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Introduc9on  Feruloyl   esterases   (FAEs,   E.C.   3.1.1.73,   CAZy   family   CE1)   and  glucuronoyl  esterases  (GEs,  E.C.  3.1.1.-­‐,  CAZy  family  CE15)  are  involved   in   the   degrada9on   of   plant   biomass   by   hydrolysing  ester  linkages  in  plant  cell  walls,  and  thus  have  poten9al  use  in  biofuel   produc9on   from   lignocellulosic   materials   and   in  biorefinery  applica9ons  with  the  aim  of  developing  new  wood-­‐based   compounds   [1,   2].   GEs   and   FAEs   are   present   in   the  genomes  of  a  wide  range  of  fungi  and  bacteria.    Under   condi9ons   of   low   water   content,   these   enzymes   can  also   carry   out   (trans)esterifica9on   reac9ons,   making   them  promising  biocatalysts  for  the  modifica9on  of  compounds  with  applica9ons   in   the   food,   cosme9c   and   pharmaceu9cal  industry.   Compared   to   the   chemical   process,   enzyma9c  synthesis   can   be   carried   out   under   lower   process  temperatures   (50-­‐60°C)   and   results   in   fewer   side   products,  thus  reducing  the  environmental  impact.      

Cita9ons  [1]  Topakas,  E.,  Vafiadi,  C.,  and  Christakopoulos,  P.  (2007).  Microbial  produc9on,  characteriza9on  and  applica9ons  of  feruloyl  esterases.  Process  Biochemistry,  42(4),  497–509.  doi:10.1016/j.procbio.2007.01.007  [2]  Spániková,  S.,  and  Biely,  P.  (2006).  Glucuronoyl  esterase  -­‐  Novel  carbohydrate  esterase  produced  by  Schizophyllum  commune.  FEBS  Le9ers,  580(19),  4597–601.  doi:10.1016/j.febslet.2006.07.033  [3]  Chávez,  R.,  Bull,  P.,  &  Eyzaguirre,  J.  (2006).  The  xylanoly9c  enzyme  system  from  the  genus  Penicillium.  Journal  of  Biotechnology,  123(4),  413–433.  doi:10.1016/j.jbiotec.2005.12.036  [4]  Wood,  S.  J.,  Li,  X.-­‐L.,  Coka,  M.  a,  Biely,  P.,  Duke,  N.  E.  C.,  Schiffer,  M.,  &  Pokkuluri,  P.  R.  (2008).  Crystalliza9on  and  preliminary  X-­‐ray  diffrac9on  analysis  of  the  glucuronoyl  esterase  cataly9c  domain  from  Hypocrea  jecorina.  Acta  Crystallographica.  Sec9on  F,  Structural  Biology  and  Crystalliza9on  Communica9ons,  64(Pt  4),  255–7.  doi:10.1107/S1744309108004594  [5]  Thörn,  C.,  Gustafsson,  H.,  &  Olsson,  L.  (2011).  Immobiliza9on  of  feruloyl  esterases  in  mesoporous  materials  leads  to  improved  transesterifica9on  yield.  Journal  of  Molecular  Catalysis  B:  Enzyma9c,  72(1-­‐2),  57–64.  doi:10.1016/j.molcatb.2011.05.002  

Conclusions  We  characterised  new  FAE  and  GE  enzymes   from  mesophilic,   thermophilic  and  cold-­‐tolerant   filamentous   fungi   produced   in  Pichia   pastoris.   The   enzymes  were  characterised   for   both   their   hydroly9c   abili9es   on   various   model   substrates  (methyl   ferulate,   pNP-­‐ferulate)   -­‐   for   poten9al   applica9ons   in  deconstruc9on  of  lignocellulosic  materials   and   extrac9on   of   valuable   compounds   -­‐   as  well   as   for  their   biosynthe9c   capaci9es.   We   tested   and   op9mised   the   FAEs’  transesterifica9on   capabili9es   on   ferulate   esters   in   a   1-­‐butanol-­‐buffer   system,  with   the   aim   of   using   the   most   promising   candidates   for   the   produc9on   of  an9oxidant   compounds   with   improved   hydrophobic   or   hydrophilic   proper9es,  such  as  prenyl  ferulate,  prenyl  caffeate,  glyceryl  ferulate  and  5-­‐O-­‐(trans-­‐feruloyl)-­‐arabinofuranose.    

