Aspergillus oryzae is good at usage of hydrolyzing enzyme on solid-surfaces ━ Recruitment of polyesterase (cutinase) of A. oryzae by the biosurfactant protein hydrophobin RolA on plastics

*Keietsu Abe1,2, Toru Takahashi1 The New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan (TEL +81-22-795-3205, E-mail: kabe@biochem.tohoku.ac.jp)

September 8, 2011, Sapporo

日本酒 (Sake)

醤油 (soy sauce)

TOPICS • Aspergillus oryzae and Biodegradable Plastic

Recycle System – Process design – Plastic-degrading enzyme CutL1 and a novel

plastic-degrading factor hydrophobin RolA

• Novel Molecular Functions of RolA – Recruitment of soluble esterase CutL1 by RolA

attached to hydrophobic surfaces

Fungi degrade biopolymers in nature

hyphae

hydrolysis of polymers

penetration

infection 1 2

3 4

Hydrophobic barrier

Target cells

Surface active proteins

pathogens

degrading enzymes for biopolymers

PBSA mimics wax polyesters

PBSA recycling system mimics koji-fermentation

Soy bean

Wheat

PBSA

A. oryzae

O n O

O

O

HO

OH

O

O

OH HO

1,4 butandiol

Succinate

NaCl solution

filter-press

koji Moromi mush Soy sauce

Plastic koji Plastic moromi

PBSA monomers

buffer Extraction

Recycling

醤油

O

O nO

O

PBSA ; polybutylene succinate co-adipate

HO

OH

1,4 - butanediol

O

HO

OH

Osuccinate

degradation by cutinase (CutL1)(Maeda et al. 2005; AMB)

PBSA-CD Agar Plate

PBSA-CD Liquid Medium

PBSA degradation by A. oryzae RIB40

CutL1, PBSA degrading esterase produced by A. oryzae

CutL1

- + PBSA film

Maeda, H. et. al. Appl. Microbiol. Biotechnol. 65:74-83 (2005)

Filter paper

PBSA film

Purified CutL1

Incubation at 37 ˚C for 6 hours Removal of the filter paper

20

14

27 37

(kDa)

CutL1

Hydrophobic loops Active center

PBSA

Degradation of a PBSA film by CutL1 The schematic model of PBSA-degradation by CutL1

SDS-PAGE analysis of purified CutL1

What is hydrophobin?

Alignment of deduced amino acid sequences of RolA homologs

PBSAdegradation by recombinant A.oryzae

TOPICS • Aspergillus oryzae and Biodegradable

Plastic Recycle System – Process design – Plastic-degrading enzyme CutL1 and a novel

plastic-degrading factor hydrophobin RolA

• Novel Molecular Functions of RolA – Recruitment of soluble esterase CutL1 by RolA

attached to hydrophobic surfaces

Purification of RolA from culture broth of A. oryzae overexpressing RolA

・Withpre-adsorptionof RolA

・Without pre-adsorptionof RolA

purifiedRolA

PBSAmicroparticle

30℃, 16hadsorption degradation

40℃, 0-3h

purifiedCutL1

purifiedRolA

PBSAmicroparticle

purifiedCutL1

++

degradation40℃, 0-3h+

+

Effect of RolA on PBSA degradation by CutL1

Takahashi et. al. Mol. Microbiol. 2005

Purified RolA

filter paper

PBSA film

slide glass

incubate at 30ｰ for 12 h

wash with purified water

+ purified CutL1

B. Incubate at 37° for 6 h(PBSA degradation)

C. Immunostaining(anti-CutL1 antibody)

A. Immunostaining(anti-RolA antibody)

Adsorption of CutL1 onto a PBSA film pre-coated with RolA

Takahashi et. al. Mol. Microbiol. 2005

QCM electrode

A quartz crystal microbalance (QCM) is a

sensitive mass-measuring device. Its resonance

frequency decreases linearly upon the increase

of the mass on the QCM electrode at nanogram

levels. Change of 100Hz of frequency corresponds

to 3ng of protein bound to the QCM electrode.

analysis chamber

buffer

stirring bar

Molecular Interaction Analysis by QCM

CutL1 injection More injection stabilization

Electrode attached with RolA

KD values which indicate binding affinity can be analyzed by monitoring oscillation frequency change.

