ABSTRACT
Chitinases have been suggested to be involved in pathogen-antagonist interaction during biological control progress of plant pathogenic fungi. Here, a recombinant bacterial chitinase originally from Serratia marcescens B4A was produced, purified, and assayed biochemically to ascertain the activity and determine the kinetics parameters. Active enzyme was used to determine its biocontrol features against fungal phytopathogens. The results demonstrated that the optimum pH and temperature for the enzyme activity were 6.0 and 55 °C, respectively. The K(m) and V(max) values were 3.30 mg ml(-1) and 0.92 units, respectively. The recombinant chitinase was demonstrated to be highly active in controlling fungal pathogens.
Subject(s)
Antifungal Agents/isolation & purification , Bacterial Proteins/isolation & purification , Chitinases/isolation & purification , Serratia marcescens/enzymology , Alternaria/drug effects , Alternaria/growth & development , Antifungal Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Biological Products/isolation & purification , Biological Products/pharmacology , Chitin/metabolism , Chitinases/genetics , Chitinases/pharmacology , Computational Biology , DNA, Bacterial , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Enzyme Assays , Escherichia coli/genetics , Escherichia coli/metabolism , Fusarium/drug effects , Fusarium/growth & development , Hydrogen-Ion Concentration , Microbial Sensitivity Tests , Mycelium/drug effects , Mycelium/growth & development , Phylogeny , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Rhizoctonia/drug effects , Rhizoctonia/growth & development , Serratia marcescens/genetics , TemperatureABSTRACT
Cytoplasmic expression is commonly used for production of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) which most often comes with inclusion body formation. We expressed rhGM-CSF in periplasmic space of Escherichia coli and optimized its extraction by osmotic shock and purification by anion exchange chromatography. Our works show that MgCl2 at 2 mM in osmotic shock buffer improves extraction of the protein and reduces contamination with other proteins. To achieve a simplified purification procedure for rhGM-CSF, efforts were focused on the adjustment of pH of the buffers and application of proper concentration of salt. Following to measurement of the pI of 5.4 for rhGM-CSF by isoelectric focusing, the pH of dialysis buffer and buffers used in anion exchange chromatography were adjusted to 6.5 for optimal binding of the protein to the column and removal of proteins with higher pIs during washing of the column. In addition, it was found that appliance of NaCl at a concentration of 20 mM in dialysis and column washing buffers prior to elution with elution buffer containing 120 mM NaCl significantly improves purification of the protein. Starting with specific amount of total proteins obtained by osmotic shock, it was possible to recover 95% of which following to purification with a purification yield of 72% for rhGM-CSF along with appropriate biological activity.