Effective laccase production method frommushroom as a natural product

天然素材向けとなるきのこ由来酵素「ラッカーゼ」の効率的生産方法

( )

1

(Lac) …?

4 Cu

(O2)H2O

OH

R

.

O

R

Laccase

O2 H2O

4e-+ H+

4 4

2

(O2 2O)

<Lac >

Laccase

Lac (red)

Lac (ox)

Lac (red) Lac (ox)

Med(red)

Med(ox)O2

H2O (ox)

(red)

Lac

Cu2+Cu+

* ; Lac

Laccase (EC.1.10.3.2)

Cu2+

Cu2+

Cu2+

Cu+

Cu+

Cu+

; Designer laccases: a vogue for high-potential fungal enzymes?Rodgers C.J. 2010 Trends in Biotechnology vol. 28 (2) p63-72

<Lac >

3

Lac産業利用の現状

( )

<Lac >

(

( )

( ) ( )

4

< >

(Lac)

5

白色腐朽菌の生産するLac

リグニン構造

Pycnoporus cinnabarinus ( )

Trametes versicolor )

Lentinula edodes(

(Lac)

Lac高生産株のスクリーニング

Strain# ibrc05015

(U/ml)

SR-1(Lentinula edodes)

Strain#ibrc05015

(a)(U/ml)

Strain#ibrc05017

(b)

(mg/ml)

Strain#ibrc05015

SR-1(Lentinula edodes)

ABTS

Strain#ibrc05007

オツネンタケモドキの特徴

Strain#ibrc05015 ITS(Polyporus brumalis)

Polyporus brumalis( ; )

(7.72U/ml ABTS 25 )

(223mg/l 35 )

( 0.9cm/day in MYPG agar medium )

(Polyporus brumalis)

a) b)

Purification step Total protein Total Lcc Specific activity Purification Yield (mg) (unit) (unit/mg) (fold) (%)

Culture filtrate 190.4 5440 28.6 1.0 100.0 Ultra centrifusion 137.2 5404 39.4 1.4 99.3 30% ammonium 89.1 3899 43.8 1.5 71.7 HiLoad Phenyl 26.8 3328 124.1 4.34 61.2 HiLoad Sepharose Q 14.6 3101 236.0 8.25 57.0

オツネンタケモドキラッカーゼ(PbLac1)の精製

PbLac1

Table.1 Purification of PbLac1

a) Purified PbLac1 CBBb) Total Secreted proteins CBB

PbLac1

PbLac1誘導剤の検討

9

Lac

act

ivity

(U

ml-1

)

PbLac1

No added

Anicidine Catecol Guaiyacol2,5xylidine

Ferulic acid

Ethanol Galicacid

Cu2+* Mn2+* Fe2+*

* 0.25mM Cu2+,Mn2+Fe2+ 1%

H2O2

各株間における銅添加によるLac活性比較

10

Lac

case

act

ivity

(U

/ml)

SR-1

-Cu +Cu (ibrc05015) -Cu +Cu

(MAFF420194) -Cu +Cu

(MAFF460203) -Cu +Cu

(MAFF460202) -Cu +Cu

(MAFF460217) -Cu +Cu

(MAFF20150) -Cu +Cu

(MAFF420155) -Cu +Cu

P. brumalis(ibrc05015

銅添加によるibrc05015Lac活性の誘導

B) **

-Cu +CuA) Lac

**0.5mM CuSO4 14

-Cu

(ABTS)

+Cu

11

-Cu +Cu +Cu/-Cu

Lac U*/ml (Max) 11.0 (day35) 156.7 (day14) 14

Lac U/mg(Max) 92.0 (day35) 2449.9 (day14) 27

C) *** CBB

PbLac1

***0.5mM CuSO4 14

PbLac1

* ABTS

12

PbLac1特性評価

< PbLac1 >

1) 2) 3)

< PbLac1 >

1) pH2)3)

Lac

P. brumalis

a) b)

d)Incubation pH

Rel

ativ

e ac

tivi

ty (

%)

Rel

ativ

e ac

tivi

ty (

%)

c)Incubation pH

Incubation pH Incubation Temperature (

Incubation Temperature (

Rem

aini

ng a

ctiv

ity

(%)

Rem

aini

ng a

ctiv

ity

(%)

Pblac1酵素特性 (1) 　温度、pHの影響

PbLac1 pH 4.0 40 pH 6.0 60 13

14

実用化試験　(1) 　PbLac1によるRBBRの脱色実験

RBBR (Remazol Brilliant Blue R)A)

→難分解性環境汚染物質PCBを分解出来ることが示唆された

RBBR 0.02%

Reaction Time (min)

0.5U PbLac1

2.5U PbLac1

5.0U PbLac1

U PbLac1

RBBRPCB

†.

