Solvent as a competitive inhibitor for Candida antarctica lipase B

(1)

HAL Id: hal-00329734

https://hal.archives-ouvertes.fr/hal-00329734

Submitted on 13 Oct 2008

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Solvent as a competitive inhibitor for Candida antarctica lipase B

Marianne Graber, Romain Irague, Eric Rosenfeld, Sylvain Lamare, Linda Franson, Karl Hult

To cite this version:

Marianne Graber, Romain Irague, Eric Rosenfeld, Sylvain Lamare, Linda Franson, et al.. Solvent as

a competitive inhibitor for Candida antarctica lipase B. Biochimica et Biophysica Acta Proteins and

Proteomics, Elsevier, 2007, 1774, pp.1052-1057. �hal-00329734�

(2)

$&%(')+*,.-/(01/324%6587&*-:9;-:9<)+*=9,?>&9@?9A-:%(B=C%(BED1F(GIH(JHKFLF(GNMF(OQPRMJPF

'97?/(0!*TS

UWVXYVZZ[]\]XV_^`[XacbedcfhgjiKVYkZ(lXVmcno[capdcqrXYs4fhgut[Zcvw[xy:apd{zc|x~}`VYZrViKVX[capdcYkZyV4{XVZ`tgZcQVZy

6VXxcn`x~w

r`A`]`p c¡Qo¢cc;o`R£``p¤{pe:¥¦`§6pR£¨p©ªc¤:`p«¬ c®

¯

`°!±`p¤{A £²o§6o³Wp¥R¬cp °´µ`¬Q³¶p`{¥¦p·`¬c !¸o¹»º¼c½]¡Q

¨`p u¥¾`¿À¸ cÁR;`

Â

p·R_Ã§6oÄÅ]£¨p`§6_ÃÆ:

¯

`:Å]£``¤Æ: AÆ:`ÇA¬`]Å?±j`p¤Æ:

´]¢»eÈÉr`;ÆÊ¥Ro;

¯eË

©A¸ºÌÍ:¸

¯

Î`§

¯Ï

``ÐeR`c`cÑÒÓ¼ÄÌÔKÕÕ`Ö{¹Ô`ÖÄÌ¼×ÁR¿ÄÑ

ÒÓ¼ÄÌÔKÕÕ`Ö{¹Ô`¼ÄÌeÔc :§`pwÑ:Î`peØÓ¢cp``ÐkÎk`ÐÃ

Á`j`` Ñ¨Ù4¥Rc;·c`e¤+`¬`Ã`c;]Ñ±pÐÑ#ÖÖc©Õ©¼ÄÌ¼cÕ©ªÔ:ÌÖÄºÚAÅc¿ÄÑ#ÖÖc©Õ©¼ÄÌe©¼cÕ©ªÔ½©AÌeÕÐ

Ë

©Û§`p

AÑ

ÜÝÞß ÝwàáâäãeåkÝæçpÝ è

³¶Ðoéh;o¢cê#Åc¿ÄÑ#¼ÄÌe©Ô©ªÕÕ`Ö{¹©Ô`¼ÄÌeÔÐ

Ë

©Û§`pÄAÑ

ë ÞAåkåk ;àßâkA ìcæë ìcæí èïî

Ð»ð.¬ê

ñ?ò_óôáõö÷{ø

ù

YkZ[_wYs»tNúûgXm`VZcYÃstgx~}`[Ze{úÀiágjx[s_n`xVXQiKg`y[xkYZcm.ü¦tgxkYy»ýþm`V»t!^YÃgÄsVªVx~|`tYpt#ú+sgjZvwgjXiáV_wYgZVx

sÿVZcm`[]ü¦tgjx~n^YkxkY~|

(3)

lZK[Z|iK[üªsV_ªVx~|`t[y(X[Vs_wYÃgjZ`tdcwÿ[]sÿgjYs[]gjvQtgx~}j[Z» gvpª[Z(ÿVetViKVX

ù

[y [vvw[s_ gjZwÿ[X[Vs_wYgZ

gcnªsgjiá[»lªZwÿcYpt`V`[XdY~#YptRtªÿg(ZwÿV_tgx~}`[Z» [vvw[s_t sgn`xÃy6^j[][xÃVYkZ[y6^|wÿ[]V_^YxkY~|ágjv:wÿ[

tgx~}j[Z»{ªgÀVs_ V»tV]sgi`[_wY~wYï}`[YkZÿcY~^Y~ªgjX!ªg(wÿ[twn^:tÛwXV_ª[

q`[XYiK[Z»ªVxkx~|dwÿ[][vvw[s_ gvQtY+tgjxï}`[Zetd`ü`[Z»ªVZgZ[dü`[ZeªVZgZ[d`üÛiK[wÿ»|xü`ü`[Z»ªVZgjxdü

iK[_wÿe|xüü`[ZeªVZgxd`üÛiK[_wÿe|x`[Z»ªVZ[]VZyüwiK[_wÿ»|x`[Z»ªVZ[KV»trtÛnoycY[y(YkZáVtgxkYy»ým`VetQX[VsªgjX

! tViKg`y[xX[Vs_wYgZdcwÿ[#" ! %$hüQsV_ªVxï|°[ywXVZÄtVs_|xV_wYÃgjZ^j[_&á[[Z(iK[wÿ»|xXg`VZg`V_ª[]VZy('ü

Xg`VZgxdKVetRtÛnoycY[y)»lÛ*áV»tRtªÿg(ZwÿV_{^`gcwÿ ù [_ªgZ[»t YkZcÿY~^Y~ª[y6wÿ[][Z|ciá[]Vs_wY~}Y~|6ÿ[X[V»t

wÿ[4ª[XÛwYVXÛ|áVxsgjÿgjxpt VZywÿ[ÿe|ycXgÄsVXÛ^`gZÄt ycYy(Zgc+

!

xsgjÿgjxYkZÿcY~^Y~wYgZKsgZÄtwªVZ»td-,ed

y[_ª[Xªi(YkZ[yáYZX[»t[Zs[]gv*`ü`[ZeªVZgZ[dü`[ZeªVZgZ[dÄVZy.üÛiK[_wÿe|cxÃüü`[ZeªVZgxd`sgjZvYkXiK[y6wÿ[

iKVX

ù

[y(YkZcÿY~^Y~ªgXÛ|ásÿVXVs_ª[Xgv:wÿ[

ù

[_ªgZ[etVZyKVZáV_^:t[Zs[]gv{YkZcÿY~^YïwYgjZágjv/üÛiK[wÿ»|xüü

`[ZeªVZgx&

0Qÿ[iKgx[s_n`xÃVXQiKg`y[xkYkZm twnoy»|áV»t%`[XvwgjXªiK[yKgjZwÿcX[[tgjxï}`[Zetd`ü`[Z»ªVZgZ[d`üÛiK[_wÿe|xü`ü

`[ZeªVZgx`VZy`üÛiá[_wÿ»|x`[ZeªVZ[elÛtÿg1á[yKV]sx[VXQYkZcÿY~^YïªgjXÛ|á[vvw[s!vwgX!wÿ[ ù [ªgjZ[]VZywÿ[

ª[XÛwYÃVXÛ|áVxsgÿgxdc^cn#Zg.[vvw[s_#vwgX!wÿ[ÿ»|ycXg`sVXw^`gZ)32Àg.sÿVZm`[YkZK[Z4|iK[]sgZcvwgXiKV_wYÃgjZáV»t

t[[ZKy»n`XYkZm(wÿ[tYin`xV_wYgZÄt5

0Qÿ[tÛnoy»|6x[yªg(wÿ[]sgZsxïn{tYgZwÿV wÿ[][vvw[s_ gv#Vyy[yKgjXªm`VZcYs]sgji`gZ[Z» gjZ6xkY`V»t[]sV_ªVx~|4e[y

wXVZ`tVs|cxÃV_wYgZKsgcn`xy^`[][1xVYkZ[y^c|wÿ[]sgi`[_wY~wYï}`[YkZÿcY~^Y~ªgjXw|KsÿVXVsª[XgjvQtgjx~}j[Z»t#ªg1áVXyÄt

wÿ[vYXAtÛ{^YZycYkZm twn^:twwXV_ª[iK[_wÿe|xXg6YÃgjZV_ª[

(4)

