Lehe Mei

A modified NRTL equation for the calculation of phase equilibrium of polymer solutions

A local composition model based on the lattice theory, which is a modified non-random two-liquid (NRTL) equation, is developed for the representation of the Helmholtz free energy of polymer solutions. A simple temperature dependence of the model parameters is introduced to account for the oriented interactions without changing the general formulation of the free energy of mixing. The model provides a flexible thermodynamic framework for both correlating and predicting the phase equilibrium of polymer solutions. Special attention is paid to the representation of phase behavior of homologous polymer solutions and systems with or without oriented interactions.

An improved isopiestic method for measurement of water activities in aqueous polymer and salt solutions

Abstract The isopiestic method was described, and its advantages and disadvantages were discussed. A nine-legged glass apparatus was developed and the experimental procedure was improved. The time for equilibration was greatly reduced to 3 or 4 d, even for the high viscous solutions. The average relative error in the observed water activities for standard electrolyte solution was ±0.01%. The water activities of PEG1000 + H 2 O, PEG4000 + H 2 O, PEG + MgSO 4 + H 2 O and PEG + (NH 4 ) 2 SO 4 systems were measured at 25°C.

Purification and Characterization of Glutamate Decarboxylase of Lactobacillus brevis CGMCC 1306 Isolated from Fresh Milk

Abstract A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase (GAD) activity. An effective isolation and purification procedure of GAD from a cell-free extract of Lactobacillus brevis was developed, and the procedure included four steps: 30%—90% saturation (NH 4 ) 2 SO 4 fractional precipitation, Q sepharose FF anion-exchange chromatography, sephacryl S-200 gel filtration, and resource Q anion-exchange chromatography. Using this protocol, the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purification fold and activity recovery of GAD were 43.78 and 16.95%, respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37°C, respectively. The purified GAD had a half-life of 50min at 45°C and the K m value of the enzyme from Lineweaver-Burk plot was found to be 8.22. 5′-pyridoxal phosphate (PLP) had little effect on the regulation of its activity.

Prediction of liquid-liquid equilibria of polymersalt aqueous two-phase systems by a modified Pitzer's virial equation

Abstract A modified Pitzers virial equation, which combines the long-range electrostatic contribution from Fowler and Guggenheim with the short-range virial equation, is presented for the excess Gibbs free energy of some special kinds of systems. The model is used to predict the liquid-liquid equilibrium (LLE) at 298.15 K of three polymer-uni-bivalent salt-aqueous two-phase systems: polyethylene glycol(PEG)(NH4)2SO4, PEGNa2SO4, PEGNa2CO3, by correlating the parameters from the corresponding vapor-liquid equilibrium (VLE) data. The results show that the model can give excellent representation of the VLE data of PEG solutions, either binary or ternary, the model parameters being independent of the chain lengths of the polymers. The comparison between the predicted and experimental phase diagrams shows that the suggested model has good prediction ability for PEG-uni-bivalent salt systems.

Modeling the protein partitioning in aqueous polymer two-phase systems: influence of polymer concentration and molecular weight

Aqueous polymer two-phase system provides a powerful method for separation and purification of biomaterials. Among various factors, polymer concentration and polymer molecular weight are essential and have strong impact on the protein partitioning in these systems. Based on the modified Pitzers model, a simple expression has been obtained for correlating protein partitioning in aqueous polymer two-phase systems with varying polymer concentration and different polymer molecular weights. Using only one group of parameters for each target protein, the partition coefficients of five proteins (lysozyme, chymotrypsinogen-A, bovine serum albumin, transferrin and catalase) in 16 sets of polyethylene glycol (PEG)/dextran systems were correlated. In addition, partition behavior of lactate dehydrogenase in PEG/hydroxypropyl starch systems were measured, correlated and partially predicted. A comparison of calculated and experimental data indicated that the model provides good correlation and prediction abilities on the protein partitioning in aqueous polymer two-phase systems with a wide range of polymer concentration and molecular weight.

A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase

A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of L-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pK(a) to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.

Separation of nattokinase fromBacillus subtilis fermentation broth by expanded bed adsorption with mixed-mode adsorbent

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 feedstockvia 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 ofBacillus 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 betweenBacillus subtilis cells and Fastline PRO, and to demonstrate a stable expanded bed in the feedstock containingBacillus 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.

C-terminal truncation of glutamate decarboxylase from Lactobacillus brevis CGMCC 1306 extends its activity toward near-neutral pH.

Glutamate decarboxylase (GAD) from Lactobacillus brevis is a very promising candidate for biosynthesis of GABA and various other bulk chemicals that can be derived from GABA. However, no structure of GAD of this origin has been reported to date, which limits enzyme engineering strategy to improve its properties for better use in production of GABA. Bacterial GAD exhibits an acidic pH optimum and there is often a sharp pH dependence. In the present work, site-directed mutagenesis was performed to delete the C-terminal residues of GAD to generate a mutant, designated as GADΔC, which exhibited extended activity toward near-neutral pH compared to the wild type. Comparison of the UV-visible, fluorescence and Circular Dichroism spectra of the mutant with those of the wild type revealed that the microenvironment of the active site had been changed. Based on the homology model, we speculated that the substrate entrance was probably enlarged in GADΔC. These results provide evidence for the important role of C-terminal region in the pH-dependent regulation of enzyme activity, and the resulting mutant would be useful in a bioreactor for continuous production of GABA.

Affinity extraction of lactate dehydrogenase by aqueous two-phase systems using free triazine dyes

Affinity partitioning of lactate dehydrogenase (LDH) was studied in polyethylene glycol (PEG) /salt and PEG / hydroxypropyl starch (PES) aqueous two-phase systems, using free triazine dyes as their affinity ligands. The free dyes showed one-sided partition to the top PEG-rich phase and thus enhanced the affinity partitioning effect in the systems. A two-step affinity extraction process has been discussed for large scale purification of LDH from rabbit muscle.

Production of Indigo by Immobilization of E. coli BL21 (DE3) Cells in Calcium-Alginate Gel Capsules

Abstract The ability of catalyzing indole into indigo of gene engineering strain expressing P450 BM3 immobilized by entrapment in calcium-alginate gel capsules was examined, and various characteristics of immobilized cells were assessed. Optimum conditions for cells activity were not affected after immobilization, and pH and temperature for both free and immobilized cells were found to be pH 7.5 and 35°C, respectively. The immobilized cells exhibited a markedly improved thermal stability than free cells. After five repeated experiments, the yield of indigo with the immobilized cells retained over 94% of their original activity, which indicated that the operational stability for recycling in batch processes was improved.