Shrinivas colloquium 18_06_10

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Enzyme Catalysis

Transcript of Shrinivas colloquium 18_06_10

  • 1. Enzymatic Catalysis in Synthesis of fine Chemicals Research supervisor Research student Prof. G.D.Yadav Shrinivas A. Shete

2. _ Synthesis of novel support Characterization of support Immobilization of lipase Characterization of biocatalyst Synthesis of hexyl acetate Outline of project 2 3. Classification of catalysis_ Catalysis Bio- Chemo- Organo - Inorganic Organo metalic Enzyme 3 4. Biocatalysis can be either homogeneous or heterogeneous Homogeneous Heterogeneous Chemo- catalysis Bio- catalysis Organo Metalic Organic compounds Acids and bases Free enzyme Inorganic solids Organic resins Immobilized enzymes Whole cells _ 4 5. Stability in organic solvents Mild reaction conditions Do not require cofactors Eco friendly catalyst Higher reaction rates Possess broad substrate specificity Exhibit high enantioselectivity Lipase (3.1.1.3)_ Lipase can be employed in the production of pharmaceuticals, cosmetics, leather, detergents, foods, perfumery and other organic synthetic materials. 5 6. They are soluble catalysts Usually very unstable They may be strongly inhibited by substrates and products work well on natural substrates and under physiologica l conditions High cost Limitations of enzyme in addition to their excellent catalytic properties _ 6 7. Engineering of enzymes from biological to chemical industry Screening of enzymes with suitable properties Improvement of enzyme properties via techniques of molecular biology improvement of enzyme properties via reaction and reactor engineering _ Improvement of enzyme properties via immobilization 7 8. Development of Biocatalyst Factors to be considered in design of a biocatalyst. A Reuse of Enzyme B Immobilization method C Enzyme stability _ Cost effectiveness & Simplicity D Development of Biocatalyst_ 8 9. Support for enzyme immobilization Support Natural Synthetic Inorganic 1. cellulose 2. dextran 3. agar 4. chitin 1. polyacrylate 2. polymethacrylates 3. polyacrylamide 1. silica 2. bentonite 3. glass _ 9 10. Silica support Porous silica Microporous 50nm MCM-41 HMS SBA-15 MCF _ 10 11. Mesocellular foam [MCF]_ B High pore volume, up to 2 ml/g Large surface area, up to 1,000 m2/g C A 3D pore system A Connected by uniform windows (9-22 nm) D Large spherical cells (24-42 nm) 11 12. P123-4g + H2O-65ml + HCl-10ml Stir at 40 C for 2 hours Static at 40 C for 20 hours Age at 100 C for 24 hours Filter, dry & Calcination at 550 C for 6 hours TMB TEOS NH4F Synthesis of MCF_ 13. Characterization of MCF_ 1. FT-IR 2. ASAP 3. SEM 13 14. 4000.0 3000 2000 1500 1000 400.0 -1.5 5 10 15 20 25 30 35 40 45 50 55 61.9 cm-1 %T 3465.76 1652.48 1086.56 972.84 804.97 462.88 FT-IR of MCF_ 14 15. ASAP of MCF_ 15 16. Silica Surface Area (m2/g) Pore volume (cm3/g) Pore size (nm)Single Point BET BJH Adsorption BJH Desorption Single point BJH Adsorption BJH Desorption MCF 431.74 447.79 471.62 739.11 1.92 1.90 1.93 17.19 ASAP results_ 16 17. SEM of MCF_ 17 18. Advantages of Immobilization_ Immobilization 18 19. Methods of enzyme immobilization Methods Adsorption Entrapment Micro- encapsulation Entrapment Cross linking Covalent binding _ 19 20. Total protein estimation y= 0.026x R =0.998 0 0.05 0.1 0.15 0.2 0.25 0.3 0 2 4 6 8 10 12 Absorbance Microgram of Protein/100ul BradfordCalibrationCurve(Microassay) Bradford Calibration Curve (Macroassay) y = 0.007x R2 = 0.9912 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 20 40 60 80 100 120 micro gram protein/100ul absorbance _ 20 21. Olive oil Tributy rin P-nitro phenol Lipase assay_ Various assay methods are used to determine the enzyme activity. 22. Tributyrin method_ 188l tributyrin + 1062l of 0.1M phosphate buffer + 250l enzyme Mix thoroughly on cyclomixer for 1 min Kept on shaker for 14 min Released acid titrated with 0.05N NaOH 22 23. 0.2 g of Calcined MCF + 20 ml ethanol Appropriate amount of APTES Mixture reflux for 850C for 8 h. Wash with DI water & ethanol Filter white solid & Dry at 600C for 24 h Covalent binding Functionalization with APTES _ 23 24. Procedure_ 300mg FMCF + 10ml sod. phosphate buffer. equilibration for 1 hour 10ml of 0.1% gluteraldehyde soln Kept in shaker for 1 hr at room temp Add. of diluted enzyme Washed three times with buffer Protein content & enzyme activity was checked Kept in shaker for 6 hr at room temp 24 25. Covalent binding results Immobilization Method Dilution % Immobilization Activity u/g Covalent binding 10 27 100 100 31 40 1000 80 15 _ 25 26. 