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Isolation and characterization of recombinant helicobacter pylori arginase inhibitor were investigated with the aim of decreasing systhesis of urea that is known for essential requirement for H. pylori growth and increasing bacterial survival inhibition by nitric oxide. So the rocF gene, which encod...

Isolation and characterization of recombinant helicobacter pylori arginase inhibitor were investigated with the aim of decreasing systhesis of urea that is known for essential requirement for H. pylori growth and increasing bacterial survival inhibition by nitric oxide. So the rocF gene, which encodes a H. pylori arginase was amflified from H. pylori genomic DNA using PCR, and cloned into E.coli BL21(DE3) using pET-16b vector. H. pylori arginase had very high activity in standard assay system and purified by Ni-NTA affinity chromatography. The molecular weight of protein was 37 kDa and the purified recombinant arginase was not stable at high temperature. The optimum temperature and pH for reaction was 35℃ and 6.0, respectively. The enzyme activity was increased by Co^(2+), Ni^(2+) and Mn^(2+) ion and had high substrate specificity for L-arginine. To find a inhibitor against H. pylori arginase, I screened various edible seaweed extracts. Detailed studies on the effect in methanol extract of Undrania pinnatifida, Hizikia fusiforme, Acrosorium polyneurum, Enteromorpha intenstinalis were performed by using in vitro assay system and the Hizikia fusiforme extract in methanol showed particularly high H. pylori arginase inhibitory activity. So the methanol extract of Hizikia fusiforme was solvent partitioned with hexane, chloroform and ethyl acetate, respectively, the activity component was separated by silica gel 60 open column chromatography. Then active fraction was separated by TLC and Toyopearl HW-40S gel column. Finally the obtained sample was dissolved in methanol and was performed on HPLC using Waters Symmetry Prep^(TM) C_(18) column. The peak appeared at 225 nm and a significant single peak was seen at 46 min. The isolated sample was analyzed by MALDI-MS and ^(1)H-NMR spectrum to predict chemical structures. Inhibition pattern of PG2b-I purified from Hizikia fusiforme on H. pylori arginase showed non-competitive inhibition and concentration dependent inhibition. The value of Km, Vmax, Ki each is 8.61 mM, 16.9 unit/mg, 2.95×10 ^(-1)mM. The present results suggest that Hizikia fusiforme could play a role in controlling H. pylori survival by inhibiting H. pylori arginase.

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