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Why to Decide for Why to Decide for Extremophile Extremophile

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									                                                               Institut
                                                               Technologies du vivant




    Why to Decide for Extremophile
  Mediated Industrial Biotransformation
             Presentation for the : « Forum sur les extrémophiles »
                                       by
                                Fabian Fischer
                              Sion March 3, 2009
03.03.2009                         Extremophiles                                   1
                                        Institut
                                        Technologies du vivant

     d      h i l industry d biotechnology need
WHY does chemical i d t and bi t h l          d
             enzymes?
extremophile enzymes?

  Chemical, Agro, Food, Detergent, Textile, Pulp
  &Paper Energy Industry …
  &Paper,        Industry,



    New products
    Employment
    E    l      t
    Profit
    Sustainability
    S t i bilit
                                                                                                        Institut
                                                                                                        Technologies du vivant


     The crude oil era

             „…It is expected that the first effects of
             the depletion of fossil fuels will be felt
                         future
             in the near future, particularly of crude
             oil …
             Therefore there is good reason to
             believe that the development of
             alternative raw materials…will be
             strongly stimulated…the only question
             is when and how fast…“




Royal Belgian Academy Council of Applied Science; Industrial Biotechnology and Sustainable Chemistry. 2004.
03.03.2009                                                       Extremophiles                                              3
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                                                                       Technologies du vivant


    Analogy in processing of raw material




  C. Wandrey. Weisse Biotechnologie: warum gerade jetzt? 2007.
03.03.2009                                             Extremophiles                       4
                                           Institut
                                           Technologies du vivant


    Processing of raw material




             Employment of extremophiles possible

03.03.2009                 Extremophiles                       5
                                                                      Institut
                                                                      Technologies du vivant

    REACH
         g
  The regulation will after a transitional
  period replace the current rules….. After
             force
  entry into force, the deadline for
  substance classification according to the
  new rules will be 1 December 2010 and
  for mixtures 1 June 2015.[1]
  Biotransformation permits the synthesis of
                    chemicals,
  well defined fine chemicals which are
  natural and may easily be registered with a
    lli REACH L b l
  selling REACH-Label.
[1]The European Commission, Brussels, 1st December 2008.
                                                      Extremophiles                       6
                                                                           Institut
                                                                           Technologies du vivant

    Point of view of academia for the successful use of
    extremophiles
             Bioreaction engineering for “White Biotechnology” with extremophiles may improve
             existing process and allow new processes to become established.

             Faster technical evolution based on directed evolution of enzymes from
             extremophiles for faster product development in the low price segment.

             Extremophiles should equally work beside natural resources using non-natural
             substrates:
                   non-natural reaction conditions,
                   non natural
                   non-natural products,
                   non-natural concentrations,
                   non natural
                   non-natural compartimentisation (reaction cascades)

             Extremophiles should enhance the biotechnology based paradigm shift in the
             chemical industry.
             (better match of biology and chemistry)

03.03.2009                                       Extremophiles                                  7
                                                    Institut
                                                    Technologies du vivant

    The basic options on becoming an extremophile
    biotransformer




         Biotransformation with:
         -      t     hil   ll in fermenters
             extremophile cells i f       t
         -   pure extremophile enzymes
         -   extremophile catalytic antibodies, a new starting point ?


  Bildquelle Lonza
03.03.2009                        Extremophiles                         8
                                                                  Institut
                                                                  Technologies du vivant


    Why to decide for industrial biotransformation?
   Potential f Bi t    f    ti
   P t ti l of Biotransformation                       Vit i B2
                                                       Vitamin
                               low                                Biotech




                                  Environmental
                                              l
   Traditional Process:    -40%
   8 Step synthesis




                                   mpact
                                                  Traditionel


                                  im
                                  high
   Biotransformation:                             high          Cost          low
   1 Step fermentation


                                                           40%
                                                          -40%
 BASF and Oeko Institute
03.03.2009                           Extremophiles                                    9
                                                                  Institut
                                                                  Technologies du vivant


