Biological Route in Breakdown and Depolymerization

of Biomass

Igor Polikarpov

IFSC/USP

_______________________________________________ Igor Polikarpov, (e): ipolikarpov@ifsc.usp.br 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

Thank you !

Structural similarities between Celulomonas fimi endoglucanase C and ThEG3 carbohydrate recognition

CfCBM ThEG3