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Shrinivas colloquium 18_06_10

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Enzyme Catalysis

Enzyme Catalysis

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Shrinivas colloquium 18_06_10 Shrinivas colloquium 18_06_10 Presentation Transcript

  • Enzymatic Catalysis in Synthesis of fine Chemicals
    Research supervisor Research student
    Prof. G.D.YadavShrinivas A. Shete
  • Outline of project
    _
    Synthesis of novel support
    Characterization of support
    Immobilization of lipase
    Characterization of biocatalyst
    Synthesis of hexyl acetate
    2
  • Classification of catalysis
    _
    3
  • Biocatalysis can be either homogeneous or heterogeneous
    _
    Homogeneous
    Heterogeneous
    Organo Metalic
    Inorganic solids
    Chemo-
    catalysis
    Organic compounds
    Organic resins
    Acids and bases
    Immobilized enzymes
    Bio-
    catalysis
    Free enzyme
    Whole cells
    4
  • Lipase (3.1.1.3)
    _
    • 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 can be employed in the production of pharmaceuticals, cosmetics, leather,
    detergents, foods, perfumery and other organic synthetic materials.
    5
  • Limitations of enzyme in addition totheir excellent catalytic properties
    _
    6
  • 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
  • Reuse of Enzyme
    A
    B
    Immobilization method
    Enzyme stability
    C
    D
    Cost effectiveness & Simplicity
    Development of Biocatalyst
    Development of Biocatalyst
    _
    _
    Factors to be considered in design of a biocatalyst.
    8
  • 1. cellulose 2. dextran 3. agar 4. chitin
    1. polyacrylate 2. polymethacrylates 3. polyacrylamide
    1. silica 2. bentonite 3. glass
    Support for enzyme immobilization
    _
    9
  • Silica support
    _
    10
  • 3D pore system
    High pore volume, up to 2 ml/g
    A
    A
    B
    Large surface area, up to 1,000 m2/g
    D
    Large spherical cells (24-42 nm)
    C
    Mesocellular foam [MCF]
    _
    Connected by uniform windows (9-22 nm)
    11
  • Synthesis of MCF
    _
    TMB
    TEOS
    NH4F
  • 1. FT-IR
    2. ASAP
    3. SEM
    Characterization of MCF
    _
    13
  • FT-IR of MCF
    _
    14
  • ASAP of MCF
    _
    15
  • ASAP results
    _
    16
  • SEM of MCF
    _
    17
  • Advantages of Immobilization
    _
    Immobilization
    18
  • Methods of enzyme immobilization
    _
    19
  • Total protein estimation
    _
    20
  • Lipase assay
    _
    Various assay methods are used to determine the enzyme activity.
  • 188µl tributyrin + 1062µl of 0.1M phosphate buffer + 250µl enzyme
    Reaction stopped by methanol
    Released acid titrated with 0.05N NaOH
    Kept on shaker for 14 min
    Tributyrin method
    _
    Mix thoroughly on cyclomixer for 1 min
    22
  • Covalent binding
    _
    Functionalization with APTES
    23
  • 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
    Protein content & enzyme activity was checked
    Kept in shaker for 6 hr at room temp
    Washed three times with buffer
    Procedure
    _
    Add. of diluted enzyme
    24
  • Covalent binding results
    _
    25
  • 300mg MCF
    10ml phosphate buffer
    300mg MCF + 10ml sod. phosphate buffer. equilibration for 1 hour
    10ml enzyme solution
    Kept it for 1hr in incubator shaker
    0.1% of Gluteraldehyde solution
    Centrifugation & washing with buffer
    Stirred at 4ºC overnight
    Gluteraldehyde crosslinking method - I
    _
    26
  • Gluteraldehyde crosslinking method – I results
    _
    27
  • 10ml sod. Phosphate buffer
    300mg MCF
    300mg MCF + 10ml sod. phosphate buffer. equilibration for 1 hour
    10ml enzyme solution
    Vortexed for 30 sec & then sonicated for 10sec
    0.1% gluteraldehyde sol.
    Kept on shaker for 30min
    Kept on shaker for 30min
    Centrifugation & washing with buffer
    Gluteraldehyde crosslinking method – II
    _
    28
  • Gluteraldehyde crosslinking method – II results
    _
    29
  • Comparison of results of immobilization methods
    _
    30
  • Synthesis of hexyl acetate
    31
  • Reaction scheme
    _
    Hexylacetate is a significant green note flavor and widely used in food industry.
    32
  • ……..Experimental
    pitched blade glass stirrer
    glass reactor
    water bath
    hexanol + vinyl acetate + biocatalyst in organic solvent
    Contd...
    Gas Chromatography
    t0…………….t1………….tn
  • Analysis
  • Effect of acyl donors
    _
    Reaction parameter
    35
  • Effect of speed of agitation
    _
    Reaction parameter
    36
  • Effect of solvents
    _
    Reaction parameter
    37
  • Effect of catalyst loading
    _
    Reaction parameter
    38
  • Effect of temperature
    _
    Reaction parameter
    39
  • Second order plot
    _
    40
  • Arrhenius plot
    _
    Activation energy
    = 52.6KJ/mol
    =12.58Kcal/mol
    41
  • Effect of mole ratio
    _
    Reaction parameter
    42
  • Substrate study
    _
    Reaction parameter
    43
  • Lineweaver-Burk plot
    44
  • Kinetic parameters
    45
  • Conclusion…
    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
  • 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
  • Acknowledgment
    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
    48
  • Thank You !