#gXVxVXm`[Zen`i^`[Xgjv#[Z4|iK[»td`VZy [et`[sYÃVxkx~|6vwgjXRtgjiá[xkY`V»t[»tRtÿg16YkZm twn^:twªVZ»wYVx`Vs_wY~}Y~|(YZ

Z[VXx~|áVZcÿe|ycXgn{trtgx~}`[Zetdcwÿ[4n{t[]gjv#gXm`VZcYÃstgx~}`[ZetV»tQX[Vs_wYgZ(iK[ycY~n`i `gut;t[»t;t[»t mX[V_

Vy»}jVZ»ªVm`[et 40 ÿgut[]Vy»}`VZeªVm`[»tQYZsx~noy[tgjxïn^YkxkYï|Kgv{ÿ»|ycXgÿgc^Ystwn^:tÛwXV_ª[»tdYkZsX[V»t[y

wÿ[XiKg:tÛªV_^YkxkY~|ágjv:wÿ[][Z|iK[dÄVZyKVtÿcYvpQgv:wÿ[4wÿ[XiKg`y»|ZVi(YÃs][»n`YkxkY~^XY~n`i YkZ6vwV_}`gcn`Xgjv

tÛ|Zewÿ[»tªYptgc}`[XQÿe|ycXgjx~|`tªYpt 40 ÿ[][Z|ciáV_wYs]Vs_wYï}Y~|6YkZKgXm`VZYstgx~}`[Z»t!}jVXY[»trtYkmZYkvYsVZewx~|-(Y~wÿ

wÿ[tgjxï}`[Ze n{t[ydc^cn{n`ZcvwgXÛn`ZVª[x~|6Y~#Yptgvpª[Z(xgK[X!wÿVZ(YZKVª[XRtgx~nwYgZÄt50Qÿ[X[vwgjX[Y~#Ypt

Yki`gXÛªVZe{ªg iKV

ù

[]Vm`g`g`y sÿgYs[]gv tgjxï}`[Ze+Äz`[_}j[XVx:twnoycY[»t%`[XªvwgjXiá[y(YkZ(xkY»n`YÃy gXm`VZYsiK[ycYÃV

ÿV}`[4^`[[ZKy[_}jgª[yªg(wÿ[Xgx[]gv tgjxï}`[Ze wÿ[xV»twQy[sVy[»t+'üdc^cntÛwYkxxcwÿ[X[YtQZgKm`[Z[XVx

n`Zy[XAtÛªVZycYkZcm.gv:wÿ[tgx~}j[Z» [vvw[stRt[[Z)40 ÿ[tÛn`mm`[»twª[yKgjXYmYkZÄtgvQtgx~}`[Z» [vvw[s_t sVZ^j[

ycY~}Yy[y(YkZ»ªg(wÿcX[[iKVYkZKsV_ª[m`gXY[»t

'

! #"%$

ò'&

" ò

")(

ó+*&ò',õ

".- õö/*

10

&õ

"2 #"

÷)3wõö

-54 ò768&:9%&

4&0

ó/0Qÿ[]sgjZvwgjXiáV_wYgZKVZy(vx[ Y~^YkxkY~|

gv!VZK[Z|ciá[]VX[]sXÛnosYVxvwgX[vvYsY[Z» sV_ªVxï|ÄtªYpt<; ! Xg`[XsÿgYs[]gvQtgx~}`[Z»*(Yxkx`g6cwYi(Ye[

wÿ[et[ `VXViK[_ª[XAt vwgjXVt `[sYkvYsX[Vs_wYgZ)40Qÿ[tÛn`mm`[etÛwYgZ(ÿVet!^`[[Z(YkZ»}j[»tÛwYm`V_ª[yÀt[_}j[XVx

wYkiK[et (YïwÿKycYkvvw[X[Z» gnªsgjiK[et>=V_nwÿgjXAt VmX[[YZcm?+A@#QVZyB '/C#ªdÄV_nwÿgjX;tycYptVmX[[YkZm?+''/

VZyD+' E5F

*

! #"%$

òøG&

"

øAõ

4IH 9!&

4&0 ó #"

÷&÷{òø;õ

4KJ+ 10

&põ

"

gjvQtwn^:tÛwXV_ª[»tVZyXgÄy»nost ! sÿVZm`[]gjv

tgx~}j[Z»*6Ykxkx`Vvvw[s wÿ[tgx~n^YxkY~|áVZyKy[»tgjx~}jV_wYgZKgv twn^:twwXV_ª[»td6Xg`y»nos_tdÄVZyK[Z|iK[d

x[VycYkZcm(ªgÀV]sÿVZm`[yKsgji6iKgjZá[»n`YkxkY~^XYVvwgXVxkx`VXwwYsY`VZet`[sYÃ[»t 40 ÿYpt 6Ykxkx`sÿVZcm`[4wÿ[

V`VX[Z»tÛn^otÛwXVª[ X[vw[X[Zs[]gv:wÿ[][Z4|iK[(YïwÿgcnQsÿVZmYkZcm(wÿ[YZ»wXYkZ`tYs Xg6`[XwwY[»t

gvuwÿ[X[VswYgjZ Äz`gx~n^YxkY~|á[vvw[s_tVX[]gjvpª[ZK[X[»tAt[yKV»tVX[xÃV_wYgZ^`[_&á[[Zá[Z|iK[

(5)

E;

õ#*{ò

0 &0 &J ò'&

"%

&I9%&

0

&õ

"

;{z`gjxï}`[Ze!iágjx[s_n`x[»t!^YkZyÄt YkZ»ªg(wÿ[]Vs_wYï}`[tYïª[d16ÿ[X[4wÿ[_|-6Ykxkx

Vs_ V»t sgji `[_wY~wY~}j[YkZcÿY~^Y~ªgXAt!ªg(wÿ[twn^:tÛwXV_ª[(Ykvvw[X[Z» ù YkZyÄtgv tgjxï}`[Ze!iágjx[s_n`x[»tQÿV_}`[

ycYkvvw[X[Z» V_^YkxkY~|ªgá^YkZyªgÀVmYï}`[ZKVs_wY~}`[tªY~ª[d`VZywÿYptycYkvvw[X[Zs[]sV_n{t[»trtgx~}`[Ze [vvw[s_t

'3ü'E+

z`gjxï}`[ZeQ[vvw[s_tVX[]ycYkvvYs_n`x~{ªg.V»tAt[»tAtQYZ(xkY»n`Yy(iK[ycYVd:tYkZs[4wÿ[4wÿ[XiKg`y»|ZVi(YÃs]Vs_wY~}Y~wY[»tgv

tÛn^otÛwXVª[»tdXg`y»nos_td`VZyÀtgjx~}j[Z»t sVZ(Zg{^`[4}jVXY[yáYZycY~}Yy»noVxkx~| 40 ÿ[X[6Ykxkx`Vxtgá^j[]V

sgZsgi(Y~ªVZ» sÿVZcm`[»tQYZÀtÛn^otÛwXV_ª[]VZyXg`y»nos_tgx~n^YkxY~|6Y~wÿÀtgjxï}`[ZeªdÄVZywÿ[]sgXX[swYgjZ6vwgjX

tÛnosÿKsÿVZcm`[»tQYtQvwVXQvXgi tÛwXVYkmÿ»wvwgXáVXy)40 ÿ[»t[xkYi(Y~ªV_wYgZÄt sVZ^j[]g}j[XsgiK[4n{tYZcm.VtgxkYy»ýþm`V»t

