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We approve the thesis of Ece YAPA AN, ,Assoc Prof Dr Talat YAL IN. Supervisor, ,Assist Prof Dr Alper ARSLANO LU,Co Supervisor. ,Assoc Prof Dr Ahmet KO ,Committee Member, ,Assist Prof Dr G l ah ANLI. Committee Member,19 December 2008, ,Prof Dr Levent ARTOK Prof Dr Hasan B KE. Head of the Chemistry Department Dean of the Graduate School of. Engineering and Science, ACKNOWLEDGEMENTS, First of all I would like to thank my supervisor Assoc Prof Dr Talat YAL IN .
for his excellent guidance understanding encouragement and help throughout this. study , Also I would like to express my grateful thanks to my co supervisor Assist Prof . Dr Alper ARSLANO LU for his help and valuable comments during thesis study . Special thanks are extended to Assist Prof Dr G l ah ANLI for her valuable. suggestions comments and kind helps I want to thank to Molecular Microbiology . Molecular Genetics and Biological Chemistry Laboratory members . I express my thanks to my searching team P nar BAYDARA a da . TA O LU and Ahmet Emin AT K for their advise and helps . I am also grateful to my laboratory friends Erhan BAL Melda G RAY Burcu. NSAL Burcu ENGEZ and Aysun ADAN for their sincere help and kindness during. studies , Finally I want to express my gratitude to my family Thanks for all their. motivation encouragement and their support throughout not only this study and also all. through my life Lastly I express my special thanks to my fianc e for his patience . supports and technological helps during thesis study . ABSTRACT, PARTIAL PURIFICATION AND CHARACTERIZATION OF LIPASE. ENZYME FROM A Pseudomonas STRAIN, Lipase is a triacylglycerol hydrolyzing enzyme which is catalyzed the hydrolysis. of water insoluble free fatty acid and glycerols and also a wide range of chemical. reactions Beside microbial lipases show regiospecificity and enantioselectivity. properties Therefore microbial lipases gain the great importance for industrial. applications and organic synthesis In this study investigation partial purification and. characterization of lipase enzyme from a Pseudomonas strain was studied by using. different analytical approach , Purification step was done by size exclusion chromatography The molecular.
weight of partial purified lipase was determined by SDS PAGE Spectrophotometric. lipase assay applied to find out the enzyme characterization Kinetic study of enzyme. was also investigated varying the substrates concentrations Specific activity staining on. gel procedures applied after native gel process After electrophoresis lipase activity. responsive protein bands were appeared on gel , After screening for the presence of lipase activity in Pseudonomas strain which. was isolated from soil it was decided to choose intracellular enzyme sample for. characterization and purification studies The enzyme gave the highest lipase activity. when p nitrophenyl laurate used as a substrate The optimum pH range for activity of. lipase was alkaline pH ranges about pH 8 0 and 9 0 The optimum temperature was. dedicated as 25oC In the presence of metal salts and organic solvents while some. additives sharply decreased enzyme activity some additives were not effect the enzyme. activity Approximate molecular mass of partially purified enzyme was between 29 kDa. and 43 kDa , iv, ZET, Pseudomonas SU UNDAN L PAZ ENZ M N N KISM . SAFLA TIRILMASI VE KARAKTER ZASYONU, Lipazlar enzimi tria ilgliserol hidrolaz bir enzim olup suda z nmeyen serbest. ya asitlerin ve gliserollerin hidrolizini ve bir ok kimyasal reaksiyonu katalizlerler . Dahas mikrobial olarak retilen bir lipase enzimi organik sentezlerde enantiomerik. se icili i ve zel b lge ba lanmalar ya da k r lmalar se icili i g sterir Bu y zden. mikrobial olarak retilen lipaz enzimi end striyel uygulamalarda ve organik sentez. tasar mlar nda nem kazanm t r Bu al mada Pseudonomas su undan elde edilen. lipaz farkl analitik y ntemler kullan larak varl kan tlam k smen safla t r lm ve. karakterizasyonu yap lm t r , Safla t rma i lemi boyut d lamal kromatografi kullan larak yap lm t r K sm . olarak safla t r lan enzimin molek ler a rl poli akrilamit jel elektroforezi. kullan larak tespit edilmi tir Spektrofotometrik al ma ile enzim karakterizasyonu. yap lm t r Ayr ca bunun yan nda enzimin kinetik al mas da yap lm t r . Elektroforez i leminden sonra potansiyel lipase activitesi g steren protein bantlar gel. zerinde g r nt lenmi tir , Topraktan izole edilmi Pseudonomas su unun farkl denemelerde ortaya kan.
