Production and characterization of staphylokinase enzyme from Staphylococcus aureus ASIA4

Six clinical Staphylococcus aureus strains isolated from different clinical samples. Isolates ASIA1 and ASIA2 isolated from urine samples of urinary tract infected patients; ASIA3 isolated from swab samples of burn abscess patients at Assiut University hospital as well as ASIA4, ASIA5 and ASIA6 obtained from blood samples of different cancer patients at South Egypt Cancer Institute. All isolates showed varied abilities to produce halo zones of hydrolysis with different diameters on blood agar, heated plasma agar, casein agar and skim milk agar plates along with different clot lyses percent. Staphylococcus aureus ASIA3, ASIA4 and ASIA6 produced 4.83, 5.98 and 2.08 U/mL of staphylokinase on tryptone soy broth reduced to 1.95, 2.08 and 1.70 U/mL on casein hydrolysate yeast extract broth, respectively. On the other hand, Staphylococcus aureus ASIA1, ASIA2 and ASIA5 gave 2.20, 2.93 and 3.65 U/mL on CYEB compared to 2.10, 1.88 and 3.41 U/mL on TSB as production medium. The staphylokinase yielded from the hyperactive producer Staphylococcus aureus ASIA4 was increased for 7.64-fold (from 2.08 U/mL to 15.88 U/mL) on the optimized fermentation medium composed of 5.0 g sucrose as carbon source, 10.0 g soy bean as nitrogen source, 5.0 g NaCl, K2HPO4 5.0 g and pH 7.0 that inoculated with isolate ASIA4 and incubated for 24 h at 35 °C. Moreover, Staphylokinase activity reached its peak at the optimal enzymatic reaction conditions which were reaction time 25 min, casein as substrate, reaction pH 8.0, reaction temperature 40 °C. In addition it retained 100% of its activity at temperature ranged between 15 and 45 °C and pH ranged from pH 6.0 to 9.0. EDTA inhibited the enzyme activity by 3.0% to 32.2% with increasing its values from 30.0 to 90.0 mM. MgCl2 at a concentration of 30 mM increased the enzyme activity by 4% and then slightly decreased at higher concentrations but NaCl was potent staphylokinase activator at concentrations lower than 90 mM.