biological route in breakdown and depolymerization of ... · 12 c2 of aromatic carbons guaiacyl in...
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Biological Route in Breakdown and Depolymerization
of Biomass
Igor Polikarpov
IFSC/USP
_______________________________________________ Igor Polikarpov, (e): [email protected] Instituto de Física de São Carlos
UNIVERSIDADE
DE SÃO PAULO
Brazilian ChemComm Symposium –
Chemistry and Sustainable Energy
(FAPESP, SP)
UK
France
Belgium Holland
Norway Sweden
Cellulose and glucose Hemicellulose and Pentose
sugars
Lignins
Marcos Buckeridge & Wanderley dos Santos
Exo-
glucanases
Endo-
glucanases
Beta-
glucosidases
Expansins GH61
Xylanases Xylosidases
• Pretreatment of Biomass
Efficiency of enzymatic hydrolysis of alkaline pretreated cellulignin increases with severity of pre-treatment
-3 0 3 6 9 12 15 18 21 24
-5
0
5
10
15
20
25
30
35
40
45
Glu
cose
(g
/L)
Time (h)
0.10
0.25
0.50
1.00
2.00
4.00
Bagasse
Cellulignin
Maeda, Serpa, et al. (2011) Proc. Biochem. 46:1196 - 1201
NaOH
concentration,
%
EF
FIC
IEN
CY
OF
PR
ET
RE
AT
ED
SU
GA
R
CA
NE
BA
GA
SS
E H
YD
RO
LY
SIS
Composition of bagasse samples after pretreatment steps
Rezende, et al., Biotechnology for Biofuels (2011) 4:54
CPMAS-TOSS NMR spectra of
sugarcane bagasse: (a) untreated; (b)
bagasse treated with H2SO4 1.0% and
(c) bagasse treated with acid and NaOH
4.0%. The spectra were normalized by
the intensity of line 10 (C1 carbon of
cellulose).
ssNMR
H L
Solid vs Hydrolisate fractions
The solid fraction spectra (a) exhibit a
progressive decrease of the lignin lines
with pretreatments using increasing NaOH
concentrations (note particularly the
methoxy carbon at 56.2 ppm on the
highlighted region).
The cellulose signals at 62.5, 64.8, 72.5,
83.5 and 105 ppm (indicated by arrows in b)
are not observed in samples pretreated with
NaOH concentrations below 0.5%, but
these lines clearly show up for higher NaOH
concentrations.
C C
C C
Line Number Chemical Group 13C Chemical Shift
(ppm)
1 CH3 in acetyl groups of hemicelluloses 21.5
2 Aryl methoxyl carbons of lignin 56.2
3 C6 carbon of non-crystalline cellulose, C6 carbon of
hemicelluloses, OCH2 carbons of lignin
62.5
4 C6 carbon of crystalline cellulose 64.8
5 C2,3,5 of cellulose, OCH2 carbons of lignin 72.5
6 C2,3,5 of cellulose and hemicelluloses 74.4
7 C4 carbon of non-crystalline cellulose and hemicelluloses,
OCH2 carbons of lignin
83.5
8 C4 carbon of crystalline cellulose 87.9
9 Shoulder of C1 carbon of hemicelluloses 101.8
10 C1 carbon of cellulose 105.0
11 C2 and C6 aromatic carbons of Syringyl and C5 and C6
aromatic carbons of Guaiacyl in lignin
110-115
12 C2 of aromatic carbons Guaiacyl in lignin 126.6
13 C1 and C4 aromatic carbons of Syringyl (e) 134.5
13 C1 and C4 aromatic carbons of Syringyl (ne) 136.9
14 C3 and C5 aromatic carbons of Syringyl (ne) and C1 and C4
aromatic carbons of Guaiacyl in lignin
148.0
15 C3 and C5 aromatic carbons of Syringyl (e) in lignin 153.5
16 Carboxyl groups of lignin 163.0-180.0
17 Carboxyl groups of hemicelluloses 173.6
ssNMR
Morphology of untreated and acid pre-treated bagasse (SEM)
Untreated
Acid pre-treated
Morphology of acid+alkaline pre-treated bagasse
SEM surface images of the sugarcane bagasse sample treated with alkaline
solutions: (a) NaOH 0.5% with bundles starting to come apart; (b) and (c) NaOH 2%,
(unstructured and unattached bundles); and (d) NaOH 4%, (individual fibers).
0,5
% N
aO
H
2%
NaO
H
2%
NaO
H
4%
NaO
H
Rezende, et al., Biotechnology for Biofuels (2011) 4:54
Crystallinity
Combined pretreatment and enzymatic hydrolysis yields of sugar cane bagasse saccharification
Enzymatic hydrolysis yields of eucalyptus bark
• Enzymatic Hydrolysis
Efficiency of biomass saccharification by commercial and home-made enzymatic cocktails.