lipaz aktivite sonu lar g zlendikten sonra enzimin h cre i inde al an bir enzim. oldu una ve yap lacak safla t rma ve karakterizasyon al malar n n bu bilgi g z n ne. al narak yap lmas na karar verilmi tir Enzim p nitrofenil laurate in substrat olarak. kullan ld al malarda en y ksek enzim aktivitesini g stermi tir Lipaz enziminin. al mas i in en uygun pH aral n n pH 8 0 9 0 civar nda alkali pH aral nda oldu u. tespit edilmi tir Uygun s cakl k de eri ise 25oC olarak belirlenmi tir Metal tuzlar ve. organik z c ler varl nda enzim aktivitesi kimi katk bile ikleri varl nda h zl bir. ekilde d erken kimi katk bile ikleri varl nda etkilenmeden sabit kalm t r K sm . olarak safla t r lm lipaz enziminin yakla k molek l a rl 23 kDa ve 49 kDa. aral nda bulunmu tur , v, TABLE OF CONTENTS,LIST OF FIGURES ix. LIST OF TABLES x,CHAPTER 1 INTRODUCTION 1, 1 1 Amino acids peptides and proteins 1. 1 1 1 Physical and Chemical Properties 1, 1 1 2 Protein purification 2. 1 1 3 Size exclusion chromatography 4, 1 2 Enzymes 5. 1 3 Lipases 6, 1 3 1 General Description 6, 1 3 2 Lipase as biocatalysts 6.
1 3 3 Bacterial Lipases 7, 1 3 4 Fermentation conditions 9. 1 3 5 Purification of bacterial lipases 9, 1 3 6 Properties of lipases 10. 1 4 Lipase assay 10, 1 4 1 Activity staining for lipases 12. 1 5 Pseudomonas lipases 13,CHAPTER 2 PROTEIN SEPERATION 15. 2 1 Electrophoresis 15, 2 1 1 Electrophoretic support media Polyacrylamide gels 15.
2 2 Protein separation 16, 2 2 1 Two dimensional SDS PAGE 17. 2 2 2 One dimensional SDS PAGE 18, 2 3 Stains and dyes 18. 2 4 The Aim of the Study 19, vi, CHAPTER 3 EXPERIMENTAL 21. 3 1 Bacteria Growth Conditions 21, 3 2 Monitorize Lipase Activity on Agar Plates 21. 3 3 Protein Sample Preparation 22, 3 4 Enzyme Characterization 22.
3 4 1 Substrate specificity of the Bacterial Lipase 22. 3 4 2 Effect of Temperature and pH on Activity 23. 3 4 3 Organic solvent and Metal Ion Effect 23, 3 5 Kinetic study 24. 3 6 Molecular Weight Determination 24, 3 7 Enzyme Activity Calculation 24. 3 8 Bradford Total Protein Assay 25, 3 9 SDS PAGE 26. 3 9 1 Activity Staining on Gel 28, 3 10 Size exclusion Chromatography 29. CHAPTER 4 RESULT AND DISCUSSION 31, 4 1 Investigation of lipase activity on agar plates 31.
4 2 Activity Staining on Gel 32, 4 3 Size exclusion Chromatography 34. 4 4 Visualization of Lipase Activity 34, 4 5 Enzyme Characterization 34. 4 5 1 Substrate Specificity 34, 4 5 2 Optimum pH and Temperature 36. 4 5 3 Effect of Metal Ion on Enzyme Activity 38, 4 5 4 Effect of Organic Solvents on Enzyme Activity 39. 4 6 Kinetic Study of Lipase Enzyme 40, 4 7 Molecular Weight Determination 41.
CHAPTER 5 CONCLUSION 43,REFERENCES 44, vii, APPENDICES. APPENDIX A BRADFORD ASSAY CALIBRATION GRAPH 49, viii. LIST OF FIGURES,Figure Page,Figure 1 1 General structure of amino acids 1. Figure 1 2 Levels of structure in proteins 2, Figure 1 3 a shematic representation of size exclusion chromatography b . schematic representation of ion exchange chromatography 3. Figure 1 4 Shematic representation of dialysis process 4. Figure 1 5 Hydrolysis or synthesis of a triacylglycerol substrate catalysed by. a lipase enzyme 7, Figure 1 6 Lipase positive colonies on agar containing Rhodamine B dye.
and olive oil 11, Figure 2 1 Reaction of polyacrylamide gel formation 16. Figure 2 2 Two dimensional electrophoresis 17,Figure 2 3 Polyacrylamide gel electrophoresis 18. Figure 2 4 Coomassie brilliant blue G 250 19, Figure 3 1 Enzyme activity calculation equation 25. Figure 3 2 Size exclusion chromatography system 30. Figure 4 1 Photos of colonies under UV light grown at 4oC 31. Figure 4 2 Photos of a Pseudomonas strain coloniesin the presence of Tween 80 32. Figure 4 3 Gel images 33, Figure 4 4 Absorbance values of substrate specificity test 35. Figure 4 5 a Enyzme activity and b relative enzyme activity values aganist. substrates containing different number of carbon chain leghts 35. Figure 4 6 a Relative enzyme activity and b enzyme activity values at different. pH Values 36, Figure 4 7 a Absorbance and b enzyme activity values at diffferent temperatures 38.
