Purification and Characterization of Hyaluronidase Produced from Staphylococcus aureus Local Clinical Isolates

A total of one hundred clinical samples were collected from two different hospitals (Al Yarmouk Teaching Hospital and Child Central Hospital) and a number of Al- Harthiya laboratories in Baghdad. The primary diagnostic results showed that 34 bacterial isolates were Gram positive bacteria and 66 were Gram negative according to their morphological and cultural characteristics. From the 34 Gram positive isolates, 25 were confirmed by the biochemical test as Staphylococcus aureus, 6 isolates S. epidermidis and 3 isolates S. saprophyticus. S.aureus isolates were tested for their ability to produce hyaluronidase enzyme. Results showed that all the isolates were hyaluronidase producer; among them, S. aureus isolate S2 obtained from urine was the most efficient in hyaluroniase production, when the enzyme specific activity of its crude filtrate was 96 U/mg protein. According to these results, S. aureus S2 isolate was selected to determine the optimum conditions for hyaluronidase production. Result of the optimum conditions for hyaluronidase production for the clinical isolate of S. aureus (S2) were showed that the maximum hyaluronidase production was achieved after supplementing the production medium of pH7 with 0.5% of both of lactose and yeast extract, an inoculums size of a fresh bacterial culture of 107cell/ml, and an incubation temperature of 37oC for 24 hours. Under these conditions, specific activity of hyaluronidase produced in the culture medium was increased to 125 U/mg protein. Hyaluronidase produced at the obove optimum conditions was purified in three purification steps, first by precipitation with 90% of saturated ammonium sulfate and then dialyzed by (dialysis tube 10000 MW), ion exchange chromatography by (DEAE-Cellulose), and the last step use gel filtration chromatography throughout Sephacryl S-300. Specific activity of the purified enzyme was 930 U/mg protein with 7.4 folds of purification and 46.5% recovery. Some characteristics of the purified enzyme was determined and the results showed that hyaluronidase enzyme has an average molecular weight of about 69 kDa, with an optimum pH of enzyme activity and stability at pH 7, and optimum temperature for activity was found to be 37oC. The enzyme was stable with full activity at a temperature ranged between (30-40)oC.
The effect of chelating and reducing agents and heavy-metal ions on purified when hyaluronidase activity was investigated, results indicated that the enzyme activity was inhibited in different ratio by using inhibitor and metal ions. The effect of both purified hyaluronidase enzyme and its crude extract was studied against cancer cell Hep G2 type at different concentrations (10, 5, 2.5, 1.25, 0.625, 0.352) using different exposure times (24, 48, 72 hrs). The result showed that the inhibition rate of both crude and purified enzyme increased by increasing the concentration, while no change was noticated at 24hr, and the inhibition rates at both 48 and 72 hrs were the same for purified enzyme but were different for the crude extract.