X[Vs_ªgjX5»lZáVtgxkYy»ým`VetQX[VsªgjXQY~#Ypt`gut;tY~^x[4ªgÀsgZ»wXgjxd`VZyáYZy[`[Zy[Z»wxï|KVy wn{tÛªdcwÿ[

wÿ[XiKg`y»|cZVi(Ys]Vs_wY~}YïwY[»tgvQtÛn^:twwXV_ª[»t VZy(ZgZüwX[Vs_ªVZ»tVXgn`Zy6wÿ[][Z|ciá[dutgáwÿV_ VtYkZmx[

X[Vs_wYgZ`VXViá[_ª[XsVZ^`[]sÿVZcm`[y6Y~wÿgcn Vvvw[s_wYZcm.VZ»|ágcwÿ[XAt 'ü'# 40 ÿen{td(ÿ[Zn{tªYkZcm.V

tgx~}j[Z»ýþm`V»tQX[Vs_ªgjXdÄVxxcwÿ[XiágÄy»|ZVi(Ys]Vs_wYï}Y~wY[et (Yxkxc^`[]sgZÄtwªVZ» y»n`XYZcm.VX[VswYgjZdÄVZywÿ[X[

6Ykxkxc^`[ZgÀ[vvw[s_tQX[xÃV_ª[yªgÀsÿVZcm`[etQYkZÀtwn^:tÛwXV_ª[tgx~n^YxkY~|

lZwÿYptRtwnoy»|dá[][_}`VxïnoV_ª[yÀtgx~}`[Z» sgi`[_wY~wY~}j[YkZcÿY~^YïwYgjZáV»tVZK[ xVZV_wYgZKgv tgjxï}`[ZeQ[vvw[s_t

q`[XYiK[Z»ªVx`yV_ªVvXgi VtgxkYy»ým`VetQX[VsªgjXd`V»t;tÛn`XYkZm(wÿ[]V_^ot[Zs[]gjvQtgjx~}j[Z» [vvw[s_tdKVet

sgi^YZ[y6Y~wÿ(iKgx[s_n`xÃVXQiKg`y[xkYkZm twnoycY[»tdÄVZy." ! %$hüQsVªVx~|4e[ywXVZ`tVs|cxÃV_wYgZ^`[_&á[[Z

iK[_wÿe|xXg6`VZg`V_ª[]VZy 'üXg6`VZgjxáV»t!n{t[yKV»tViKg`y[x:tw|Ätwª[i 40 ÿ[tÛnoy»|6YkZsx~noy[yÀtY gjXm`VZcYs

tgx~}j[Z»t>= `ü`[Z»ªVZgZ[dü`[ZeªVZgZ[d`üÛiK[wÿ»|xü`ü`[Z»ªVZgjxdüÛiK[wÿ»|xüü`[Z»ªVZgjxd`ü

iK[_wÿe|x`[Z»ªVZ[]VZyüwiK[_wÿ»|x`[Z»ªVZ[7Fgjv{Xgcn`mÿcxï|wÿ[tViK[iágjx[s_n`xVXRtªYe[dc^cn*6Y~wÿKycYkvvw[X[Z»

(6)

!#"

"RÿcYkXV|iK[4 =Ûx~|gÿcYkxkYe[y(xkY`V»t[vXgi

¥r`KoÃ°p

|4`[$F áV»tVmYkvp#vXgi fhgÄsÿ[

lZy»n{tÛwXY['=ALQ[Z^j[Xm`d`\[XªiKVZ»|F

!

xkx:twn^:tÛwXV_ª[»t á[X[]gv:wÿ[ÿcYmÿ[»tw cn`XªY~| = @#@%$ i(YkZcYin`iF

"Rÿ[i6YsVxpt/K[X[]ycXY[yXYgX!ªgán{t[*'üL#Xg6`VZgxKV»t ycXY[y^c|6X[vxïnYkZm 6Y~wÿ(iáVmZ[»tªY~n`i VZy6wÿ[Z

ycYptwwYkxkx[y)UW[wÿ»|xXg`VZg`V_ª[áV»t ycXY[y 6Y~wÿ#2.V

"&(' VZyKycYptwwYkxkx[y(vXgi L

&*) 0Qÿ[]ycXYÃ[y

tgx~}j[Z»t á[X[twªgjX[yn`Zy[XVXªm`gjZKVwiKgut ÿ[X[]VZyKgc}`[XQiKgx[sn`xVXRtYÃ[_}`[»t5

+ %",%-/.10,2,43!.1%",0,",!5"67..1,%-80

qrZ|iK[]VyÄtgjX cwYgjZ-KV»t`[XvwgXiK[yKgZ»ªg "RÿXgjiágutgjXÛ^L

!:9

U "µz`diK[»tªÿ #CüC = ! sYy

9 V»tÿ[y (Y~U [_wÿ»|x"RÿxgjXg zYkxVZ[7F>=L#XgxV_^jgÄdc{XVZs[7FVety[»tsXY~^`[yáYZKV X[_}Ygn{t/KgX

ù '7;#&^c|

n{tYZcm #C(i(m.gjv{x~|gÿcYkxkYe[yK[Z|ciá[ X[`VXVwYgjZ `[X (mÀgvQtgxkYyÀtÛn`gXÛ"RÿcXgiKg:tgXÛ^*

=<.1>-50!?"8067.3@,%-1"8,!5AB%"05!@"8#"

0Qÿ[4^YÃgjX[Vs_ªgX!n{t[y(YkZwÿYptRtwnoy»|(Ypt y[»tsXªY~^`[y(YkZ X[_}YÃgn{t%cn^xkYsVwYgjZ`t 'd' ! |YÃsVx

[1`[XYkiK[Z»*KVetQXÛn`ZKV_C;#CD+"4d6Y~wÿKV_^jgn 'i(m.gjv#VyÄtgjXw^`[yK[Z4|iK[d%'4i6mÀgv#VyÄtgXÛ^j[yK[Z|iK[

X[`VXV_wYgZKsgXX[»t `gjZycYkZcm(ªg FE`m.gjv{x~|gÿcYkxkYe[y(xkY`V»t[40 ÿ[4ªgªVxvxÃg1 `V»tAtªYkZcm(wÿcXgn`mÿwÿ[

X[Vs_ªgjX áV»tRt[_ V_ 'i(iKgxi(YkZ%Ga`mY~}YZcm.V4}`gx~n`iK[_wXªYsvxg gv i i(YkZHGaI6Zy[X!wÿ[»t[

sgZycY~wYÃgjZ`tdx[»tAt#wÿVZEC%$3gv:wÿ[twn^:tÛwXV_ª[»t á[X[]sgZ»}j[XÛª[yªg.XgÄy»nostVxkxg6YkZcm(wÿ[

V»t;tÛn`icwYgZwÿV_#YkZcY~wYÃVxXV_ª[»t =[X[»tAt[y(YkZEÄiágjx[etgjv*Xg`y»nos_#vwgXiK[y`[XQi(YZ»nª[]VZy `[XQi(m.gjv

(7)

@-,0,B@-8 .1%" "8 @"

0Qÿ[4}`V3`gn`X%ÿV»t[x[V_}YkZm(wÿ[4^YÃgjX[Vs_ªgX KV»trtVixÃ[yn{tYkZmÀV'C) i xg`g6.gZKVtY`ü áV_|}`Vx~}j[

=6Vxsg#FQiKVYZ»ªVYkZ[yKV_ 'CD "#`zÄVix[»t á[X[]VnªgjiKVwYsVxkxï|(YkZ [s_ª[yáYZwÿ[t xkY~#YkZ [s_ªgXgjv#Vm`V»t

sÿXgjiáV_ªgjmXVÿ = !