Figure 4 8 Relative enzyme activity values in the presence of metal salts 39. Figure 4 9 Relative enzyme activity values in the presence of organic solvents 40. Figure 4 10 Absorbance values of kinetic study 40,Figure 4 11 Graph of kinetic study 42. Figure 4 12 Gel image under white light 43, ix, LIST OF TABLES. Table Page, Table 1 1 Chromatography techniques which used for protein purification. according to their properties 2, Table 1 2 Pseudomonas lipases for biotechnological applications 14. Table 3 1 List of used p nitrophenly fatty acyl esters at various chain leghts 23. Table 3 2 Preparation of BSA standars 26, x, ABBREVIATIONS.
BSA Bovine Serum Albumin,CBB Commasie brillant blue. CE Capillary electrophoresis,DNA Deoxyribonucleic Acid. EDTA Ethylenediamine tetra acetic acid,HPLC High performance liquid chromatography. IPG Immobilized pH gradient,IEF Isoelectric focussing. LB Luria Bertani,MM Minimal medium,Mr Molecular weight.
PAGE Polyacrylamide gel electrophoresis,RNA Ribonucleic acid. SEC Size exclusion chromatography,SDS Sodium dodecyl sulfate. TEMED Tetramethylethylenediamine,UV Ultra violet,1 D One dimension. 2 D Two dimension,3 D Three dimension, xi, CHAPTER 1. INTRODUCTION,1 1 Amino Acids Peptides and Proteins.
1 1 1 Physical and Chemical Properties, A major role for many sequences of DNA is to encode the sequences of proteins . Proteins are the most abundant biological macromolecules They are produced in all. cells Proteins also occur in great variety different kinds ranging in size from relatively. small peptides to polymers Moreover proteins exhibit variety of biological function . All proteins are constructed from the same ubiquitous set of 20 amino acids These. amino acids covalently linked each other in a characteristic linear sequences Cells can. produce proteins with different properties and activities by joining the same 20 amino. acids in many different combinations and sequences Amoung these protein products . the enzymes are the most varied and specialized All cellular reactions are catalyzed by. enzymes All 20 of amino acids are common in structure carboxyl group and an amino. group bonded to the same carbon atom the carbon They differ from each other in. their side chains or R groups R groups define structure size electric charge polaritiy. and the solubility of the amino acids , Figure 1 1 General structure of amino acid . The peptides and proteins are the polymers of amino acids Two or more amino. acid molecules can be covalently joined through a peptide bond to yield a dipeptide . oligopeptide and polypeptide Four levels of protein structure are commonly defined A. description of all covalent bonds peptide bonds and disulfide bonds linking amino acid. residues in a polypeptide chain is its primary structure Secondary structure is. 1, particularly stable arrangements of amino acid residues giving rise to recurring. structural patterns Tertiary structure describes the three dimensional folding of a. polypeptide When a protein has two or more polypeptide subunits their arrangement in. space is called as quaternary structure Nelson and Cox 2008 . Figure 1 2 Levels of structure in proteins, Source Nelson and Cox 2008 . 1 1 2 Protein Purification, A pure protein sample is important before determining protein s properties and.
activities Different kinds of methods are used for purification process according to. protein s properties such as size charge and binding properties Nelson and Cox 2008 . Biomolecules are purified using chromatography techniquies that seperate them. according to differences in their specific properties The application of chromatography. to the study of both large and small biological molecules gain great importance in recent. years Chromatography allows the isolation of substance in very small amounts and in. non denaturating conditions , Table 1 1 Chromatography techniques which used for protein purification according to. their properties , Property Technique, Gel filtration size exclusion . Size chromatography, Charge Ion exchance chromatography. Hydrophobicity Hydrophobic interaction chromatography. Reversed phase chromatography, Ligand specificity Affinity chromatography. 2, A porous solid material with convenient chemical properties the stationary.
phase is packed in a column and a buffered solution the mobile phase elute through. it Individual proteins migrate faster or more slowly through the column depending on. PARTIAL PURIFICATION AND CHARACTERIZATION OF LIPASE ENZYME FROM A Pseudomonas STRAIN Lipase is a triacylglycerol hydrolyzing enzyme which is catalyzed the hydrolysis of water insoluble free fatty acid and glycerols and also a wide range of chemical reactions Beside microbial lipases show regiospecificity and enantioselectivity properties

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