Chávez  et  al.,  2006  

Feruloyl  esterases  (FAEs)  

Glucuronoyl  esterases  (GEs)  

Figure   1.   Schema'c   representa'on  of   secondary   cell   wall   polymers   in  wood     (courtesy   of   Gunnar  Henriksson,  WWSC).  

Figure   2.   The   structure   of   xylan   and   site   of  ac'on  of  the  enzymes  of  the  xylanase  complex.  1:  endoxylanases;  2:  α-­‐l-­‐arabinofuranosidases;  3:  glucuronidases;   4:   feruloyl   and   coumaroyl  esterases;   5:   acetyl   xylan   esterases.   Adapted  from  Chávez  et  al.,  2006  [3].    

Figure   3.   Proposed   cross-­‐link   structure   between   lignin   and  glucuronoxylan   in  plant  cell  walls.  MeGlcA  =  4-­‐O-­‐methyl-­‐D-­‐glucuronic  acid.   The   arrow   indicates   the   ester   bond   hydrolyzed   by   GE.   Adapted  from  Wood  et  al.,  2008  [4].  

                   

                   

                   

Hydroly'c  assays  

GEs  

MFA     FA    FAE    

pNPF     pNP    FAE    

Benz-­‐D-­‐GlcA     GlcA  

GE    

Methods  &  Results  

FAEs  

For   screening   of   they   hydroly9c   ac9vity   of   FAEs   and   GEs,  spectrophotometric  assays  were  used  

Immobilisa'on  on  mesoporous  silica  par'cles  (SBA-­‐15)    

Figure  8.  Enzyme  loading  on  mesoporous  par'cles  at  different  pH  values.  

Immobilisa9on  of  enzymes  can  improve  the  stability  and  reusability  of  the  biocatalyst,   and   can   also   change   the   substrate   specificity   [5].   SBA-­‐15  (Santa   Barbara   Amorphous   material)   is   a   well-­‐ordered,   hexagonal  mesoporous   silica   (MPS)   matrix   with   a   controllable   pore   size.   It   is   an  akrac9ve  support  material,  as  it  has  a  very  large  surface  area  that  can  be  func9onalized   if   desired,   and   proper9es   such   as   pore   structure   can   be  adjusted  to  individual  needs.    Immobilisa9on  on  MPS  is  done  by  simple  adsorp9on.  The  enzyme  loading  is  es9mated  by  measuring  protein  concentra9on  in  the  supernatant  aser  immobilisa9on.   Adsorp9on   is   mediated   mostly   by   electrosta9c   and  hydrophobic  interac9on,  thus  the  pH  of  the  used  buffer  is  very  important  for  immobilisa9on  efficiency.    

wash (5x)

add enzyme

incubate overnight

wash (3x)

dry & store

add MPS

0"

0.002"

0.004"

0.006"

0.008"

0.01"

0.012"

0.014"

0.016"

0.018"

pH"3" pH"4" pH"5" pH"6" pH"7" pH"8" pH"9"

Enzyme'load

ing'(m

g'En

gzym

e'/'mg'

MPS)'

0.1"M"citrate"phosphate"

0.1"M"TrisHCl"

Genome  mining  

Produc'on  of  fungal  enzymes  in  Pichia  pastoris  

Homologues   of   known   FAEs   were   found   in   the   genomes   of   two  cold-­‐tolerant   Aspergillus   strains   (A.   glaucus   and   A.   zonatus),  several  candidate  genes  were  cloned  and  subsequently  expressed  in  Pichia  pastoris.  

