ABSTRACT
A direct acting, extracellular, fibrinolytic enzyme, ~50 KDa from marine Pseudomonas aeruginosa KU1 (PEKU1), was purified. It was found to be a metalloprotease. 60% of the total activity of the purified PEKU1 was retained at 70 °C and the enzyme was practically denatured at 80 °C, 2 h. Metal ions, such as Na+, K+ and Co2+, were found to enhance slightly the fibrinolytic activity, while Fe2+, Mn2+ and Zn2+ were found to be inhibiting. The enzyme showed only less than 5% haemolysis, suggesting its thrombolytic administration safe. Tryptic digestion revealed its similarity to serralysin like alkaline protease of P. aeruginosa. In silico studies showed its binding of protease substrates and fibrin D-dimer in its active site. High affinity binding of bradykinin to the active site of PEKU1, confirmed by in vitro cleaving, suggested its future use as an analgesic. The purified enzyme with Na+, K+ and Co2+, and without Fe2+, Mn2+ and Zn2+ showed thrombolysis in vivo in carrageenan induced murine tail thrombolytic model. The enzyme PEKU1, a novel protease from marine isolate Pseudomonas aeruginosa KU1 has great potential to be developed as a therapeutic agent to combat cardiovascular diseases, as well as analgesic and anti-inflammatory drug in appropriate sites.
Subject(s)
Aquatic Organisms/enzymology , Bacterial Proteins , Hemolysis/drug effects , Peptide Hydrolases , Pseudomonas aeruginosa/enzymology , Thrombolytic Therapy , Thrombosis , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/pharmacology , Cattle , Disease Models, Animal , Male , Mice , Mice, Inbred BALB C , Peptide Hydrolases/chemistry , Peptide Hydrolases/isolation & purification , Peptide Hydrolases/pharmacology , Thrombosis/chemically induced , Thrombosis/drug therapyABSTRACT
Phytase production was studied by three Mucor and eight Rhizopus strains by solid-state fermentation (SSF) on three commonly used natural feed ingredients (canola meal, coconut oil cake, wheat bran). Mucor racemosus NRRL 1994 (ATCC 46129) gave the highest yield (14.5 IU/g dry matter phytase activity) on coconut oil cake. Optimizing the supplementation of coconut oil cake with glucose, casein and (NH(4))(2)SO(4), phytase production in solid-state fermentation was increased to 26 IU/g dry matter (DM). Optimization was carried out by Plackett-Burman and central composite experimental designs. Using the optimized medium phytase, alpha-amylase and lipase production of Mucor racemosus NRRL 1994 was compared in solid-state fermentation and in shake flask (SF) fermentation. SSF yielded higher phytase activity than did SF based on mass of initial substrate. Because this particular isolate is a food-grade fungus that has been used for sufu fermentation in China, the whole SSF material (crude enzyme, in situ enzyme) may be used directly in animal feed rations with enhanced cost efficiency.