About QCM

oscillation frequency down indicate Molecular interaction

RolAimmobilizedQCM electrode

BSA (control)CutL1

CurveA CurveB

※ Concentrations in the figure indicatefinal concentrations of CutL1and BSA in QCM step-wise analyses.

RolA (ligand) – CutL1 (analyte) interaction

Takahashi et. al. Mol. Microbiol. 2005

CutL1 immobilizedQCM electrode

Curve C Curve D

BSA (control)RolA※ Concentrations in the figure indicate

final concentrations of RolAand BSA in QCM step-wise analyses.

CutL1 (ligand) – RolA (analyte) interaction

Takahashi et. al. Mol. Microbiol. 2005

Preparation of CutL1 mutants for RolA-CutL1 binding assay

Six acidic amino acid residues (Glu31、Glu109、Asp142、Asp145、Asp171、Asp203) were substituted with Ser, and all variants produced by A. oryzae were purified. .

CutL1mutant のSDS-PAGE

Spin-down

SDS-PAGE

Purified CutL1 and its variants

Incubated at 30ºC for 1h

Spin-down

Free CutL1

Teflon-RolA complex

4 5 6 7 8 9

pH

CutL1

RolA＋ N a C l

RolA-CutL1 binding assay

0

0.1

0.2

0.3

0.4

0.5

Cut

L1(m

ol /

mol

of

Rol

A)

RolA

CutL1

E31, D142, D171 are important residues for CutL1-RolA interaction.

Acidic amino acid residues (Asp,Glu) of CutL1 involved in CutL1-RolA interaction

180° D142 D171

Active center

E31

60 min 10 sec

Ser-substitution variants

Schematic model of RolA-CutL1 interaction Active center

180°

D142 D171 E31

His32& Lys34

Hydrophobic side

Hydrophilic side

CutL1

RolA

Abe et al. unpublished results

Models of PBSA degradation by RolA with CutL1

SUMMARY

• RolA adsorbed to the PBSA surface recruits CutL1 to the surface, resulting in condensation of CutL1 and stimulation of PBSA hydrolysis.

• Glu31, Asp142 and Asp171 of CutL1 are critically required for the RolA-CutL1 interaction by multivalent effect.

• H32 and K34 of RolA are important for the RolA-CutL1 interaction.

ACKNOWLEDGEMENT • RolA

Muragaki K., Uehara K., Ohataki S., Maeda H, Yamagata Y., Gomi K., Hasegawa F. (Tohiku Univ.)

• A. oryzae genomics Machida M. and Asai K.( AIST)

• Recycle of PBSA Ishioka R. and Okino Y.(Showa High Polymer)

• CD analysis of RolA Wang X. and Robillard G.T.(Groningen Univ.)

• FRAP analysis Kato M., Komura M. and Sato M. (Olympus Corp.)

• QCM analysis Jitsukawa T. (Inisium Corp.)

• AFM analysis Hondo H. (Ritsumeikan UNIV)

REFERENCES • Takahashi T., H. Maeda, S. Yoneda, S. Ohtaki, Y. Yamagata, F.

Hasegawa, K. Gomi, T. Nakajima and *K Abe, The fungal hydrophobin RolA recruits polyesterase and laterally moves on hydrophobic surfaces, Mol. Microbiol., 57, 1780-1798 (2005)

• Ohtaki S., H.Maeda, T. Takahashi, Y. Yamagata, F. Hasegawa, K. Gomi, T. Nakajima and *K Abe, Characterization of a novel hydrophobic surface binding protein HsbA produced by Aspergillus oryzae and its role in degradation of polybutylene sucuccinate-co-adipate, Appl. Environ. Microbiol., 72, 2407-2413 (2006)

• *Abe K., K. Furukawa, T. Fujioka, D. Hagiwara, H. Maeda, J. Marui, O. Mizutani, T. Takahashi, A. Yoshimi, Y. Yamagata, K. Gomi, F. Hasegawa, “Novel Industrial Applications of Aspergillus oryzae Genomics.” Aspergillus: Molecular Biology and Genomics, Chapter 10, pp. 199-227, Machda M. and Gomi K. edts., Caister Academic Press (2010)

laserbeam(lowpower)

PBSAfilm

FITC labeled RolA

A. pre-Bleach

Time

laserbeam(high power)

B. Bleach

100%

0%

C. post-Bleach D. post-Bleach

A

BC

D

Fluorescence recovery after photo-bleach (FRAP)

Cover-glass

Buffer

Slide-glass

PBSA-film

FITC labeling of RolA ↓ Immobilization of FITC-RolA on PBSA film ↓ PBSA surface is overplayed with (A)buffer, (B) CutL1, (C) BSA, or (D) anti-RolAantibody ↓ FRAP analysis

FRAP analysis of FITC-RolA adsorbed to PBSA films

Takahashi et. al. Mol. Microbiol. 2005

・RolA is laterally mobile on PBSA.