    Why to decide for industrial biotransformation?
      Potential f Biotransformation
      P t ti l of Bi t    f    ti                Antibiotika Cephalexin
                                                 A tibi tik C h l i

                              Traditional Process:          Environment
                               10 Step synthesis
                                                            65% less use of material
                                                            65% less use of energy

                                                            Economy
                                                            50 % less variable cost
                              Biotransformation:
                           Combination of fermentation
                              and enzyme catalysis




 DSM and Oeko Institute
03.03.2009                            Extremophiles                                    10
                                                                                                                                                    Institut
                                                                                                                                                    Technologies du vivant


                             115°
    Enzymatic biorefining at 115°C

                       CH2OH              CH2OH                                                          Method used by several companies
                  O
                       OH
                            O

                                     O
                                          OH
                                               O
                                                                                                         1,4-α-D-glucan glucanhyrdolyse
             CH2OH
                            OH
                             CH2OH
                                               OH
                                                 O
                                                CH2              CH2OH
                                                                                                         glucoamylase,
                                                                                                         glucoamylase amyloglucosidase
             OH
                  O
                                OH
                                     O
                                                   OH
                                                        O
                                                                 OH
                                                                      O
                                                                                                         Aqueous media
        O              O                  O                  O               O

                  OH                 OH                 OH            OH




                                                                                                                                                                      Fructose
                                                                           oil, germ,
                                                                          bran, fiber                                                                  protein fat

                                                                                        H2O                      H2O                     H2O   E2
                                                                                                  E1                     E1                                            CH2OH
                       SO2
                                                                                                                                                                            O
                                                                                                                                                                       OH         OH
                                                                                                                                                                     OH
                                                                                                                                                                            OH
                                                                       centrifuge

                                                                                              1. liquefaction          2. liquefaction
                                                                                                 100-115°C
                                                                                                 100-115 C                90-95°C
                                                                                                                          90 95 C
                                                                                                    pH-6
                                                                                                                                                                     Bioethanol




  A. Liese, K. Seelbach, C. Wandrey. Industrial Biotransformations. 2000.

03.03.2009                                                                                                      Extremophiles                                                          11
                                                                                              Institut
                                                                                              Technologies du vivant

      I d t i l biotransformation at 70°C f 6 months
      Industrial bi t   f    ti    t 70° for     th
                        2-azabicyclo[2.2.1]hept- en-
      Deracemisation of 2-azabicyclo[2.2.1]hept-5-en-3-one

                                                                                              This lactam can be
                                                                                              converted to carbovir
                                                                                              an HIV-1 inhibitor
              O
                    H
                    N                                                                 -OOC   NH3+



                                                                   acetone
                                      E                                                                    O
                                                                                                       H
                                                                                                       N
                                                                    crystallization




S.C. Taylor. Chirality in Industry 1997. Process used by Chirosience Ltd.


 03.03.2009                                                       Extremophiles                                       12
                                                                                               Institut
                                                                                               Technologies du vivant


                                                  5°
    Industrial enzymatic acrylamide production at 5°C

                                                                                          Acylamide is the
  nutrients
              inducing agent
                                                                                          monomer for SDS-PAGE
                                                                                          gels and a versatile
                                                                      CN                  basic chemical for
                                                                                          numerous polymers.
                                       fermentation
                                       f     t ti                          analytics
                                       medium             H2O


                                                             E
                                                                                                                O
                               cells
                                       immobilization                                           decoloring          NH2




                                                                                       spent
                                                                                       cells




H. Yamada, Y. Tani. Nitto Chemical Industry Co., Ltd., US 4637982.
03.03.2009                                                      Extremophiles                                        13
                                                                                                                                      Institut
                                                                                                                                      Technologies du vivant

       Comparison of glycoside formation by
       biotransformation and chemical synthesis
                                              OH

