long-time batch reactions and lower cost; the whole process was suitable for industrial ethyl oleate production.     

Separation of endo-polygalacturonase using aqueous two-phase partitioning

The partitioning of endo-polygalacturonase (endo-PG) in polyethylene glycol (PEG)-polyvinyl alcohol (PVA10000) and PEG-hydroxypropyl starch (Reppal PES100) aqueous two-phase systems was studied, and revealed the possibility of using aqueous two-phase extraction to purify and concentrate endo-PG from its clarified fermentation broth. For the PEG8000-PVA10000 system, endo-PG presented in the fermentation broth (at concentration that is more than 40% of total protein) mainly dominates in the top phase with a partitioning coefficient of 6, while total protein concentrates in the bottom phase. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: a first extraction in polyethylene glycol (PEG8000)-polyvinyl alcohol system, followed by a second extraction in PEG8000-(NH4)2SO4 system. This allowed the separation of endo-PG from polymer and the recycling of PEG polymer, since endo-PG was very strongly partitioned into the bottom phase of the PEG8000-(NH4)2SO4 system. Laboratory-scale experiments were performed to test the efficiency of this scheme. It was found that enzyme recovery was up to 91% with a total purification factor of about 1.9 and a concentration factor of more than 5. About 90% of the total PEG added into the systems can be recovered, and no reduction was obtained in the purification factor using recycled PEG.     

发酵液中谷氨酰胺提取新工艺

本文根据谷氨酸和谷氨酰胺的电离常数的差异，采用一种新型的离子交换工艺，通过单根阴离子交换柱，从谷氨酰胺发酵液中提取谷氨酰胺。粗晶溶解液通过阴离子交换柱后，谷氨酰胺最高收率达到83％，谷氨酸去除率90％。该法在显著提高产品收率的同时，还大大减少了酸碱用量。     

Nondispersive extraction for recovering lactic acid

Applied Biochemistry and Biotechnology - A nondispersive extraction process for recovery of lactic acid from fermentation broth is being developed. The criteria for selection of solvent,...     

pH Neutralization while Succinic Acid Adsorption onto Anion-Exchange Resins

Succinic acid is a useful chemical and its purification from fermentation broth by ion-exchange resins has widely drawn attention. In this study, pH neutralization in the process of adsorption of succinic acid from model solutions and fermentation broth by anion-exchange resin NERCB 04 has been tested. Adsorption capacity of NERCB 04 was about 0.41 g succinic acid/g resin at concentrations of succinic acid in the range of 10–50 g/L in packed column. In the process of succinic acid removal, pH of the system could also be neutralized. The neutralizing ability of the resin as a neutralizing agent has also been studied in the model cycle system and in the real fermentation cycle process. It was found that NERCB 04 showed stable adsorption capacity and pH neutralization ability after each regeneration. A certain amount of anion-exchange resin could neutralize the low pH values (pH 2–5) and maintain the system around pH 7.0. This means the anion-exchange resins have the function of neutralizing reagent in the process of adsorbing succinic acid.     

Immobilized metal-ion affinity chromatography of recombinant Fab protein OPG C11 in the presence of EDTA-Mg(II)

Undesired adsorption of host cell proteins poses a big challenge for immobilized metal-ion affinity chromatography (IMAC) purification. In this study, by using His6-tagged protein Fab OPG C11 from Escherichia coli fermentation as a model, we found that the presence of low concentrations of EDTA-Mg2+ in feed streams weakens the adsorption but makes it more specific towards polyhistidine tag. By combining EDTA-Mg2+ treatment and periplasmic extraction, we developed a one-step purification procedure for His6-tagged recombinant Fab OPG C11 using Ni-IDA (iminodiacetic acid) chromatography. This procedure eliminated the buffer exchange step after periplasmic extraction, which is usually required before IMAC in order to remove EDTA. In addition to savings on time and cost, this procedure eliminates undesired adsorption of most host cell proteins thus significantly improves the purity of polyhistidine-tagged recombinant proteins. The strategy of EDTA-Mg2+ treatment may have general application potentials.     

A hollow-fiber membrane extraction process for recovery and separation of lactic acid from aqueous solution

An energy-efficient hollow-fiber membrane extraction process was successfully developed to separate and recover lactic acid produced in fermentation. Although many fermentation processes have been developed for lactic acid production, and economical method for lactic acid recovery from the fermentation broth is still needed. Continuous extraction of lactic acid from a simulated aqueous stream was achieved by using Alamine 336 in 2-octanol contained in a hollow-fiber membrane extractor. In this process, the extractant was simultaneously regenerated by stripping with NaOH in a second membrane extractor, and the final product is a concentrated lactate salt solution. The extraction rate increased linearly with an increase in the Alamine 336 content in the solvent (from 5 to 40%). Increasing the concentration of the undissociated lactic acid in the feed solution by either increasing the lactate concentration (from 5 to 40 g/L) or decreasing the solution pH (from 5.0 to 4.0) also increased the extraction rate. Based on these observations, a reactive extraction model with a first-order reaction mechanism for both lactic acid and amine concentrations was proposed. The extraction rate also increased with an increase in the feed flow rate, but not the flow rates of solvent and the stripping solution, suggesting that the process was not limited by diffusion in the liquid films or membrane pores. A mathematical model considering both diffusion and chemical reaction in the extractor and back extractor was developed to simulate the process. The model fits the experimental data well and can be used in scale up design of the process.     

Analysis of the Production Process of Optically Pure <Emphasis Type="SmallCaps">d</Emphasis>-Lactic Acid from Raw Glycerol Using Engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains

Glycerol has become an ideal feedstock for producing fuels and chemicals. Here, five technological schemes for optically pure d-lactic acid production from raw glycerol were designed, simulated, and economically assessed based on five fermentative scenarios using engineered Escherichia coli strains. Fermentative scenarios considered different qualities of glycerol (pure, 98 wt.%, and crude, 85 wt.%) with concentrations ranging from 20 to 60 g/l in the fermentation media, and two fermentation stages were also analyzed. Raw glycerol (60 wt.%) was considered as the feedstock feeding the production process in all cases; then a purification process of raw glycerol up to the required quality was required. Simulation processes were carried out using Aspen Plus, while economic assessments were performed using Aspen Icarus Process Evaluator. D-Lactic acid recovery and purification processes were based on reactive extraction with tri-n-octylamine using dichloromethane as active extractant agent. The use of raw glycerol represents only between 2.4% and 7.8% of the total production costs. Also, the total production costs obtained of D-lactic acid in all cases were lower than its sale price indicating that these processes are potentially profitable. Thus, the best configuration process requires the use of crude glycerol diluted at 40 g/l with total glycerol consumption and with D-lactic acid recovering by reactive extraction. The lowest obtained total production cost was 1.015 US$/kg with a sale price/production cost ratio of 1.53.