RBBR Polychlorinated biphenyl (PCB)

†Novotony et al. (1997)Folia Microbiol. 42 136-140+PbLac1 -PbLac1

Dec

olor

izat

ion

rate

(%

)

15

実用化試験(1) 　PbLac1-Med系によるPoly R 478の脱色実験

Poly R 478

Reaction Time (min)

0.5mM VA

1.0mM VA

2mM VA

mM VA

5mM VA

B)

Poly R-478

Poly R-478Benzo[a]pyren †

Abs

orba

nce

ratio

(A

531/

A36

2)

Benzo[a]pyren

†Field et al. (1992)Appl. Environ. Microbiol. 58;2219-2226

-VA +VA

+PbLac1

O NH NHAc

SO3H

NH

O

n

CH

O

NOH

ONH

O

HN

Violuric acid (VA)

→発癌性物質Benzo［a］pyrenを分解出来ることが示唆された

Poly R-478 0.02% 5U/ml PbLac1

16

実用化試験 (2)　PbLac1による環境ホルモン(BPA)の分解

Bisphenol A

Bisphenol A (BPA)*

Blank +PbLac1 Blank+PbLac1-PbLac1

A) PbLac1 BPA

BPA(500ppm)PbLac1 (0.5U/ml)at 30ºC. 48hr,

GC/MS BPA

Rel

ativ

e ac

tivity

of

BPA

(%

)

Rel

ativ

e ac

tivity

of

estr

ogen

rec

epto

r ac

tivity

(%

)

*Bisphenol A

B)

*

PbLac1はBPAのホルモン活性を減少させる

Blue Copper redox protein 0v /mv Source

Plastocyanin Spinach

Azurin Pseudomonas eruginousa

Stellacyanin Rhus vernicifera

Psudoazurin Achrombactor cycloclastes

Multi Copper Enzyme

Bilirubin Oxidase Myrothecium verrucaria

CotA Bacillus subtilis

Laccase Rhus vernicifera

Coprinus cinereus

Trametes hirsute

Trametes versicolor

811 Polyporus brumalis

17

実用化試験(3)　PbLac1の酸化還元電位

* .

< *>

<PbLac1 > E=811mV vs. NHE†

PbLac1は既存の銅配位酵素の中で最も酸化還元電位が高い

< ‡>

†NHE; normal hydrogen electrode

Buffer; McIlvain buffer(pH5.0 ion intensity=0.3).

lectrode; g/AgCl E=550-600mVvs.Ag/AgCl=747-797mV v.s NHE†

18

高酸化還元電位Lacの産業的利用例

PbLac1

* PbLac1

SONY 0.5mW ( Trametes Lac)

T. versilcolor 780mV vs. NHEP. brumalis 811 mV vs. NHE

…

PbLac1*

オツネンタケモドキラッカーゼの有用性

RBBR, olyR-478)

BPA

(E=811mV vs. NHE)

19

PbLac1

(2009 )IBRC05015

PbLac1の評価

20

P. brumalis( )

T. Versicolor( )

P. Cinnabarinus( )

Ibrc05015( ) Ha1(New Zealand) ATCC200478(Australia)

PbLac1 LACA Lcc3

0.81 0.78 0.72

30-60 30-60 30-80

40 55 65

PH 4 5.5 4.5

200-250mg/L Cu PbLac1

200-250mg/L400mg/L-1.0g/L ( )

130-300mg/LMax 1.2g/L (EtOH )

NR (not reported) P. Methanolica (12.6U/ml)P. pastoris (17-64.9mg/L)S. cerevisiae (2-9.95mg/L)Y. lipolytica (2.5mg/L)

A.niger (70-80mg/L)P.pastoris (8mg/L)Y. lipolytica

,NP( )

,

( )

)

*PbLac1

(SONY)0.73mV

Pentachlorophenol(PCP)

)

高い酸化還元電位、高い蛋白質生産能を活かして産業化へ

A

21

< >

2009-295057

< >Characterization of an extracellular laccase, PbLac1, purified from Polyporus

brumalis.Keiko Nakade, Yuichi Sakamto, Yuko Nakagawa, Akira Yano and Toshitsugu Sato.Fungal Biology(2010): 114 609-618

< >Purification and characterization of Polyporellus brumalius laccase (PbLac1).

Keiko Nakade, Yuichi Sakamoto, Yuko Nakagawa, Akira Yano and ToshitsuguSato.The 5th Meeting of East Asia for Mushroom Science (Sep. 2008)

Polyporus brumalis