                                    HO                O
                                      HO                     OH
                                                     OH                   protection and
              protection and
              derivatization                                              derivatisation
                                                                                                                       Chemical Syntheses
                                                                                      OBz
              OAc
                                                                                             O
   AcO               O                               HCl                  BzO
                                                                            BzO
                                                                                                                       1)   Smiths
                                                     R2OH                                         OR1
     AcO                   OR1                                                              OBz
                    OAc                              R2=CH3 or C2H5

 1 (87%)             2 (65%)                         3 (35%)           4 (49%)                    5 (56%)              2)   Fischer
                                                                              Base                  BuLi or NaH
R1=Br or Cl          R1=OAc
Ag+ or Hg2+          BF3.Et2O                                                 Cl3C-CN                                  3)   Koenigs Knorr
R2OH                 R2OH
                                                             OBz                                     OBz
CHCl3                CH2Cl2
or CH2Cl2                                                          O
                                                                                                                       4)   Trichloroacetamine
                                               BzO                               NH         BzO             O
           OAc                                   BzO                      O                   BzO
                                                                   OB
                                                                   OBz                                            OM
                                                                                                          OBz
                 O
                                                                                 CCl3
                                                                                                  M=Li or Na
                                                                                                                       5)   1-O-Alkylation
 AcO
   AcO
                          OR2
                 OAc

                                                            Lewis acid
                                                            R2OH                                   R2X or R2OTf
                                                                                                   Benzene or THF
         deprotection


                                  OH                                              OBz
                                         O                  p
                                                          deprotection
                         HO                                              BzO                O
                           HO                                              BzO
                                                OR2                                               OR2
                                         OH                                                 OBz
                                R2 = alkyl or aryl

  B. M. de
 03.03.2009Roode The Integrated Enzymatic Production and Down Stream Processing of Glucosides. Thesis Wageningen University, 2001.                        14
                                                                                                                                                                                     Institut
                                                                                                                                                                                     Technologies du vivant

                                     Comparison of glycoside formation by
                                     biotransformation and chemical synthesis
                                                                OH                                                                                                               OH
                                                                     O                                       β -Glucosidase                                                             O
                                                  HO                                      +   ROH                                                                  HO                                  +          H2O
                                                       HO                                                                                             50°C              HO                      OR
                                                                     OH       OH                                                                                                       OH

                                                            R= -(CH2)nCH3 or other aglycon

                                     100.00                                                                                                           100.00

                                      90.00                                     3 [%, v:v] H2O 0 [%, v:v]                                              90.00                     1   [%, v:v] H2O   30 [%, v:v] DMF 4 [g/l] Glc
                                                                                DMF 4 [g/l] Glc                                                                                  1   [%, v.v] H2O   30 [%, v:v] DMF 32 [g/l] Glc




                                                                                                                                  cylglucosides [%]
                                      80.00                                                                                                            80.00
                  ylglucosides [%]




                                                                                3 [%, v.v] H2O 10 [%, v:v]                                                                       1   [%, v:v] H2O   20 [%, v:v] DMF 4 [g/l] Glc
                                                                                DMF 4 [g/l] Glc                                                                                  1   [%, v:v] H2O   30 [%, v:v] DMF 32 [g/l] Glc
                                      70.00                                                                                                            70.00
                                                                                3 [%, v:v] H2O 20 [%, v:v]
                                      60.00                                     DMF 4 [g/l] Glc                                                        60.00

                                                                                  [%              [%
                                                                                3 [%, v:v] H2O 30 [%, v:v]




                                                                                                                Production en dodec
                                      50.00
                                      50 00
 Production en octy




                                                                                                                                                       50.00
                                                                                DMF 4 [g/l] Glc
                                      40.00                                                                                                            40.00
                                                                                3 [%, v:v] H2O 0 [%, v:v]
                                                                                DMF 8 [g/l] Glc
                                      30.00                                                                                                            30.00
                                                                                6 [%, v:v] H2O 0 [%, v:v]
                                      20.00                                     DMF 4 [g/l] Glc                                                        20.00
                                      10.00                                                                                                            10 00
                                                                                                                                                       10.00
                                       0.00                                                                                                             0.00
                                              0        10         20             30              40            50                                              0   1         2            3             4                 5        6
                                                                 Temps d'incubation [h]
                                                                                                                                                                                 Temps d'incubation [h]




