Shrinivas colloquium 18_06_10

794 views

Published on

Enzyme Catalysis

Published in: Education, Technology, Business
  • Be the first to comment

  • Be the first to like this

Shrinivas colloquium 18_06_10

  1. 1. Enzymatic Catalysis in Synthesis of fine Chemicals Research supervisor Research student Prof. G.D.Yadav Shrinivas A. Shete
  2. 2. _ • Synthesis of novel support • Characterization of support • Immobilization of lipase • Characterization of biocatalyst • Synthesis of hexyl acetate Outline of project 2
  3. 3. Classification of catalysis_ Catalysis Bio- Chemo- Organo - Inorganic Organo metalic Enzyme 3
  4. 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. 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. 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. 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. 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. 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. 10. Silica support Porous silica Microporous <2nm Mesoporous 2-50nm Macroporous >50nm MCM-41 HMS SBA-15 MCF _ 10
  11. 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. 12. P123-4g + H2O-65ml + HCl-10ml Stir at 40 ºC for 2 hours Static at 40 ºC for 20 hours Age at 100 ºC for 24 hours Filter, dry & Calcination at 550 ºC for 6 hours TMB TEOS NH4F Synthesis of MCF_
  13. 13. Characterization of MCF_ 1. FT-IR 2. ASAP 3. SEM 13
  14. 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. 15. ASAP of MCF_ 15
  16. 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. 17. SEM of MCF_ 17
  18. 18. Advantages of Immobilization_ Immobilization 18
  19. 19. Methods of enzyme immobilization Methods Adsorption Entrapment Micro- encapsulation Entrapment Cross linking Covalent binding _ 19
  20. 20. Total protein estimation y= 0.026x R² =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. 21. Olive oil Tributy rin P-nitro phenol Lipase assay_ Various assay methods are used to determine the enzyme activity.
  22. 22. Tributyrin method_ 188µl tributyrin + 1062µl of 0.1M phosphate buffer + 250µl enzyme Mix thoroughly on cyclomixer for 1 min Kept on shaker for 14 min Released acid titrated with 0.05N NaOH 22
  23. 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. 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. 25. Covalent binding results Immobilization Method Dilution % Immobilization Activity u/g Covalent binding 10 27 100 100 31 40 1000 80 15 _ 25
  26. 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 4ºC overnight Centrifugation & washing with buffer 26
  27. 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. 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. 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. 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. 31
  32. 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. 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. 34. Analysis
  35. 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 ºC Solvent Toluene Enzyme loaded MCF 20mg hexanol:acyl donar 1:2 35
  36. 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 ºC Solvent Toluene Enzyme loaded MCF 20mg hexanol:vinyl acetate 1:2 Reaction parameter 36
  37. 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 ºC Enzyme loaded MCF 20mg hexanol:vinyl acetate 1:2 37
  38. 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 ºC Solvent toluene hexanol:vinyl acetate 1:2 Reaction parameter 38
  39. 39. Effect of temperature 0 10 20 30 40 50 60 70 80 90 100 0 30 60 90 120 150 %Conversionofhexanol Time (min) 30ºC 40ºC 50ºC _ Speed of agitation 300RPM Enzyme loaded MCF 20mg Solvent toluene hexanol:vinyl acetate 1:2 Reaction parameter 39
  40. 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) 30ºC 40ºC 50ºC Second order plot_ 40
  41. 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/T×103 (1/K) Arrhenius plot Activation energy = 52.6KJ/mol =12.58Kcal/mol _ 41
  42. 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. 40ºC Reaction parameter 42
  43. 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. 50ºC Reaction parameter 43
  44. 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. 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. 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. 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. 48. • Prof. G. D. Yadav • Prof. A. M. Lali • DBT Govt. of India • Novo Nordisk • Dr. Reddy’s lab. • Chem. Engg. Dept. ICT, Mumbai • Lab mates Acknowledgm ent 48
  49. 49. Thank You !

×