mYkxÃ[Z»#iKg`y[x )@#C 2 F[»n`Y`[y6Y~wÿKVvxViá[YgjZYeV_wYÃgjZKy[ª[s_ªgX>= {l FQvwgjX

y[_ª[swYgjZágjv#VxkxXgÄy»nos_t5° gjX!wÿ[][`[XªYkiK[ZetQYkZX[et[Zs[]gjv*`ü`[Z»ªVZgjZ[d ü `[Z»ªVZgjZ[d ü

iK[_wÿe|x`[Z»ªVZ[d`üÛiK[_wÿe|xü`ü`[ZeªVZgx`VZyüwiK[_wÿ»|xü ü`[Z»ªVZgxdwÿ[]sgx~n`i(Zn{t[yKV»t VZ& C '

=i C)Ki(i Y&ïy) C)FE`i vYkxki wÿYs

ù

Z[etAtd"RÿcXgi`Vs

ù

d{XVZs[/F ° gjX!wÿ[][`[XªYkiK[ZetQYkZ

X[»t[Zs[]gjv*`üÛiK[wÿ»|x`[ZeªVZ[d`V -$ '7;#C '¨sgx~n`i(ZáV»t!n{t[y =#C(i C)Ki(i Y&ïy) C) E`i

vYkxki wÿYs

ù

Z[»tAtd

9

z`sY[ZewYkvYs7F5»lZáVxkx`sV»t[»t!wÿ[txkY~#XVwYg áV»t#'/@ %&' 0Qÿ[YkZ [s_ªgXVZy6wÿ[

y[_ª[sªgjX á[X[ ù [c V_/ECD+"VZy CD "ÓX[»t`[s_wY~}`[x~| 2KY~wXgm`[ZáV»t!n{t[yKV»tV]sVXªXY[XQm`V»t/(Y~wÿáV

vxg XVª[]gjv ' &'i i(YkZ%Ga`YkZwÿ[]sgjxïn`i(Z)|ycXgm`[ZKVZyKVYkX á[X[twnxkY[yªgáwÿ[4{l TV_ (VZy

C(i i(YkZ GadX[»t `[s_wY~}j[x~|

#gX!wÿ['»noVZewYkvYsVwYgjZágjv{iK[_wÿVZgxdXg`VZgxdiK[_wÿ»|xXg`VZgÄV_ª[]VZyXgc|cxXg6YgZVª[djYZwÿ[

X[»t[Zs[]gjv*`ü`[ZeªVZgZ[]VZy.ü`[ZeªVZgZ[dcwÿ[YZcY~wYVx`sgjxïn`i(Zª[i`[XV_n`X[áV»t ECD+"40Qÿ[

ª[i`[XV_n`X[áV»t

ù

[c V_ ECD+"y»n`XYZcm.Ki(YkZ»nª[etVZywÿ[Z(YkZsX[V»t[yªg HD+" n{tªYkZcm.VXV_ª[]gv

C)HD "Ói(YkZHGa#gX!wÿ['»noVZewYkvYsV_wYÃgjZKgv:wÿ[twn^:tÛwXV_ª[»tVZyXgÄy»nostdYkZwÿ[ X[»t[Zs[]gv ü

iK[_wÿe|x`[Z»ªVZ[dcwÿ[]sgx~n`i(Zª[i`[XVn`X[KVetQÿ[xÃy V ECD+" vwgX%Ki(YkZdwÿ[ZXgmXVi(iá[yªg

YkZsX[V»t[]V_ C)&'7D "#äi(YkZ%Gaªg %'7D+"#gX!wÿ['»noVZewYkvYsVwYgjZágjv:wÿ[twn^:twwXV_ª[»tVZyXg`y»nos_tdYkZwÿ[

X[»t[Zs[]gjv*`üÛiK[wÿ»|x`[ZeªVZ[dcwÿ[]sgjx~n`i6Zª[i`[XV_n`X[áV»tQÿ[xyKV_ #CD "ÓvwgjX#'i(YkZdwÿ[Z

XgmXVi(iá[yªg YkZsX[V»t[]V_ '7D "#äi(YkZHGajªgF;D+"#noVZ»wYïªV_wY~}`[]yVªVK[X[]g^ªVYkZ[y Vvpª[X

YkZeª[mXV_wYgZKgZKVZ

!

\]lÛQq2 0 #@ Àt[XYÃ[»tQlllQYZ»ª[mXVªgjX

!

ZK[ª[XZVx:tÛªVZyVXy(iK[_wÿgÄyáV»t!n{t[y

(8)

,!56!%- ,!5 B

!

xkxiKg`y[xkYZcmKVetygjZ[]gjZáV(zÄ\]lQvpno[x:twªV_wYgZ =AzYkxkYsgjZK\]XVÿYs»tQlªZs7F!n{tªYkZcm(wÿ[(zc|e^c|x ;)CB '/@#

`Vs

ù

Vm`['=0 XªY`gut lZs7F5°#gX!wÿ[iágjx[s_n`xVXy»|ZVi(Ys»tRtªYkin`xVwYgjZ`tdcwÿ[46gxkxkiKVZ ! xkx ! ªgi ECd%'/

vwgXs[vY[xÃy áV»t!n{t[y)

!

sXÛ|`tÛªVx:twwXÛnos_n`X[]gv:wÿ[][ic|á[Z4|iK[d1L -$ sg`y[ '30 " ! E áV»t

X[_wXªY[_}`[yávXgji f "]z$1yV_ªV4^jVZ

ù #ªdÄVZyX[`VX[yávwgXQvpn`XÛwÿ[XQiKg`y[xkYkZmÀVet%X[_}Ygn{tªx~|

y[»tsXY~^j[yB E

#gX[Vsÿ.tgx~}`[ZeªdÄVtgjxï}`[Ze!iágjx[s_n`x[áV»tQiKVZenoVxkx~|6iKg`y[xkx[yáYZ»ªgáwÿ[]Vs_wY~}j[tY~ª[YZKV

sgZcvwgXiKV_wYÃgjZnwYxkYYkZm.V_}`VYxV_^xÃ[ÿ»|ycXgm`[Z^`gZycYZcm.`gut;tY~^YkxkY~wYÃ[»t 40 ÿ[iKVZenoVxkx~|6vwgn`Zy`gutªY~wYgZ

áV»tgcwYki(Ye[y6^|KVt[XY[etgjvQtªÿgXÛ y»|cZVi(YsetRtYkin`xV_wYgZÄtVZyK[Z[Xmc|(i6YkZcYki6YeV_wYgZÄt ?cvwgxkxgá[y^c|

VZK[Z[Xmc|6i(YkZYki(Y°V_wYgZKgjv)6ÿgx[tÛwXwnos_n`X[{YkZVxkx~|áV]y»|ZVi(YÃs»tRtYin`xV_wYgZ ='3#C:t F KV»t XVZKgZ

wÿ[6ÿgx[twwXÛnos_n`X[d:tÛªVXÛwYkZcm6Y~wÿKV##C:t áVXiKünÿV»t[40Qÿ[]y»|ZVi(Ys»tRtªYkin`xVwYgjZ-KV»t

tVix[yK[_}j[XÛ| C :tdjmYï}YkZm EC#CÀtªZV:tªÿgc¦twwXÛnos_n`X[et]gv:wÿ[][Z4|iK[ü<tgjx~}j[Z» sgix[ *40 ÿ[

6ÿgx[ Xg`s[y»n`X[áV»t!wÿ[Z(YkZy[`[Zy[Z»wx~|6X[`[Vª[y(vXgji wÿ[tgx~}`[Z» ygÄs ù YkZcm.ÿVet[dmY~}YZcm.V

