Process Development for the Production and Purification of PEGylated RhG-CSF Expressed in Escherichia coli.

BACKGROUND AND OBJECTIVES: Recombinant human granulocyte-colony stimulating factor (rhG-CSF) and its PEGylated form (PEG-GCSF) are used in cancer therapy. Thus, developing a more cost-effectively method for expressing rhG-CSF and the PEGylation optimization of rhG-CSF by reaction engineering and subsequent purification strategy is necessary. METHODS: RhG-CSF expression in Escherichia coli BL21 (DE3) was carried out by auto-induction batch fermentation and improved for maximizing rhG-CSF productivity. After that, purified rhGCSF was PEGylated using methoxy polyethylene glycol propionaldehydes (mPEG20-ALD). The various conditions effect of extraction and purification of rhG-CSF and PEG-GCSF were assayed. RESULTS: The assessment results revealed that the auto-induction batch cultivation strategy had maximum productivity, and rhG-CSF purity was more than 99%. The obtained data of rhG-CSF PEGylation displayed that the optimized conditions of rhG-CSF PEGylation and purification enhanced homogeneity PEG-GCSF and managed reaction toward optimal yield of PEG-GCSF (70%) and purity of 99.9%. Findings from FTIR, CD, fluorescence spectroscopy, and bioassay revealed that PEGylation was executed exactly in the rhG-CSF N-terminus, and products maintained their conformation properties. CONCLUSION: Overall, the developed approach expanded strategies for high yield rhG-CSF by simplified auto-induction batch fermentation system and rhG-CSF PEGylation, which are simple and timesaving, economical, and high efficiency.

Continuous biodegradation of parathion by immobilized Sphingomonas sp. in magnetically fixed-bed bioreactors and evaluation of the enzyme stability of immobilized bacteria.

Magnetically-modified Sphingomonas sp. was prepared using covalent binding of magnetic nanoparticles on to the cell surface. The magnetic modified bacteria were immobilized in the fixed-bed bioreactors (FBR) by internal and external magnetic fields for the biodetoxification of a model organophosphate, parathion: 93 % of substrate (50 mg parathion/l) was hydrolyzed at 0.5 ml/min in internal magnetic field fixed-bed bioreactor. The deactivation rate constants (at 1 ml/min) were 0.97 × 10(-3), 1.24 × 10(-3) and 4.17 × 10(-3) h(-1) for immobilized bacteria in external and internal magnetic field fixed-bed bioreactor and FBR, respectively. The deactivation rate constant for immobilized magnetically modified bacteria in external magnetic field fixed-bed bioreactor (EMFFBR) was 77 % lower than that of immobilized cells by entrapping method on porous basalt beads in FBR at 1 ml/min. Immobilized magnetic modified bacteria exhibited maximum enzyme stability in EMFFBR.

Immobilization of magnetic modified Flavobacterium ATCC 27551 using magnetic field and evaluation of the enzyme stability of immobilized bacteria.

The magnetic modified Flavobacterium sp. was prepared by covalently binding carboxylate-modified magnetic nanoparticles, and also, ionic adsorption of magnetic Fe(3)O(4) nanoparticles on the cell surface. The magnetic modified bacteria were immobilized by both internal and external magnetic fields. The pH stability and inherent resistance of the enzyme activity of the immobilized bacteria under acidic and alkaline conditions were increased. Immobilization of the magnetic modified bacteria using an external magnetic field improved the enzyme thermal stability. The results revealed that immobilization of the magnetic modified bacteria by an external magnetic field keeps 50% of the enzyme activity after 23.4, 16.6 and 6 h of incubation at 55 °C for the covalently binding of magnetic nanoparticles, the ionic adsorption of magnetic nanoparticles and the free cells, respectively. The results demonstrated the negative effect of various magnetic beads on the enzyme thermal stability of immobilized magnetic modified bacteria using an internal magnetic field.