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Adsorption Performance of Proteins to CM Sepharose FF and DEAE Sepharose FF Adsorbents
Yao, Shan-Jing,Guan, Yi-Xin,Yu, Li-Hua 한국화학공학회 2003 Korean Journal of Chemical Engineering Vol.20 No.1
Adsorption equilibrium and kinetic were studied for the binding of proteins to CM Sepharose FF and DEAE Sepharose FF. The influence of temperature pH, viscosity, initial concentration and the volume of adsorbents on the adsorption characteristics was investigated in detail. The results showed that the isotherms of lysozyme to CM Sepharose FF were well described by a Langmuir-type correlation. The two phase resistance model describing the dynamic adsorption process of lysozyme, papain, BSA to CM Sepharose FF was presented, and the pore diffusion coefficients were determined by using this model and the dynamic adsorption data.
Shan-Jing Yao,Miao-Hua Lu,Dong-Qiang Lin,Yuan-Chun Wu,Jun-Xian Yun,Le-He Mei 한국생물공학회 2005 Biotechnology and Bioprocess Engineering Vol.10 No.2
Mixed-mode hydrophobic/ionic matrices exhibit a salt-tolerant property for adsorbing target protein from high-ionic strength feedstock, which allows the application of undiluted feedstock via an expanded bed process. In the present work, a new type of mixed-mode adsorbent designed for expanded bed adsorption, Fastline PRO, was challenged for the capture of nattokinase from the high ionic fermentation broth of Bacillus subtilis. Two important factors, pH and ion concentration, were investigated with regard to the performance of nattokinase adsorption. Under initial fermentation broth conditions (pH 6.6 and conductivity of 10 mS/cm) the adsorption capacity of nattokinase with Fastline PRO was high, with a maximum capacity of 5,350 U/mL adsorbent. The elution behaviors were investigated using packed bed adsorption experiments, which demonstrated that the effective desorption of nattokinase could be achieved by effecting a pH of 9.5. The biomass pulse response experiments were carried out in order to evaluate the biomass/adsorbent interactions between Bacillus subtilis cells and Fastline PRO, and to demonstrate a stable expanded bed in the feedstock containing Bacillus subtilis cells. Finally, an EBA process, utilizing mixed-mode Fastline PRO adsorbent, was optimized to capture nattokinase directly from the fermentation broth. The purification factor reached 12.3, thereby demonstrating the advantages of the mixed-mode EBA in enzyme separation.
Shan-Jing Yao,Yi-Xin Guan,Hai-Xue Pan,Yong-Gui Gao,Man-Gi Cho 한국생물공학회 2005 Biotechnology and Bioprocess Engineering Vol.10 No.2
A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon- (rhIFN-) at a high concentration. The rhIFN- was overexpressed in E. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the buffer-exchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-, with protein recovery of 67.1% and specific activity up to 1.2 107 IU/mg.
Hu, Hong Bo,Yao, Shan Jing,Zhu, Zi Qiang,Hur, Byung Ki 한국화학공학회 2001 Korean Journal of Chemical Engineering Vol.18 No.3
The static and kinetic adsorption characteristics of Streamline DEAF and DEAF-Sepharose FF were studied under various operating conditions. The adsorption isotherms for the two types of adsorbents were obtained and found to fit well to a Langmuir-type expression. The adsorption kinetics of Streamline DEAF at different concentrations, temperatures, and viscosities were studied and a mathematical model including particle size distribution was developed to describe the adsorption performance of Streamline DEAE. Comparing the uptake curves of Streamline DEAF with DEAF-Sepharose FF, it could be concluded that Streamline DEAF achieves equilibrium faster to get equilibrium than DEAE-Sepharose FF, indicating that Streamline DEAF could be used in higher flow rate systems.
Wimonrat Phottraithip,Dong-Qiang Lin,Fei Shi,Shan-Jing Yao 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.6
New hydrophobic charge-induction chromatography(HCIC) resin coupled with 2-mercaptoimidazole(MI) as the ligand was prepared and used to purify IgGfrom porcine plasma. The cellulose matrix was activatedby divinylsulfone (DVS), and was then coupled with MI asthe functional ligand. The reaction conditions wereoptimized as pH: 11, volume ratio of DVS to matrix: 1.0,reaction time: 4 h. The ligand density reached about 100μmol/mL gel. The adsorption isotherms of porcine IgGwas determined at different pH values, and high saturatedadsorption capacities of 78.02 mg IgG/mL gel were foundat pH 8. The adsorption of IgG showed a typical pHdependentproperty of HCIC, and the adsorption capacitydecreased significantly in acidic conditions. The preparedresin was used to separate IgG from porcine plasma. Afterprecipitating the fibrinogen by salting-out, the supernatantwas loaded onto the column at pH 7 and the elution pHwas optimized. The results indicated that acidic elution pHwas necessary to recover the IgG. The purity of IgG in theelution fractions was in the range of 81 ~ 90%, whichdemonstrated that HCIC with the new ligand showed theexcited separation performs and is a potential effectivetechnique to purify IgG from the complex feedstock.
Directed Evolution of Beta-galactosidase from Escherichia coli into Beta-glucuronidase
Xiong, Ai-Sheng,Peng, Ri-He,Zhuang, Jing,Liu, Jin-Ge,Xu, Fang,Cai, Bin,Guo, Zhao-Kui,Qiao, Yu-Shan,Chen, Jian-Min,Zhang, Zhen,Yao, Quan-Hong Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.3
In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli $\beta$-galactosidase and $\beta$-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more $\beta$-glucuronidase activity than wild-type $\beta$-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high $\beta$-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.