ªgcªVx`gjv C#CÀtZV:tÿgtwwXÛnos_n`X[»t5

!

xx:tZV3:tÿgctÛwXÛnosn`X[»t á[X[twn`[XYki`g:t[y gZwÿ[vYkXAtÛ gZ[

n{tYZcm.Vxkx`[Z4|iK[]V_ªgiÀtd`VZy6wÿ[X[»twn`x~wYkZcmKvYkxÃ[KV»t#n{t[yªgá}YptÛnoVxkYe[4wÿ[tgx~}`[Ze#iKgjxÃ[s_n`x[

iKgcwYgZ(YkZwÿ[]Vs_wY~}j[tY~ª[]y»n`XYZcm(wÿ[iKgx[sn`xVXy»|ZVi6Ys»tRtªYkin`xV_wYÃgjZ

)(

Y`V»t[üsVªVx~|4e[ywXVZ`tVs|cxÃV_wYgZ(X[VswYgjZ`tQvwgxkxg V YkZm`ü`gjZmKiK[sÿVZcYptªi (YïwÿKVZKVxÃsgjÿgjx

y[Vyüª[Zy(YkZÿcY~^Y~wYgZB '`d'7;d »lªZwÿ[ X[et[ZeQsV»t['=Az`sÿ[iK[ ' F!wÿ[xkY`V»t[]sVZ^YkZyK[Y~wÿ[X

iK[_wÿe|xXg6`VZg`V_ª[4|Y[xycYkZm.VxkY`V»t[üÛiK[_wÿe|xXg6`VZg`V_ª[]sgix[.gX!^YZy 'üXg`VZgxc|Y[xycYZcm

(9)

sVZ XgÄs[[yªgÀVZáVs_|xü[Z4|iK[YkZeª[XiK[ycYÃV_ª[]VZywÿ[vYkXAtÛ/Xg`y»nos_ =ÛiK[wÿVZgjxKF ágcn`xy^`[

X[xÃ[V»t[y)2.[cªdcwÿ[t[sgZyÀtÛn^otÛwXVª[d6Xg6`VZgjxÃdjYkZeª[XVst (Yïwÿwÿ[]Vs|cxÃü[Z|ciá[X[»tÛn`xïwYkZcmKYkZwÿ[

wÿ[ XgÄy»nos_/Xgc|xXg`VZg`V_ª[]VZy VvX[[xkY`V»t[elZwÿ[ X[»t[Ze#YkZ»}j[»tÛwYkm`VwYgjZdwÿ[ YkZcm`ü `gjZm

iK[sÿVZYpti áV»tsgZcvYXiK[y(YkZwÿ[ X[»t[Zs[]gv*`ü`[ZeªVZgZ[dü`[ZeªVZgZ[]VZy.üÛiK[_wÿe|xüü

`[ZeªVZgxd`Vxkx`V_ VvYj[ywÿ[XiágÄy»|ZVi(Ys]Vs_wYï}Y~|ágjvC);) ! tVZK[jVix[dwÿ[X[»tÛn`xïtg^ªVYkZ[y(YkZ

X[»t[Zs[]gjv*`ü`[ZeªVZgZ[]VX[tÿg16Z(YkZuYkmcn`X[ *

06 2,4303 3@0@,433 3@>-80@,%- B5,.1,6 %" , + 0550 B%" ,6 " .1 %"8

, 0! "#" <.1>-50#"

qrZ|iK[]gZKVtgxkYyÀtwn`gjXÛ*áV»t%xVs[y(YkZwÿ[tgxkYy»ým`Vet!^YgX[Vs_ªgjXdÄVZyKV»t`[XsgxV_ª[y6Y~wÿ

ZcYïwXgjm`[ZKsVXXw|cYZcm(wÿ[4&ág twn^:tÛwXV_ª[»tQiK[wÿ»|xXg`VZg`V_ª[]VZy('ü Xg6`VZgxd16ÿg:t[

wÿ[XiKg`y»|cZVi(Ys]Vs_wY~}YïwY[»t á[X[ ù [c sgZÄtwªVZ» = C)&' F elZKVyycY~wYgZd`VZgZ(X[VsªVZ» gXm`VZcYÃs

sgi`gZ[Ze!i6Yki(Ys

ù

YZcm.Vtgx~}`[Z»*KVetVyy[yd(Yïwÿwÿ[XªiKgÄy»|ZVi(Ys]VswY~}Y~wYÃ[»t!^`[&K[[Z CKVZy C))

lZuYkmcn`X[ 'Rwÿ[4wXVZÄtVs_|xV_wYgZKVs_wYï}Y~|ágjv/" ! %$ YptRtÿg16Z(YkZwÿ[ X[»t[Zs[]gjv{mXVy»noVxkx~|

YkZsX[V»tªYkZcm(wÿ[XiKg`y»|ZVi(YÃs]Vs_wY~}Y~wY[»tgv*`ü`[ZeªVZgZ[dü`[ZeªVZgZ[d6`üÛiá[_wÿ»|xü`ü^cnªVZgjxd`ü

iK[_wÿe|xü`ü`[ZeªVZgxdüÛiK[_wÿe|xüü`[ZeªVZgxd`üÛiK[_wÿe|x`[Z»ªVZ[]VZyüwiK[_wÿ»|x`[Z»ªVZ[0Qÿ[4&ág

ù

[_ªgZ[»t VZy`üÛiK[wÿ»|xü`ü ^nªVZgx`sx[VXx~|6YkZcÿY~^Yïª[ywÿ[][Z4|iK[]Vs_wY~}Y~|dutªYkZs[4wÿ[YkZY~wYVxX[Vs_wYgZ

XV_ª[]y[sX[V»t[y(YïwÿKVZ(YkZsX[VetYkZm ù [ªgjZ[4wÿ[XiágÄy»|ZVi(Ys]Vs_wYï}Y~| #gjX!wÿ[]gwÿ[XRtÛn^otÛwXVª[»tdZg

YkZÿcY~^Y~wYgZKVetRt[[Z)

0>-805, ,435 05,2,@"00",43. -, .1,! . ->"8,43.10,

.10, 0!.105 ,!

lZwÿ[ X[»t[Zs[]gv#Vtgx~}`[Z»ªdc^`gcwÿwÿ[]V`VX[Z» VZyXg`VZgjxYkZÿcY~^Y~wYgZKsgZÄtwªVZ»t!-,°sVZ^j[

y[_ª[Xªi(YkZ[yávXgji [1`[XYkiá[Z»ªVx`yVªVd16ÿ[X[4wÿ[X[Vs_wYgZ(YkZY~wYVxXVª[d}1,ädYpt%xgcª[y}`[XAtÛn{t!wÿ[

(10)

Xg`VZgx`V ! = {Ykm.#F ]fh[xgcwYkZcm(wÿ[]yVªV]gjZeªg X[sYXg`sVx`Vj[»td%'Aýp} , }`[X;tÛn{t#'Aý V !=uYkm 1F!|cYÃ[xy[y

VmXVÿ-(Y~wÿ.tÛwXVYmÿ»#xkYkZ[»tQYkZeª[XAt[s_wYkZcm.V_ 'Aý#V "!