M=Multifect
Maeda, Serpa, et al. (2011) Proc. Biochem. 46:1196 - 1201
M+P+T M+T
M+P
Some of our glycosyl hydrolases structural studies Aparicio, R. et al. (2002) Biochemistry 41: 9370-9375.; Rojas, A.L., Nagem, R.A.P. et al. (2004) J. Mol. Biol. 343: 1281-1292; Golubev, A.M. et al. (2004) J. Mol. Biol. 339: 413-422; Nagem, R.A.P. et al. (2004) J. Mol. Biol., 344: 471-480; Rojas, A.L. et al. (2005) Biochemistry 44: 15578-15584; Watanabe, L., et al. (2007) Acta Cryst. F63: 780-783; Kim, K.-Y., Nascimento, A.S. et al. (2008) BBRC 371: 600-605; Golubev A.M., et al. (2008) Prot. Pept. Lett. 15:1142-1144; Nascimento, A.S., et al. (2008) J. Mol. Biol. 382:763-778; Zamorano, L.S., et. al. (2008) Biochimie 90: 1737-1749; Watanabe, et al. (2010) J. Struct. Biol. 169: 226-242; Colussi, F., Textor, L.C., et al. (2010) Acta Cryst. F66: 1041-1044; Bleicher, L., Prates, E., et al. (2011) J. Phys. Chem. B 115: 7940–7949; Textor, L.C., Santos, J.C. et al. (2011) Acta Cryst. F67: 1641-1644; Hidalgo-Cuadrado, N., Arellano, J.B. et al. (2011) Curr. Topics Biochem. Res. 13:67-79; Santos, C., Paiva, J. et al. (2012) Biochem. J. 441:95–104; Liberato, V. M., et al. (2012) Acta Cryst. F68: 306-309; Colussi, F., Garcia, W. et al. (2012) Eur. Biophysics J. 41: 89-98
A-gal (T. reesei) Exo-Inul (A. awamori)
TrAsP (T. reesei) Lamin (Rhodothermus
marinus)
Peroxidase
(Roystonea regia)
B-man
(T. reesei)
B-gal (Penicilium sp)
Endo-Inul
(Arthrobacter sp.)
• Exoglucanases (T. harzianum CBHI/Cel7A)
0
10
20
30
40
50
60
70
80
90
100
0
500
1000
1500
2000
2500
3000
0 50 100 150 200C
on
cen
tração
B (
%)
Ab
s (
mA
u)
Volume (mL)
Native gel electrophoresis of CBHI (6, 3, 1.5 e 1 mg/mL)
CBHI
66 kDa
Topt=50ºC, pHopt=5
Colussi, F., Textor, L.C., et al. J. Microbiol. Biotech. (2011) 21: 808–817
CBHI from Trichoderma harzianum
A
Loop 6
Loop 5
Loop 6
B
Loop 6
Loop 5
Loop 5
C
A384
V216
Y371
I203
A386
T216
Tr_CBHI
Th_CBHI
Ph. crys_CBHI
A384
Y260
Y371
V216
I203
Y247
A B
Loop 5
Loop 6
T. reesei CBHI T. harzianum CBHI
L3
L3
L5
L5
A384
Q101
-6 -5
Catalytic side loops movements
are strongly anticorrelated!
DYNAMIC CROSS-CORRELATION MATRIX & ESSENTIAL DYNAMICS
• Endoglucanases (T. harzianum EG3/Cel12)
3D structure of EG3 (Cel12, T. harzianum): A cellulase without CBM
3D structure of EG3 (Cel12, T. harzianum): A cellulase without CBM
Substrate Binding Cleft
Comparison between Celulomonas fimi endoglucanase C and ThEG3
• Thermostable enzymes
Bleicher, L., et al., & Polikarpov, I. J. Phys. Chem. B (2011) 115: 7940–7949
Hyperthermostable Rhodothermus marinus β-1,3-glucanase
Topology of the salt bridges
2 C L 2
H 2 Y K
L a m R
2 5 o C 9 0 o C
2 C L 22 C L 2
H 2 Y KH 2 Y K
L a m RL a m R
2 5 o C 9 0 o C2 5 o C 9 0 o C
Rodothermus
Nocardiopsis
P. chrysosporium Salt bridges within the hydrophobic
environment facilitate water
penetration
(not every salt bridge favors
thermal stability)
Water penetration into the
hydrophobic layer of LamR
is reduced relative to less
thermostable proteins.
COLAPSE OF THE ACTIVE SITE FOR P. CHRYSOSPORIUM LAMINARINASE,
WHILE IT IS PRESERVED IN LAMINARINASE RH. MARINUS SIMULATIONS AT
HIGHER TEMPERATURE
Collapsed active site
Solvent-accessible active site
Rhodothermus
P. chrysosporium
Novel Enzymes
Targeted analysis of microbial lignocellulolytic secretomes -
a new approach to enzyme discovery
São Paulo State (Brazil):
- Prof. Igor Polikarpov (PI,
IFSC/USP),
- Dr. Sandro José de Souza
(Ludwig Institute),
- Prof. Eduardo Ribeiro de
Azevedo (IFSC/USP) &
- Prof. Wanius José Garcia da
Silva (UFABC)
UK, University of York
- Prof. Neil Bruce (PI),
- Profs. Simon McQueen-Mason &
- Peter Young (Co-PIs).
Acknowledgements
Thematic project & CeProBIO
Prof. Munir Skaf (UNICAMP)
CeProBIO team
Prof. Marcos Buckeridge (CTBE& IB/USP)
Prof. Paulo Seleghim Jr. (EESC/USP)
Profa. Anete P. de Souza (CBMEG/UNICAMP)
Prof. A. Augusto F. Garcia (ESALQ/USP)
Profa. Glaucia M. de Souza (IQ/USP)
Prof. Carlos Labate (ESALQ/USP)
Prof. Marcelo E. Loureiro (UFV)
Dr. Itamar Soares de Melo (EMBRAPA)
Dr. Jose Geraldo Pradella (CTBE)
Prof. Luiz Antonio Martinelli (CENA/USP)
Prof. Armando Augusto H. Vieira (UFSCar)
&
all the EMBRAPA and SUNLIBB collaborators
Structural similarities between Celulomonas fimi endoglucanase C and ThEG3 carbohydrate recognition
CfCBM ThEG3