Ini'al  screening  of  19  fungal  strains  (mesophilic  and  thermophilic)  isolated  from  soil,  wood  and  agricultural  compost  in  Vietnam    

0"

10"

20"

30"

40"

50"

60"

FEC"40"

FEC42"

FEC93"

FEC108"

FEC110"

FEC128"

FEC130"

FEC161"

FEC162"

FEC258"

FEC385"

FCH"5.2"

FCH"5.4"

FCH"5.5"

FCH"5.7"

FCH"9.4"

FCH"10.4"

FCH"10.5"

mU#

0.1M"MOPS"pH"6.0,"37°C,"30"min"

0.1M"MOPS"pH"6.0,"45°C,"30"min"

0"

10"

20"

30"

40"

50"

60"

70"

80"

FEC40"

FEC42"

FEC93"

FEC108"

FEC110"

FEC128"

FEC130"

FEC156"

FEC161"

FEC162"

FEC258"

FEC385"

FCH5.2"

FCH5.4"

FCH5.5"

FCH5.7"

FCH9.4"

FCH10.4"

FCH10.5"

mU#

0.1"M"K3phosphate"buffer,"pH"6.0,"45°C,"30"min""

Selec'on  of  novel  FAEs/GEs  

DNA  &  RNA  sequencing  

Figure  4.  Screening  of  mesophilic  (FEC)  and  thermophilic  (FCH)  fungal  strains  for  FAE  ac'vity  on  methyl  ferulate  (MFA).    

Figure  5.  Screening  of  mesophilic  (FEC)  and  thermophilic  (FCH)  fungal  strains  for  GE  ac'vity  on  benzyl-­‐D-­‐glucuronate  (Benz-­‐D-­‐GlcA).  

Two   thermophilic   fungal   strains   were   chosen   for   DNA   and   RNA  sequencing.  Mycelium  was  grown  for  48h  at  50°C  on  glucose,  washed  thoroughly  and  subsequently  divided  between  shake  flasks  containing  1%  glucose,  1%  wheat  bran,  1%  beechwood  xylan  or  1%  wheat  bran  +  0.01%   ferulic   acid   as   the   sole   carbon   source.   Samples   were   taken   at  different  9me  points,  RNA  extracted  and  sequenced.    

Comparing  the  expression  pakerns  of  genes  on  different  (inducing  and  non-­‐inducing)  media  will  allow  the  iden9fica9on  of  novel  FAEs  and  GEs.  These   enzymes   will   then   be   cloned   and   overexpressed   in   the  heterologous  host  Pichia  pastoris.    

condition 1 (e.g. growth on glucose)

condition 2 (e.g. growth

on wheat bran/xylan))

Transesterifica'on  reac'on  

Figure  6.  Example  of  a  chromatogram  obtained   from  HPLC  analysis  of   the   products   of   the   transesterifica'on   reac'on  methyl   ferulate  (MFA)   to   butyl   ferulate   (BFA)   in   a   reac'on   system   with   92.5%   1-­‐butanol  and  7.5%  buffer.  

In   a   reac9on   system   with   very   low   water   content,   FAEs   preferably  carry  out  a  (trans)esterifica9on  reac9on.    We  used  a  Myceliophthora  thermophila  FAE  to  produce  butyl  ferulate  (BFA)   from   methyl   ferulate   (MFA)   in   a   binary   reac9on   system    consis9ng   of   1-­‐butanol   and   7.5%   buffer,   pH   6.5.   At   30°C,   aser   69h  more  than  55%  of  MFA  is  converted  to  BFA,  while  only  14%  of  MFA  is  hydrolysed  to  ferulic  acid  (FA).    

Figure   7.  Ra'os   of   BFA,   FA   and  MFA   present   in   the   reac'on   at  different  'me  points.    

BFA  

FA  

MFA  

MFA    +  butanol  

(   (  

BFA    FA    FAE     FAE    

0%#

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20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

0h# 4h# 6h# 23h# 27h# 48h# 69h#

%#BFA#

%#FA#

%#MFA#

Cellulose

Glucomannan

Xylan

Lignin

Cellulose