Journal of Ecobiotechnology 2/4: 11-15, 2010 ISSN 2077-0464 http://journal-ecobiotechnology.com/ Growth Optimization Conditions for the Production of Fibrinolytic Enzyme from Ganoderma lucidum S. Kumaran 1 , L.K. Jagadish 2 , P. Palani 2 , C. Chellaram 1 , T. Prem Anand 1 and V. Kaviyarasan 2* 1 Department of Biomedical Engineering, Veltech Multi TechDr. Rangarajan Dr. Sakunthala Engineering College, Chennai- 6000 61, India 2 Center for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai – 6000 62, India *Corresponding author, Email: kumaran.1331@gmail.com Tel: +91-9840064350; Fax: +91 044 22352494 Keywords Ganoderma lucidum fibrinolytic enzyme Mushroom Abstract Ganoderma lucidum collected and isolated from south eastern part of India screened to produce fibrinolytic enzyme was conserved under laboratory condition to stablize or enhance the enzyme production. The fibrinolytic enzyme producing mushroom Ganoderma lucidum was grown under different pH, temperature, carbon and nitrogen sources to optimize the growth conditions required for enzyme production. The optimized conditions for fibrinolytic enzyme production was found to be pH at 5.5 (106 U/g. w.wt), Temperature at 30°C (88.17 U/g. w.wt), Carbon source - Glucose (143.43 U/g. w.wt) and Nitrogen source- Peptone (162.17 U/g. w.wt). 1. Introduction Fibrinolytic enzymes are agents that dissolve fibrin clots extracted from microbes were considered to be a potential thrombolytic agent for the treatment of clot-dissolving in the cases of Myocardial infarction [1]. Over the past decades, many derived microbial fibrinolytic enzymes have been discovered in various traditional fermented foods [2, 3 and 4], in which mushrooms have captured the attention of a large number of investigators, because of their nutritional and economic value. In addition to that they have high protein content and a variety of proteins that are yet to be discovered. Lectins [5], Laccase [6] and Haemolysin [7] were some examples of biologically active mushroom proteins that have potential application in both medicinal and industrial sectors. Fibrinolytic enzyme from mushrooms are reported from a few medicinal and edible mushrooms such as, Flammulina velutipes [8], Cordyceps militaris [4], and Pleurotus ostreatus [9], Grifola frondosa and Armillaria mellea [10] respectively. Thus, it would be worthwhile when undertaking a work to isolate fibrinolysin from another species like Ganoderma lucidum. Ganoderma lucidum is an important non-toxic medicinal mushroom that has been used in folk medicine for hundreds of years and commercially cultivated for preparation of health tablets [11]. The present investigation would add up biological data to this medicinal mushroom that increase its economic importance. Hence, in the present study Ganoderma lucidum isolated from south eastern part of India was optimized for its fibrinolytic enzyme production under the laboratory condition could be used for characterization in detail. 2. Materials and Methods Mushroom The basidiocarps of Ganoderma lucidum collected from south eastern part of India, Chennai. Identification and classification were carried out and the specimens were deposited at the laboratory, CAS in Botany, University of Madras, Guindy Campus, Chennai - 25. Assay of fibrinolytic activity Fibrinolytic protease activity was carried out according to the method described by Greenberg [12]. The reaction mixture contained 8 mg bovine fibrin, 500 µl mycelial extract in phosphate buffer, (0.05 M, pH.6.8) in a total volume of 1 mL. This mixture was incubated for 30 min at 37°C in a water bath. The reaction was stopped by adding 0.5 mL of 15% cold trichloro acetic acid (TCA) in glass distilled water. The mixture was centrifuged at 3,000g for 10 min to remove precipitated fibrin. To 0.5 mL of acid soluble filtrate 2.5 mL of 0.3 N sodium hydroxide and 2.9% (w/v) sodium carbonate in glass distilled water was added, followed by 0.75 mL of Folin’s phenol reagent (diluted 1:3 with glass distilled water). The mixture was incubated for 25 min at room temperature and the color developed was read at 650 nm [13]. The above said procedure was followed with heat killed enzyme and kept as blank. One unit of enzyme activity was calculated as the amount of enzyme which releases 1 µmol of tyrosine/ min under the specified reaction conditions. Effect of different ph and temperature on fibrinolytic activity Fibrinolytic activity of Ganoderma lucidum grown under different pH and temperature in Modified