'dÄVZywÿ[4}jVx~no[]gvuwÿ[]gjXycYkZV_ª[]V wÿYpt

YkZeª[XAt[s_wYgZ`gjYZ» [»noVxpt 'Aý +°uXgji wÿ[X[sYXgÄsVxxgcªdÄycYpt;tg`sYV_wYÃgjZKsgjZ`tÛªVZetdc-,äd`gjv

Xg`VZgxYkZwÿ[ X[»t[Zs[]gv*`ü`[ZeªVZgZ[]sVZ^`[]gc^cªVYZ[y)

9

ÿ[Zwÿ[txg`[»tgv:wÿ[]sgZ»}j[XmYkZm

xkYkZ[»tQYkZwÿ[X[sYXg`sVxxgc#Ypt%xgcª[yKVm`VYkZ`tÛ{wÿ[4wÿ[XiKg`y»|ZVi6Ys]Vs_wY~}Y~|ágjv*Xg`VZgxd`V !dwÿ[

X[»twn`x~#YptVtwwXVYkmÿe!xYkZ[6ÿg:t[YkZeª[XAt[s_wYgZ6Y~wÿwÿ[]V^:tsYptAtV][»noVxptü

, = {Ykm #F

9 Y~wÿKVvYj[yÀtgx~}`[Ze Vs_wY~}Y~|KgvC);dwÿ[]ycYptAtgÄsYÃV_wYgZKsgZÄtÛªVZ»tQvwgX%`ü`[ZeªVZgZ[dü`[ZeªVZgZ[

VZyüwiK[_wÿ»|xü ü`[Z»ªVZgxK[X[]y[_ª[Xi(YZ[yªg C)C C)C ''dEC)C C)C#C#@ VZy C)CC)C ' `d

X[»t`[s_wY~}j[x~| 40 ÿ[»t[4}jVx~no[»t á[X[YZKVssgXyVZs[6Y~wÿwÿ[4}jVXÛ|YkZm YZcÿcYï^Y~ªgXÛ|ásÿVXVs_ª[Xgv:wÿ[

ycYkvvw[X[Z» sgi`gcn`ZyÄt

!

tV]sgi`VXªYptgZdcwÿ[

î

g^ªVYkZ[y(YkZKV_^ot[Zs[]gjv#VZe|KVyy[y(ZgZü!X[Vs_ªVZ»

sgi`gZ[Ze!YZwÿ[m`V»t[gcn{tRtÛwX[Vi KVetQvwgn`Zyªgá^`[][ »noVxcªg C)C '/@B '7;# 40 ÿ[

î

gc^cªVYkZ[y(YkZwÿ[

X[»t[Zs[]gjv:wÿ[4&ág

ù

[ªgjZ[»t á[X[]V_^jgn{&6Ys[]V»tQÿYkmÿdYkZycYsV_wYkZm.V]sgji `[_wY~wYgZ^`[&K[[Zwÿ[

ù [_ªgZ[»t VZyXg`VZgxvwgX!wÿ[YkZeª[XVs_wYÃgjZ6Y~wÿ(xY`V»t[]Vs_wY~}`[tªY~ª[40 ÿ[»t[4}jVx~no[»t g^ªVYkZ[yávwgX

ù

[_ªgZ[»t VX[Z[VX!ªgáwÿ[ X[_}YÃgn{tªx~|Ky[ª[Xi(YkZ[y

î

gc^cªVYkZ[y(YkZwÿ[ X[»t[Zs[]gjv*`üÛiK[wÿ»|xü`ü

^cnªVZgxE=[»noVxcªg C)C Fd16ÿcYsÿKVetQvwgn`Zyªgá^`[]VZ(YkZcÿY~^YïªgjXgv/" ! %$

,!56!%- ,!5 B

lZeª[XVs_wYÃgjZ`t!^`[_&á[[Zwÿ[xkY`Vet[]VZy6wÿ[tgx~}`[Z»t%`ü`[Z»ªVZgjZ[d6`üwiK[_wÿ»|xü`ü`[Z»ªVZgxdÄVZy`ü

iK[_wÿe|x`[Z»ªVZ[K[X[tÛnoycY[y 6Y~wÿ(iágjx[s_n`xVXQiKg`y[xkYZcm*40 ÿ[

ù

[_ªgjZ[]VZywÿ[4ª[XÛwYVXw|KVxÃsgjÿgjx

tÿg1á[yKV]sx[VXVvvYkZY~|(vwgX!wÿ[]Vs_wY~}`[tªY~ª['= {Ym V]üQs/F n`XYkZm.V '/C#C :trtYkin`xÃV_wYgZdc^`gcwÿÀtÛªV_|[y

YkZwÿ[]Vs_wY~}j[tY~ª[4^jgn`Zy(Y~wÿ6ÿ»|ycXgm`[Z^`gZyÄt#ªgáwÿ[]gc|VZYgZ(ÿgx[]VZy»ýwgX!wÿ[]sV_ªVx~|ewYst[XªYkZ[

lZwÿ[][ZyKgv:wÿ[tYin`xV_wYgZdcwÿ[

ù

[ªgjZ[xgutw#Y~tQÿ»|ycXgm`[Z^jgjZyÄtVZyûtwªVXÛª[yK[1xgXYkZcm(wÿ[]Vs_wY~}j[

ù

(11)

tÛªVXÛª[yªg iKgc}`[]VXgn`Zywÿ[X[Vvpª[XQx[vp{wÿ[]Vs_wY~}j[tY~ª[40Qÿ[][Z4|iK[tÛwXÛnosn`X[]ycYy(Zgctÿg VZ»|

tYmZcYkvYsVZ» sgZcvwgXiKV_wYÃgjZVx`sÿVZcm`[]y»n`XYkZm.VZ»|ágjv:wÿ[]y»|cZVi(YsetRtYkin`xV_wYgZÄt>=wX[»tÛn`xïtQZgctÿg6Z1F

E #

%"" 3@,%-1"8,!0@3 3@0"

q`[XYiK[Z»t YkZKVtgxkYy»ýþm`V»tQX[Vs_ªgXRtÿgá[ywÿV_{wÿ[]Vs_wY~}Yï|Kgv/"

!

%$W}`VXY[y6Y~wÿÀtgx~}j[Z»

X[»t[Z»#YkZ6m`V»t%ÿVet[d`VZyá[ÿV_}j[][_}`Vx~noV_ª[ywÿ[ `g:tAtªY~^YxkY~|ágjv:wÿ[tgjxï}`[ZetV»t!^j[YkZcm.sgi`[_wYïwY~}`[

YkZÿcY~^Y~ªgjX;t!ªgáwÿ[tÛn^:twwXV_ª[40Qÿ[YkZÿcY~^Y~ªgjXw|K[vvw[s_ gjv

ù

[_ªgZ[»t/KV»t sx[VXªx~|Ky[iKgjZ`tÛwXVª[y^c|

YkZsX[V»tªYkZcm(wÿ[YkX!wÿ[XiKg`y»|cZVi(Ys]Vs_wY~}YïwY[»t>=uYkm ' F 0Qÿ[YkXQYkZcÿY~^YïªgjXÛ|á[vvw[s áV»tQX[xV_ª[yªg wÿ[

[vvw[sQgZwÿ[]ycYptAtgÄsYÃV_wYgZKsgZÄtÛªVZ» gjv*Xg`VZgjxd

î

djYZwÿ[ Xg`VZgjxüª[Z|iK[]sgjixÃ[*

! Z

YkZsX[V»t[YkZ

î

iK[VZ`t!wÿV wÿ[YkZcÿY~^YïwYgjZ^c| Xg6`VZgxKV»t xg1á[X[ydÄgX!wÿV_{wÿ[

ù

[_ªgjZ[»t!ªg`g

ù

gc}`[X

wÿ[Xgjx[]Vety[VyK[ZyáYZcÿcYï^Y~ªgXAtQZg1Tsgji `[_wYkZcm6Y~wÿ(iK[wÿ»|xXg`VZg`V_ª[4ªgá^YkZyªg(wÿ[vX[[

[Z4|iK[elZX[_}Ygcn{tRtÛnoycY[et!wÿ[tViK[4^`[ÿV_}Ygn`X áV»tQvwgcn`Zy(vwgX KVª[XVZywÿ[iKgÄy[XV_ª[x~| `gxVX

sgi`gcn`Zy`üÛiK[_wÿe|xü`ü ^nªVZgxE= {Ykm ' F 'd no[4ªgáwÿ[tªYki(YkxÃVXY~|^`[_&á[[Zwÿ[4ª[XÛwYÃVXÛ|