O. Tordion, F. Fischer Poject HEVs.
                                                                                                           Institut
                                                                                                           Technologies du vivant

    Comparison of glycosid formation by
    biotransformation and chemical synthesis
                                                                                                 Space-time        Waste in
                                     Sugar
             Method                                             Product                            yield           kg per kg
                                     donor
                                                                                                   g/(l·d)
                                                                                                   g/(l d)          product
                                                                OH
                                                                     O
        Koenigs-Knorr               Glucose              HO
                                                          HO
                                                                              O                      36                  20
                                                                      OH
                                                                OH
            Di
            Direct                                                   O
                                    Glucose              HO
                                                                              O                      10                   2
         glucosylation                                    HO
                                                                      OH

                                                                 OH
            Direct                                                       O
                                   Glucose                HO                                         21                  0.7
         l      l ti
        glucosylation                                      HO
                                                                         OH
                                                                                  O


                                                                 OH
            Trans-                                                       O
                                    Lactose               HO
                                                                                  O                 580                   4
         glycosylation                                     HO
                                                                         OH

                                                                 OH
            Direct
                                   Glucose                HO
                                                                         O
                                                                                                    5.6                  0.5
        glucosylation                                      HO
                                                                         OH
                                                                                  O
                                                                                      (CH2)6OH
                                                                 OH
            Trans
            Trans-                                                       O
                                    Lactose
                                    L                     HO
                                                                                  O                 2380                  3
         glycosylation                                     HO
                                                                         OH           (CH2)6OH

 B. M. de Roode The Integrated Enzymatic Production and Down Stream Processing of Glucosides. Thesis Wageningen University, 2001.
03.03.2009                                                       Extremophiles                                                      16
                                                                     Institut
                                                                     Technologies du vivant


    Biotransformation in ionic liquids
     Ionic liquids provide novel                     N
                                                         +
                                                             BF4-                          N
                                                                                               +   PF6-
     options:
                                                 N                                     N
              No vapour pressure
              Good solubility properties
              Biphasic processes
                                           [bmim][BF4]                        [bmim][PF6]
     .



                                                         (CH2)5CH3                     (CH2)5CH3
                                    CH3(CH2)13        +
                                                                        (
                                                                     CH3(CH2)13        +
                                                  P                                P  (CH2)5CH3
                                                        (CH2)5CH3
                                                     (CH2)5CH3                     (CH2)5CH3


                                                 BF4-                             PF6-

                                              [thtdp][BF4]                        [thtdp][PF6]




03.03.2009                            Extremophiles                                                       17
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    Biphasic catalysis using ionic liquids


                                               Recycling of enzymes
                                               Recycling of i i li id it lf
                                               R     li    f ionic liquid itself
             Substrate     Product             Green Processes
                                               Simplified DSP



                                                     Ionic liquid
                     Enzyme
                  dissolved in IL



03.03.2009                           Extremophiles                                      18
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                                                                                                                 Technologies du vivant


    β-Glucosidase activity in ionic liquids
             y                                                 q
      Activity measured at different concentrations of ionic liquids at 50 °C.


