Optimization of media for mass production of Xanthine Oxidase from endophytic fungus. A Thesis submitted In partial fulfillment of the requirement of the degree of MASTER OF SCIENCE IN BIOTECHNOLOGY Submitted By: Under the supervision of Pallavpreet kaur Dr. Sanjai Saxena Department Of Biotechnology And Environmental Sciences Thapar University, Patiala July, 2012 1 2 3 Contents S.no Contents Page no. 1. Executive summary 2. Introduction 3. Review of literature 4. Aim of study 5. Materials and methods 6. Results & discussion 7. Conclusion 8. Bibliography 4 List of Tables S.no Table No Page no. 1. Composition of reaction mixture for NBT assay 40 2. Composition of Lowry Reagent 43 3. Estimation of uric acid at 292 nm 52 4. Estimation of Uric acid by phosphotungstate assay 54 5. The optimized nutritional and physical parameters 55 6. The Placket –burman design 56 7. ANOVA for placket – burman design 59 8. Response Surface methodology 62 9. ANOVA for Response surface methodology 62 10. Coefficient estimate and standard error in RSM 63 11 XO activity in unoptimized and optimized media 65 12 The values of optimized parameters 65 13 Purification of XO from Endophytic fungus #19 NOBASVNP 69 14 The kinetic parameters of Purified XO. 69 5 List of figures S.no Figure no. Page no. 1. Crystallographic structure of XO 16 2. Purine degradation pathway 18 3. Template of microtiter used for standard curve of XO 39 activity 4. Template of microyiter for XO inhibition by allopurinol 41 5. Pure culture of Endophytic fungus strain #19NOBASVNP 46 o 6. Culture filtrate fermented at 29 C 49 7. The Pareto Chart 58 8. The Range of XO activity 60 9. The effect of Xanthine concentration on XO activity 61 10. 3 D plot of XO activity Vs pH and sucrose percentage 64 11. Prediction of the XO activity 65 12. XO inhibition assay using culture filtrate 66 13. XO inhibition assay using purified XO 71 6 List of Graphs S.no Graph no. Page no. 1. Optimization of Xanthine concentration 47 2. Optimization of sucrose percentage 48 3. Optimization of Potato extract 48 4. Optimization of temperature 49 5. Optimization of temperature 49 6. Optimization of pH 50 7. Optimization of inoculums size ( cells/ml) 50 8. Standard Curve of uric acid 51 9. XO inhibition assay in presence of allopurinol 53 10. Standard curve of uric acid by phosphotungstate. 67 11. XO activity in presence of allopurinol 67 12. XO activity of eluates from gel –filtration column 68 13. Line-weaver burk plot 70 14. The effect of Xanthine concentration on XO activity 70 15. The effect of temperature on XO activity 71 16. XO activity in presence of allopurinol 72 17. XO activity in absence of allopurinol 72 7 Abbreviations BSA Bovine Serum Albumin FAD Flavin Adenine Dinucleotide KDa Kilodalton mg milligrams PDA Potato Dextrose Agar ROS Reactive oxygen pecies rpm Revolutions per minute SDW Sterile distilled water PAGE Polyacrylamide gel electrophoresis PDB Potato Dextrose Broth NBT Nitroblue Tetrazolium µl Microlitre µM Micromoles XA Xanthine Agar XB Xanthine Broth XO Xanthine Oxidase XDH Xanthine Dehydrogenase XOR Xanthine Oxidoreductase nm Nanometer RSM Response surface Methodology PBD Placket Burman Design ABTS 2, 2 –azino –di (3- ethylbenthiazoline-6- sulphonate ) 8 Executive summary Xanthine Oxidase has been in lot of demand in clinical industry due to its crucial role in purine metabolism pathway. Various ailments like gout, hyperuricemia, myocardial infarction and reperfusion injury are related to the levels of expression of Xanthine Oxidase .A plethora of questions that need to be answered regarding the implication of genes in XO related disorders and mechanism of action and metabolic pathway of oxidoreductase class of enzymes. Xanthine Oxidase can also find application in the development of biosensors for monitoring substrate utilization in cell culture media. #19 NOBASVNP, an Endophytic fungus isolated from Nerium oleander , demonstrated the maximum potential to produce Xanthine Oxidase. The pure strain of this Endophytic fungus was grown on synthetic media to assess the yield and activity of Xanthine Oxidase. The natural media demonstrated higher ability to produce XO than synthetic media. The natural media was optimized by conventional methods optimizing one parameter at a time and by use of design expert version 8. The optimized media gave higher XO activity than unoptimized media. The enzyme activity of crude enzyme precipitate obtained from culture filtrate was assessed by Nitro blue tetrazolium assay. The crude enzyme was purified by gel filtration chromatography to achieve 34% fold purity. The various kinetic parameters of the partially purified enzyme were also deduced.Further studies on full- purification and characterization of enzyme would allow the commercial exploitation of enzyme from Endophytic fungus. 