・Proteins that can interact with RolA inhibits the mobility.

A phylogenetic relationship between HsbA and 4MeS

1 ATGCTTGCCAAACACGTCCTTGCTGTCCTTCTGTCTGTTGGCGCTTCCGCCATTCCTTTC 6 0 1 M L A K H V L A V L L S V G A S A I P F 20

61 GACAAGCGAGATGCATCCGCTGTCCTCGCTGATTTTAACACACTCTCAACCGACCTGTCG 1 2 0 21 D K R D A S A V L A D F N T L S T D L S 40

121 GCTCTCGGCTCTGCTATTTCGAGCTTCGATGGAACGCTCAATGGTGCCTTGGGCGTCCAG 1 8 0 41 A L G S A I S S F D G T L N G A L G V Q 60

1 8 1 CAGAAGGAAGGCCAAGTAGAGACGGCGTTGAAGCAAACCGTCAGCGATGTGAAAGCGTCT 2 4 0 61 Q K E G Q V E T A L K Q T V S D V K A S 80

241 ACTGCGTTCAGTGCTGCTGACAGCACAAGCGTGACCAATGCTGTGACTGGTCTGGAGCCT 3 0 0 81 T A F S A A D S T S V T N A V T G L E P 100

301 AGCATTGTGAACGTTCTCAACGATCTCGTTTCCAAGAAATCCGGTTTTGACTCTGTTGGC 3 6 0 101 S I V N V L N D L V S K K S G F D S V G 120

361 GTTACCAGCATTGTCGTATCGGATCTCAACTCCCTCCATGATCTTACTGGCCAGCTGTCA 4 2 0 121 V T S I V V S D L N S L H D L T G Q L S 140

421 ACTGAGCTCCAGTCGAAGGTCACTTCGGGTGACGCATCCACTATTTCTGATGAGGCTGCG 4 8 0 141 T E L Q S K V T S G D A S T I S D E A A 160

481 CGACTTGATGCGGAATACAAGAAGGCCATCGCTGCCTATTCCTAG 525 161 R L D A E Y K K A I A A Y S * 175

0.1

66.8

100

95.6

M. anisopliae 4MeS

A. oryzae HsbA

A. oryzae HsbB

A. nidulans hypothetical protein AN6795.2

A. fumigatus hypothetical protein Afu2g17630

A. fumigatus hypothetical protein , Afu4g00870

■ Produced by a entomopathogenic fungus ■ Transcribed in the fungal cells growing on the insect curticle ■ Function unknown

The phylogenetic tree of HsbA and its orthologues

N-terminal sequence

M. anisopliae growing on the surface of a insect

4MeS Hydrophobic Surface Binding protein A (HsbA)

Expression and purification of recombinant HsbA

14

20 27

(kDa)

DASAV 14

20

27

(kDa)

Recruitment of CutL1 on HsbA adsorbed on the PBSA surface

Anti-HsbA

Anti-CutL1

HsbA + -

PBSA film

HsbA

CutL1

1

2

3

4

immobilization

Detected by immunostaining using anti-HsbA or anti-CutL1 antibody

Interaction between CutL1 and HsbA Ligand; CutL1

Ligand; HsbA

BSA

CutL1

HsbA

BSA

CutL1

HsbA

WB anti-HsbA WB anti-CutL1

Immunoprecipitation

anti-H

sbA

anti-C

utL1

anti-H

sbA

anti-C

utL1

Interaction between soluble CutL1 and HsbA adsorbed on the hydrophobic

surface by QCM analysis

Soluble HsbA did not interact with soluble CutL1. (immunoprecipitation)

Ohtaki et al., Appl. Environ. Microbiol. 2006