Vxsgjÿgjxpt`üÛiK[_wÿe|cxÃü`ü ^cnªVZgx`VZy`üÛiK[wÿ»|xü`ü`[Z»ªVZgjxY~ sgcn`xy^`[]VetAtÛn`iá[ywÿV_{wÿ[|ágn`xÃy

[ÿcY~^Y~ VtYki(YxVXQYkZeª[XVs_wYÃgjZ6Y~wÿwÿ[][Z|ciá[djZg [»n`Yï}`Vx[Z»{ªgáwÿ[][`[XªYkiK[ZeªVxvYkZycYZcm:t

0Qÿ[][1`[XYkiK[Z»ªVxvYkZycYkZcm:t gjv#VZ(YZcÿcYï^Y~ªgXÛ|á[vvw[s_ gv

ù

[ªgjZ[»tVZy6ª[XÛwYVXÛ|áVxsgjÿgjxpt#^cn#Zgc gjv

Vx

ù

VZ[»tgZ."

!

%$hüsV_ªVx~|4e[y(X[VswYgjZ`t á[X[tÛn`gXÛª[y^|(iKg`y[xkYZcmKX[»twn`x~t `ü L [Z»ªVZgZ[]VZy `ü

iK[_wÿe|xü`ü`[ZeªVZgxá[X[4^`gcwÿKV_^x[4ªgá^YkZy(YkZwÿ[]Vs_wYï}`[tYïª[ X[_}`[Z»wYkZm iá[_wÿ»|xXg`VZg`V_ª[4ªg

X[Vs_+%&]Zwÿ[]gwÿ[XQÿVZyd6`üwiK[_wÿ»|x`[ZeªVZ[ÿVy(Zg+tÛnosÿK[vvw[s_+n`XYZcmKiKgx[s_n`xVXy»|cZVi(Yset

Vvpª[X!^j[YkZmcn#YkZwÿ[]Vs_wYï}`[tYïª[djYï¦twªVXÛª[y(iKgc}YZcm.VZy(vYkZVxkx~|6x[vp{wÿ[]Vs_wY~}j[tY~ª[dxVs

ù

YkZcm(wÿ[

`g:tAtYï^YkxkYï|(m`[_wYZcm.VZsÿgX[y^c|6ÿ»|ycXgm`[Z^jgjZyÄt

(12)

0Qÿ[][1`[XYkiK[Z»ªVx`yV_ªVYZKsgi^YkZV_wYgZ6Y~wÿ(iKgx[s_n`xÃVXQiKg`y[xkYkZm X[»tÛn`x~trtÛwXgZcmx~|.tÛn`gXÛt

sgi`[_wY~wYï}`[YkZÿcY~^Y~wYgZKV»tVZá[xVZV_wYgZ(vwgXRtgx~}`[Z» [vvw[s_t5°uXgji Viágjx[s_n`xVXQiKg`y[xkYZcm.`gjYZ»

gvu}Y[ádwÿYptRtwnoy»|KVxtgX[»t[Zet!&ág X[VetgZÄtQvwgX%X[vw[XªXYkZcmetgx~}`[Z»#YkZcÿY~^YïwYgjZg}j[X[Z|iK[

sgZcvwgXiKV_wYÃgjZVx`sÿVZcm`[V»t V4^`V»tªYptQvwgX!wÿ[tgx~}j[Z» [vvw[stRt[[Z){YkXAtwwx~|KVZy(YkZ(vwV_}jgn`Xgv:wÿ[

ÿ»|4`gcwÿ[»tªYptgv#sgji `[_wY~wY~}j[YkZcÿY~^Y~wYÃgjZdÄ[1`[XYkiá[Z»ªVxx~|

ù

Zg16Z(YkZÿcY~^Y~ªgXÛ|Àtgjxï}`[Zet%X[vw[XªX[yªg twªV_|

YkZwÿ[]Vs_wY~}j[tY~ª[]y»n`XªYkZcm(wÿ[iágjx[s_n`xVXy»|ZVi(Ys»tRtªYkin`xVwYgjZ`td(ÿ[X[Vet!wÿ[ZgjZüÛYkZcÿY~^YïªgjXÛ|.tgx~}j[Z»

ÿVyáZgán`Xm`[4ªg+tÛªV_| `z`[sgZycx~|d`VZy(YkZKsgjZvxkYs_*6Y~wÿwÿ[twn`mm`[»tÛwYÃgjZKgv#sgZcvwgXiKV_wYgZVx`sÿVZm`[»td

Zg+tYkmZYkvYsVZ» [Z|ciá[]sgjZvwgjXªiKV_wYgZVx`sÿVZm`[»t á[X[t[[Z(YkZKVZ»|ágjv:wÿ[tªYkin`xVwYgjZ`t

z`gjxï}`[ZeQ[vvw[s_tVX[]sgji xkYsV_ª[yáYZ(ZV_n`X[dÄVZywÿ[]sgZsx~n{tYgZKgv:wÿcYptRtwnoy»|(X[xkY[»t gjZwÿ[4n{t[]gv

wÿ[tgjxYy»ým`V»t X[Vs_ªgXdmY~}YZcm(wÿ[]g`gjXwn`ZcY~|ªgÀ[jsx~noy[twn^:tÛwXV_ª[tgx~n^YxkY~|6YptAtwno[»tV»tV

sgixkYsV_wYkZcmKvwVsªgjX elZ(xY»n`YyÿV»t[4wÿ[X[YptQZg+tÛnosÿ `gut;tY~^YkxkY~|diKV

ù

YkZm YïQ[}`[Z(ÿVXy[X

iK[VetÛn`XYkZm.VZyn`Zy[XAtÛªVZycYkZcm+tgjx~}j[Z» [vvw[s_t elZÀtwn`i(iKVXÛ|dcwÿ[4wÿX[[4^`VetYs][1xVZV_wYgZÄtQvwgX

tgx~}j[Z» [vvw[stQiK[ZewYgjZ[y(YkZwÿYpt%`V`[XüQsgjZvwgjXiáV_wYgZVx`sÿVZcm`[dutgjx~n^YkxkY~|áVZy sgji`[wY~wY~}`[

YkZÿcY~^Y~wYgZKü (Yxkx:tÛwYkxxc^`[4}`VxkYyd`VZyÀtÿgn`xÃy Vxkxc^`[]sgZÄtªYy[X[y 6ÿ[Z(iKV ù YkZm.[vvwgjXwt!ªgÀ[1xVYkZ

[1`[XYkiK[Z»ªVxkxï|KVsÿcY[_}j[yÀtgx~}`[Z» [vvw[s_t YkZKgcwÿ[XRtw|Ätwª[iÀt5

'

qrXY

ù

([XZ[_#Ypt Vs

ù

Zg6x[ycm`[y(vwgXQÿcYptsgjZewXY~^cnwYÃgjZ`t!ªgáwÿ[iKgÄy[xkYkZcm*

(13)

%

'/Q\. $h[xxd8L áVxkxkYkZm`d1$ °U gÄgX[d L VXÛwXYycm`[]VZy ï\.°fh[[etd$YÃgÄsVªVx~|`tÛ{^`[ÿV_}Ygn`XQYZ

xgKü&KV_ª[XRtÛ|`tÛª[i.td

±Q£¨p``pÐ

'3 ='/@#@ F )ü ;)

E z*KgXycYs ù d8L#XYkZsYx[etVZyÀt[x[s_ª[y V3xkYsV_wYÃgjZ`tgjv{ZgjZüV»no[gcn{t[Z4|iKgjxÃgjmc|dYkZ%° 9