300mg MCF + 10ml sod. phosphate buffer. equilibration for 1 hour 10ml enzyme solution Gluteraldehyde crosslinking method - I_ 300mg MCF 10ml phosphate buffer Kept it for 1hr in incubator shaker 0.1% of Gluteraldehyde solution Stirred at 4C overnight Centrifugation & washing with buffer 26 27. Immobilization Method Dilution % Immobilization Activity u/g Gluteraldehyde ( Method I ) 10 78 222 100 79 82 1000 100 24 Gluteraldehyde crosslinking method I results_ 27 28. Gluteraldehyde crosslinking method II_ 300mg MCF 10ml sod. Phosphate buffer 300mg MCF + 10ml sod. phosphate buffer. equilibration for 1 hour 10ml enzyme solution Vortexed for 30 sec & then sonicated for 10sec Kept on shaker for 30min 0.1% gluteraldehyde sol. Kept on shaker for 30min Centrifugation & washing with buffer 28 29. Immobilization Method Dilution % Immobilization Activity u/g Gluteraldehyd e ( Method I ) 10 97 333 100 99 198 1000 100 75 Gluteraldehyde crosslinking method II results _ 29 30. Comparison of results of immobilization methods 0 50 100 150 200 250 300 350 Gluteraldehyde I Gluteraldehyde II Covalant bonding Immobilization methods Enzymeactivityu/g 10 100 1000 _ 30 31. 31 32. Reaction scheme_ CH3 OH + CH2 O O CH3 CH3 O O CH3 + CH2 OH CH3 O vinyl alcohol vinyl acetate hexanol hexyl acetate acetaldehyde Lipase Hexylacetate is a significant green note flavor and widely used in food industry.32 33. ..Experi mental Gas Chromatography Contd... water bath hexanol + vinyl acetate + biocatalyst in organic solvent t0.t1.tn glass reactor pitched blade glass stirrer 34. Analysis 35. Effect of acyl donors 0 10 20 30 40 50 60 70 80 90 100 0 30 60 90 120 150 %conversionofhexanol Time (min) vinyl acetate acetic anhydride acetic acid triacetin _ Reaction parameter Speed of agitation 300RPM Temp. 50 C Solvent Toluene Enzyme loaded MCF 20mg hexanol:acyl donar 1:2 35 36. Effect of speed of agitation 0 10 20 30 40 50 60 70 80 90 100 0 30 60 90 120 150 %Conversionofhexanol Time ( min ) 200 RPM 300 RPM 400 RPM 500 RPM _ Temp. 50 C Solvent Toluene Enzyme loaded MCF 20mg hexanol:vinyl acetate 1:2 Reaction parameter 36 37. Effect of solvents 0 10 20 30 40 50 60 70 80 90 100 0 30 60 90 120 150 %ConversionofHexanol Time (min) Toluene Benzene 1,4 Dioxane Acetonitrile _ Reaction parameter Speed of agitation 300RPM Temp. 50 C Enzyme loaded MCF 20mg hexanol:vinyl acetate 1:2 37 38. Effect of catalyst loading 0 10 20 30 40 50 60 70 80 90 100 0 30 60 90 120 150 %Conversionofhexanol Time(min) 5mg 10mg 15mg 20mg _ Speed of agitation 300RPM Temp. 50 C Solvent toluene hexanol:vinyl acetate 1:2 Reaction parameter 38 39. Effect of temperature 0 10 20 30 40 50 60 70 80 90 100 0 30 60 90 120 150 %Conversionofhexanol Time (min) 30C 40C 50C _ Speed of agitation 300RPM Enzyme loaded MCF 20mg Solvent toluene hexanol:vinyl acetate 1:2 Reaction parameter 39 40. y = 0.0027x R2 = 0.9976 y = 0.0065x R2 = 0.9855 y = 0.0098x R2 = 0.994 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 30 60 90 120 150 ln((2-Xa)/2*(1-Xa)) Time (min) 30C 40C 50C Second order plot_ 40 41. -6 -5.5 -5 -4.5 -4 -3.5 -3 0.00305 0.0031 0.00315 0.0032 0.00325 0.0033 0.00335 ln(k) 1/T103 (1/K) Arrhenius plot Activation energy = 52.6KJ/mol =12.58Kcal/mol _ 41 42. Effect of mole ratio 0 10 20 30 40 50 60 70 80 90 100 0 30 60 90 120 150 %Conversionofhexanol Time (min) 1:01 1:05 1:02 01:02.5 1:03 _ Speed of agitation 300RPM Enzyme loaded MCF 20mg Solvent toluene Temp. 40C Reaction parameter 42 43. Substrate study 0.00E+00 1.00E-05 2.00E-05 3.00E-05 4.00E-05 5.00E-05 6.00E-05 7.00E-05 8.00E-05 9.00E-05 1.00E-04 Initialrate(mols/lit.min) Mole:ratio (hexanol:vinyl acetate) _ Speed of agitation 300RPM Enzyme loaded MCF 20mg Solvent toluene Temp. 50C Reaction parameter 43 44. 0.00E+00 1.00E+04 2.00E+04 3.00E+04 4.00E+04 5.00E+04 6.00E+04 7.00E+04 8.00E+04 0 0.05 0.1 0.15 0.2 1/[initialrate] 1/[vinyl acetate] 5mM 15mM 20mM 10mM Lineweaver-Burk plot 44 45. Parameters Values refined by polymath Vmax (mol/lit.min) 0.00057 KmA (mol/lit) 0.065 KmB (mol/lit) 0.533 KiA (mol/lit) 0.083 Ki B 0.013 Kinetic parameters 45 46. MCF is the best support for enzyme immobilization Gluteraldehyde cross-linking method II (ship-in-a-bottle- approach) is the best method for lipase immobilization Selective biocatalyst for hexyl acetate synthesis Economic process as compared to other reported methods 46 47. Future plan Functionalization of MCF for effective immobilization of Enzyme Enhancement of thermo stability of enzyme by immobilization method Carry out reactions with packed bed reactor Synthesis of chiral MCF 47 48. Prof. G. D. Yadav Prof. A. M. Lali DBT Govt. of India Novo Nordisk Dr. Reddys lab. Chem. Engg. Dept. ICT, Mumbai Lab mates Acknowledgm ent 48 49. Thank You !