                                160

                                140

                                120                                                                       [bmim][PF6]: increase of 68 %
         elative activity [%]




                                                                                                          [bdmim][PF6]: increase of 10 %
                        y




                                100

                                80

                                60
        Re




                                40

                                20

                                 0
                                      0       20           40            60           80            100
                                                          % v/v ionic liquid

                                          [bmim][PF6]
                                          [b i ][PF6]           [b i ][BF4]
                                                                [bmim][BF4]         [bmim][AlCl4]
                                                                                    [b i ][AlCl4]
                                          [emim][EtSO4]         [bmim][MeSO4]       [bdmim][PF6]

 O. Clerc,
03.03.2009 F. Druey, P. Wyden, F. Fischer. Project HEVs.                        Extremophiles                                          19
                                                                                                                                       Institut
                                                                                                                                       Technologies du vivant

    Polyphenolglycosides f enhanced solubility and release
    P l h      l l    id for h       d l bilit      d l
    properties in cosmetic and medical applications
                                                                                                                                   OH
                                                 OH
                                                                                              HO               O
                HO              O                                                                                                  OGlc
                                                 OH        +       Glucosid

                                                                                                         OH    O
                       OH       O

                       Luteolin
                                                                                                  Luteolin-4'-α-D-glucopyranoside




                                                  OH                                                                              OH
                                                               +        Glucosid
                      HO                                                                            HO
                                                  OH                                                                              OGlc

                            O                                                                             O

                                caffeic acid                                                             caffeoyl-3-O-glucoside




                                    O    O                                                    O    O            OH
                                                      OH
                                                                   +   Glucosid
                                                      OH                                                        OGlc

                                         Aesculetin
                                                                                     6-ß-D-Glucopyranosyloxy-7-hydroxycumarin




 M. Yang, G. J. Davies, B. G. Davis. Angew. Chem. Int. Ed. 2007, 46, 3885 –3888 (one of several articles in the field).
 E. Zufferey, F. Fischer. Project HEVs.
03.03.2009                                                                         Extremophiles                                                           20
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                                                                                                                       Technologies du vivant

Lipase catalyzed esterification

                                                                                                    O
                                                                                             6               3'        5'        7'        9'         11'         13'
           O       O                       myristic acid                   O       O 5
                              OH                                                                 O 1'
                                                                      7        1                        2'        4'        6'        8'        10'         12'         14'
                                       Candida antarctica                                4
          HO              OH             solvent /60°C                    HO 2      3        OH
                   OH                                                              OH


    α/β-D-Methylglucose                                                                          Methyl-α/β-D-glucopyranoside




J. Mutschler, T. Rausis, I. Mohrenz, JM. Bourgeois, Ch. Bastian, F. Fischer. Project HEVs.
                                                                                                       Institut
                                                                                                       Technologies du vivant
        90


                                                        Acetone
        80
                                                                         MeCN
                               TBME

                                      THF




                                                                                                              Salt resista extremophiles ?
        70

                                                        tBOH
        60
                                                                     thtdp BF4


        50
 ield




                                                                                                                         ant
                                                      thtdp PF6
                                                          p                                      bmim PF4
                 Hexane
Yi




        40                                                             bdmim PF4

                          Toluene
                                                                     bdmim PF6                 bmim PF6




                                                                                                              S
        30



        20                                                                                mpim PF6                                           bmpyr
                                                                                                                                              PF6

        10


                          Common Solvents                                         Green Solvents
        0
             0         0.1          0.2           0.3          0.4        0.5       0.6        0.7      0.8                       0.9                1

    J. Mutschler, T. Rausis, Fischer. Project HEVs.                    Polarity
                                          Institut
                                          Technologies du vivant


    FP7 Call for robust enzymes




03.03.2009                Extremophiles                       23
                                              Institut
                                              Technologies du vivant


Does industry need extremophile enzymes?




     3-Isopropylmalate dehydrogenase from
    the extreme thermophile T. thermophilus
    Imada, Sato, Tanaka, Katsube, Matsuura,
       Oshima (1991) J. Mol. Biol. 222, 725
                                             Institut
                                             Technologies du vivant



   Thank you for your attention

    Acknowledgments
      Isabelle Mohrenz
      Jessica Emery
      Olivier Clerc
      Olivier Tordion
      Dr. Thierry Rausis
      Fabrice Druey
      Silvio Bonnomelli
      Dr. Julien Mutschler
      Pascal Wyden
      Christelle Bastian




03.03.2009                   Extremophiles                       25

								
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