9 Chapter 1 Introduction 10 1. Introduction Purines are heterocyclic aromatic organic compounds, consisting up of pyrimidine ring fused to an imidazole ring. There are many naturally occurring purines like Adenine, Guanine, uracil hypoxanthine and isoguanine. Aside from the pivotal role of purines in DNA and RNA , purines also act as significant components in a number of other important biomolecules such as ATP, GTP, cyclic AMP, NADH and coenzyme A. The purine metabolism is essential to the body, it provides components of nucleic acids, DNA and RNA, and produces ubiquitous metabolites like ATP. Purine catabolism leads to the formation of a poorly soluble compound Uric acid. The reference range of uric acid in human blood plasma is 3.6mg/dL to 8.3mg/dL. Elevated levels of uric acid leads to hyperuricemia which is linked to Gout. Xanthine Oxidase, the major enzyme of purine metabolic pathway that control the rate limiting step of the pathway i.e. the conversion of hypoxanthine to uric acid via Xanthine with the concomitant production of reactive oxygen species (ROS). Reactive oxygen species are chemically reactive species that are formed as a natural byproduct of normal metabolism oxygen and have important role in cell signaling and homeostasis. Xanthine Oxidase is highly versatile and ubiquitous complex molybdoflavoprotein that oxidizes Xanthine to uric acid and transfer electrons to NAD + (Xanthine dehydrogenase ) or oxygen (Xanthine Oxidase). Xanthine Oxidase (XO) and Xanthine Dehydrogenase (XOD) are interconvertible forms of the same enzyme Xanthine Oxidoreductase (XOR) (Hille and Nishano,1995). These different forms of enzyme are conserved in living organisms including archea, bacteria, fungi, plants and metazoans (Amit Aggarwal et al..2009). XOR is widely distributed throughout various organs including liver, intestine, kidney, lungs, myocardium, brain, plasma. The enzyme consist up of 2 monomers each independently capable of catalysis. XO and XDH are capable of detoxification of endogenous compounds apart from oxidation of purines (Borges et al., 2002). The Oxidase form of Xanthine utilizing enzyme uses oxygen as + electron acceptor whereas dehydrogenase uses NAD (although it is capable of utilizing O2.).The presence of NAD+ binding site constitutes the primary difference between the dehydrogenase and Oxidase. Xanthine Oxidase family, commonly referred to as molybdenum hydroxylases, 11 which catalyze the hydroxylation of large variety of substrates ranging from aldehydes to aromatic heterocyclic. Members of this enzyme family have the same basic constitution of redox active centers: a molybdenum centre, two 2 Fe/2S centre’s of the ferrodoxin variety and one flavin adenine dinucleotide. The elevated levels of Xanthine Oxidase can lead to various pathological conditions like inflammation, carcinogenesis, ischemia-reperfusion and hepatitis. The level of Xanthine Oxidase is elevated in injured human skeletal muscle in association with inflammatory events (Ylva Hellesten et al.,1997). Thus there is utmost need to explore various inhibitors of Xanthine Oxidase. Allopurinol, structural isomer of hypoxanthine is being used to reduce the uric acid level and for the treatment of Gout. Due to the unavailability of free XO in the blood, diverse sources were exploited for the isolation of enzyme so as to design suitable xanthine oxidase inhibitors. On the other hand , Type I Xanthinuria, a condition due to loss of Xanthine Oxidase activity is due to mutations in genes encoding Xanthine oxidoreductase. Type II Xanthinuria results from loss of both Xanthine Oxidase and aldehyde Oxidase activities. Patients with type I and type II xanthinuria are asymptotic. The condition is due to inability to synthesize unique pterin co factor cheated to molybdenum and result in loss of activity of all mononuclear molybdenum enzymes. Xanthine Oxidase have been isolated and purified from bovine milk. Bovine milk Xanthine Oxidase is commercially available and is used for determination of Xanthine, hypoxanthine and as an auxiliary enzyme for determination of guanine. It has also been suggested that Xanthine Oxidase, due to wide range of substrate specificity may play a role in detoxification. Xanthine Oxidase may detoxify potentially oncogenic and dangerous compounds such as Purine N Oxidase by oxidizing them to less toxic form. It has also been suggested that uric acid may function as anti-oxidant and radical compounds by scavenging singlet oxygen and radicals. Milk is a rich source of Xanthine Oxidase (Ball, 1938). The probable role of enzyme in milk is its antibacterial activity by virtue of its ability to produce oxygen derived free radicals (Massey et 12 al., 1968). The Xanthine Oxidase from cow’s milk crude or purified, appears as Oxidase (type O) and can be converted almost completely into a NAD+ dependant dehydrogenase (type D) by treatment with dihydrolipoic acid, but only to some extent by other thiols. The D form of enzyme is inhibited by NADH which competes with NAD+. The kinetic constants of the two forms of enzyme are similar to those of the corresponding form of rat liver Oxidase. The enzyme on electrophoresis shows a major faster band and a minor slower band on gel electrophoresis. Xanthine Oxidase has also been isolated and purified from Bovine Small intestine (G.G.Roussos et al., 1956).