红外光谱探测离交色谱分离硼同位素的树脂柱吸附机理

研究离交色谱法分离硼同位素的过程机理对于实现^(10)B同位素高效富集与分离具有重要的意义。针对前人工作提出的树脂柱吸附10B同位素过程机理无法解释盐酸洗脱过程中的耗酸量与实验现象不符的问题,本文提出一种新的硼同位素吸附过程机理。研究结果表明,Amber IRA 743树脂官能基团中的氨基可能与硼酸根离子发生反应,生成新的物种。实验采用红外光谱法证实了在1 467cm^(-1)处有新峰(N—B键)的生成。研究为深入认识硼同位素吸附-洗脱过程的分子层次行为规律奠定了基础。

Study on Separation of Phycoerythrin by Q-Sepharose Fast Flow

[Objective] This study aimed to establish an efficient process for separation of phycoerythrin by using Q Sepharose Fast Flow resin and verity its feasibility for scale-up. [Method] Elution gradient, sample volume and flow rate were optimized to determine the optimal separation condition, under which the scale-up process was verified. [Result] The optimal condition for separation of phycoerythrin by using Q Sepharose FF resin was investigated： 30 ml of laver extract was loaded to the Q Sepharose FF column with a bed volume of 8 ml; subsequently, the column was stepwise eluted with 0-0.10-0.35-1.00 mol/L NaCI solution （pH 6.0） at a constant flow rate of 1 ml/min; the elution peak under 0.35 mol/L NaCI solution was collected, and the recovery rate and purity coefficient （A565/A280） of phycoerythrin were determined as 44.3 and 1.15, respectively. Based on the established process, 75 ml of phycoerythrin extract was loaded to the Q Sepharose FF column with a bed volume of 20 ml for separation, while no significant variation was observed in the separation result. [Conclusion] Phycoerythrin can be well separated from laver extract by using Q Sepharose FF resin and the process is feasible for scale-up.

Isolation of Lysozyme from Chicken Egg White Using Polyacrylamide-based Cation-exchange Cryogel

An effective cation-exchange chromatographic method for lysozyme isolation from chicken egg white is presented, using supermacroporous cryogel grafted with sulfo functional groups. The chromatographic processes were carried out by one-step and sequential elution, respectively. Sodium phosphate buffer （pH 7.8） containing different concentrations of NaC1 is used as elution agent. The corresponding breakthrough characteristics and elution behaviors in the cryogel bed were investigated and analyzed. Purity of lysozyme in the elution effluent was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. The maximum purity of the obtained lysozyme was about 96%, and the cryogel is demonstrated as a potential separation medium for purification of high-purit lysozyme from chicken egg white.

Purification and characterization of novel κ-carrageenase from marine Tamlana sp.HC4

We isolated a bacterial strain （HC4） that is able to degrade k-carrageenan from a live specimen of the red alga Hyalosiphonia caespitosa. With 16S rRNA gene sequencing, we identified the strain as Tamlana sp., and then purified an extracellular K-carrageenase from a culture of Tamlana sp. HC4 by ammonium sulfate precipitation, Sephadex G-200 gel filtration chromatography, and DE-cellulose 52 anion-exchange chromatography. The purified enzyme yields a single band on SDS-PAGE with a molecular mass of 66.4 kDa. The optimal pH and temperature for κ-carrageenase activity are at 8.0 and 30~C, respectively. The enzyme is stable over the range ofpH 7.2-8.6 below 45℃. The enzyme activity is strongly inhibited by Zn2＋ and Cu2＋ at 1 mmol/L. The enzyme-catalyzed reaction follows Michaelis-Menten kinetics with the Michaelis constant （Kin） at 7.63 mg/ml. Analysis of the degradation products of the κ-carrageenase by ESI-MS and 13C-NMR spectroscopy indicates that the enzyme degrades κ-carrageenan down to the level ofκ-neocarrabiose sulfate.

Optimization of DsbA Purification from Recombinant Escherichia coli Broth Using Box-Behnken Design Methodolog

Disulfide bond formation protein A （DsbA） is one of the important helper proteins for folding in protein synthesis in vivo. In this study, purification of recombinant DsbA was investigated by examining four important factors with Box-Behnken design method, a statistic-based design of experiments. The optimal operation conditions were obtained by adopting the effectiveness coefficient method on the multi-objective problem, which takes the protein recovery, purification efficiency and throughput of ion-exchange chromatography into account. After the optimization, protein recovery of 96.8% and purity higher than 95% DsbA was achieved, and the productivity was （377.9±1.7） mg soluble DsbA per liter broth. The purified protein was identified by peptide mass fingerprinting matching the record of gil2624856, a mutant of DsbA. The DsbA was preliminarily applied to the refolding of denatured lysozyme in vitro.

Determination of Cordyceps Sinensis/Betaine Compound Feed Nutriment by HPLC

[Objective] The aim was to conduct HPLC analysis on Cordyceps Sinensis/Betaine compound feed nutriment. [Method] Cordyceps Sinensis/Betaine compound feed nutriment was under HPLC analysis to determine separation of Cordyceps Sinensis effluent and betaine. [Result] Different compositions in Cordyceps Sinensis/etaine compound feed nutriment would be well separated by the method. [Conclusion] The method provides a suitable platform of separation and analysis for Cordyceps Sinensis /Betaine compound feed nutriment.

Rapid Purification of Enhanced Green Fluorescent Protein from Escherichia coli

As an excellent reporter molecule, enhanced green fluorescent protein (eGFP) was widely used for gene expression and regulation and was generally expressed in Escherichia coli strain. A rapid procedure consisting of ammonium sulfate precipitation, size exclusion chromatography, and anion exchange chromatography was devel- oped for the purification of eGFP. Based on the proposed procedure, recombinant eGFP with an electrophoretic pu- rity was achieved in combination with an overall yield of 66% and a purification factor of 17.9. The fluorescent spectrometry of purified eGFP and lysate from E. coli strain expressing eGFP exhibited the same wavelength of ex- citation and emission maxima, indicating that the purification procedure did not influence the construct and fluo- rescent characteristics of desired protein. The procedure mentioned was easy to scale up for the purification of large quantities of eGFP.

Chiral ionic liquids as chiral selectors for separation of tryptophan enantiomers by ligand exchange chromatography

Chiral ionic liquids (CILs) containing imidazolium cations and L-Proline (L-Pro) anions were applied as chiral selector to separate tryptophan (Trp) enantiomers on a C18 column by ligand exchange chromatography. Several factors influencing Trp enantiomers separation, such as alkyl chain length of CILs, concentrations of Cu2+ and CILs, pH of the mobile phase, flow rate, organic solvent and temperature, were studied. Under the optimal conditions, the Trp enantiomers could be successfully separated within 21 min with the resolution of 2.30. At the same time, some thermodynamical parameters were obtained. The experimental results show that the enthalpy values of the Trp enantiomers are negative, indicating that the separation process is exothermic. And the enthalpy values of D-Trp are larger than those of L-Trp, which indicates that L-Trp could form more stable ternary complexes with tryptophan enantiomers.

Purification and Identification of a Clotting Protein from the Hemolymph of Chinese Shrimp (Fenneropenaeus chinensis)

The clotting protein(CP) plays important and diverse roles in crustaceans,such as coagulation and lipid transportation.A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis(named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography.Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+,suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase in shrimp hemocytes.The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE.CP exists as disulfide-linked homodimers and oligomers.The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon,Farfantepenaeus paulensis and Litopenaeus vannamei;and similar to that of other decapods.The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight(MALDI-TOF/TOF) mass spectrometry.Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.