European Journal of Biomedical and Pharmaceutical Sciences

EFFECT OF METAL IONS ON MICROBES FOR THE PRODUCTION OF HEPARINASE

*Saumya Gupta and Mahesh M.

ABSTRACT

Heparinase is an enzyme, known to be a coagulant that works by acting on heparin to degrade it and this property of the enzyme finds various uses and applications in medicine. In this study, the effect metal ions have on microbes for the production of heparinase was observed. For this, micro-organisms were isolated from soil samples using soil suspensions and M9 modified media containing heparin by pour plate method. The isolated colonies were screened on the basis of a coagulation test and a bioassay. The organism producing the highest amount of heparinase (2.02 units/ml enzyme) in the enzyme assay was chosen as the test organism and subjected to identification which involved Gram staining and a series of biochemical tests. Optimization of the physical parameters required for the production of heparinase was carried out for each parameter by bioassay method using broth cultures made from M9 modified media containing heparin. The optimum conditions were determined to be an incubation period of 48 hours (7.98 enzyme units/ml) and a pH of 9 (9.19 enzyme units/ml) at 35°C (10.32 enzyme units/ml). Another bioassay was then performed on broth cultures made from M9 modified media containing heparin, incubated at the optimum conditions along with the addition of ZnCl₂, MgCl₂, MnSO₄, NiCl₂ and FeCl₃ trace elements. The addition of FeCl₃ showed the most increase in production of the enzyme (11.97 units/ml enzyme) and upon testing various concentrations of the element, it was found that 500mg of the trace element increased the amount of heparinase even more (14.13 units/ml). The enzyme was then produced at optimum conditions with the addition of FeCl₃ in the broth and was purified by salt precipitation, dialysis, ion exchange chromatography and gel filtration. Characterization was done by SDS-PAGE.

Keywords: Heparinase, purification, ion exchange chromatography, gel filtration, SDS PAGE.