$¨xVZsÿd z*4"¾xÃVX

ù = q¾yÄt Fd

!

xkY[y^YÃgÄsVªVx~|`tYptdU VXs[x K[

ùù

[Xd 2.[(gX

ù

d%'/@#@ 'd6*&'ü *'

&#%"#rVVZ[d z* $]g`[X[Zdc 6gut VZy."# 6[[m`[Xdfn`x[etQvwgjXgcwYki(YeV_wYgZKgvu^Yg`sV_ªVx~|`tªYptQYkZ

gXm`VZcYÃstgx~}`[Zetd

£``pÐe£e¤{Ð

#C ='/@)H;1F *'ü<H;)

E f 6VxkY~}j[_|dÄ\.! gjÿZÄtÛªgZd"# `znos

ù

xkYkZcm.VZy L °áVxkxkYZcm`d{zÄgx~}j[Z» [vvw[stgjZ

^YÃgÄsVªVx~|`tYpt YkZKgXm`VZcYÃstÛ|`tÛª[i.td`[»n`YkxkY~^XY~n`i `g:tY~wYÃgjZKVZy(XV_ª[»t gjv{xkY`Vet[]sV_ªVx~|4e[yK[»tÛª[XYkvYsVwYgjZd

£¨p``¡&£¨pe¤{Ð

)='/@#@ ' F#''3;ü'')

!L ! °uY~ `V_wXYÃs

ù

VZy ! UxkY~^jVZgc}`dcKg sVZwÿ[tgjxï}`[ZeQVvvw[s_ [Z|ciá[][ZVZewYg:t[x[s_wY~}Yï|

Ð´h§6Ðo¥r`§(Ð

¯

Ð

''3 =3'/@#@ ' F ' ü '7; '

#%"#f

9

[»tsgcªd

!

°U °xkYï^`VZg}jd 0Qÿ[tgjx~}j[Z» y[`[Zy[Zs[]gjv#[Z4|iK[t`[sYkvYsYï|d

£¨p`§(Ð

£¨p·RÆ:Ð´]eÃ

d%' EC ='/@#@ #F#'ü @)

4;#!L KVxxkYkZcm`d$¨Yg`sV_ªVx~|`tªYptQYkZ(xÃg1áüáV_ª[XQiK[ycYÃV#°n`Zy[XAtÛªVZycYkZcm.[vvw[s_tgv X[Vs_wYgZKsgZycYïwYgjZ`td

¥R¬cAÐ:·`Ðo¥¦`§(Ðe£¨p

; =EC#C#C1F=; `üC)

(14)

5 ! "RÿV_noycÿVXw|d{z* 6ViKV_ªdcq# $h[s

ù

iKVZd 32án`Xg

ù

dcf °U °x[|cxÃ[d8L KVy»nod !

fn{tAt[xd"4gZ»wXgjx`gvQtÛn^cwYxkYptYZÀtÛn^otÛwXV_ª[t `[sYkvYÃsY~|^c|.tgx~}`[Ze [ZcmYkZ[[XYZcmKYkZKgXm`VZYstgx~}`[Z»tVZy

tÛn`[XsXY~wYÃsVxvx~nogXgvwgjXiád

Ð´§(Ðo¥¦`§(Ð

¯

Ð

''='/@#@ 1F#' @ 'ü' E@#C '

A@# 9 VªVZV_^j[d0 (g:tÿYyVd{z* Iá[Yd0Qÿ[Xgx[]gv!sgjZvwgjXiáV_wYgZVxvx[ Y~^YkxkY~|ágjv#[Z4|iK[»tQYZwÿ[

ycYptsXYki(YZV_wYgZ^j[_&K[[ZKVi(YZgÀVsYyKVZyK[»twª[XRtÛn^otÛwXVª[»tQvwgX!wÿ[tÛn^wYkxkYptªYkZüªsV_ªVx~|`t[y(X[Vs_wYÃgjZ(YZ

gXm`VZcYÃstgx~}`[Zetd

£c¤:`¥r`§(ÃÆ:

= EC#C #F C `ü *'

'/C# H&µxÃgjvt;tgZdl

!

32KYsÿgxkxptd z*

9 Yù

iKVZd0 $ !

Vs_wY~}Yï|(YkZ-KV_ª[XüÛi(YptsY~^x[]gjXªm`VZcYs

tgx~}j[Z»tcsgXX[xVwYgjZ`t!^`[&K[[ZK[Z|ciá[ `[XvwgXiKVZs[d`[ZVZewYg:t[x[s_wY~}Yï|KVZyXgª[YkZÀtÛwXwnos_n`X[

wÿcXgn`mÿÀt `[s_wXg:tsg6YstwnoycY[»td

¤{Ðe£c§(Ð:¥¦`§6Ð

=EC#C F=; Cü;

''/ 6VZcm`d `z* ágjXycYs

ù

d{z \VXy[dc|ycXVwYgjZágjv#[Z4|iK[YkZ(ZgjZV»no[gcn{tQiK[ycY~n`i Yt

sgZÄtªYptÛª[Ze*(Y~wÿ.tgx~}`[Ze y[`[Zy[Zs[]gv{Y~tVs_wYï}Y~|d

£·RÆ:Ð

Ð

H; =EC#C #F *' `ü %'

' E `z`[s_n`ZygÄd`\4"VXX[VdY`V»t[]VswY~}Y~|áVZy sgjZvwgjXiáV_wYgZ(YkZ(Z[V_ gjXªm`VZcYstgjxï}`[Zetd Ð³ pÐ

¥¦oÃ`Ð»£Ñ

Ë Æ:§op

d '/@üEC =EC#C#F>@ü3'/C*

'3# \VXsYVü

!

xx[»td c\gªgXd

!

xsgÿgxYkZcÿY~^YïwYgjZáVZyÀt`[sYkvYsYï|ÀtwnoycY[»tgv{xkY`V»t[$¶vXgi

¥¦`KoÃe

YkZKgXm`VZYstgx~}`[Z»td

£¨p``pÐe£e¤{Ð

@ =3'/@#@) F#'ü'7;#C)

'E U °U VXÛwYkZ[xkx[dc n`x~ªdcYkZ[_wYs»tgv#Vs_|xcwXVZ`tvw[XQX[Vs_wYgZ(YkZágjXm`VZcYsiK[ycYV]sV_ªVxï|Ät[y^c|

¥¦`KoÃe

xkY`V»t[$hd

£¨p`§(Ðe£¨p·RÆ:Ð´]eÃ

d%' *'='/@#@ F#'/@ 'ü'/@;)

(15)

'Rz*rViKVX[]VZyU °r[m`gc|d 9 gX

ù

YkZcm.V_ sgZ»wXgjxkxÃ[yKV_ª[XVs_wY~}Y~|(YZKV]sgZ»wYkZenogn{t%XgÄs[etAt/

wÿ[m`VetÛýtgjxYyÀtÛ|`tÛª[i VetVtgx~nwYgZd

£``pÐe£e¤{Ð

='/@#@ F)H;ü #@;)

'#Rz*rViKVX[dU r[m`g|áVZy6U c\]XV_^j[X!Uïd{z`gjxkYÃy»ým`V»t!^YgX[Vs_ªgXAt `g1á[Xvpn`xcªg`gxptQvwgX

vpn`ZyViK[Z»ªVxX[»t[VXsÿKVZyK[vvYsYÃ[Z»{ª[sÿZgxgmc|(vwgXQYkZy»n{twwXYVx`V3xkYsV_wYÃgjZ`t

éhû¥¦`§(Ð

=EC#C #F ü #

'7;# U L $]gcn{t<»no[ªün^jgnosÿdcU\µXV^`[Xd 2 Äz`gcn{tVd{z*°rViáVX[]VZy6U r[m`g|d

!

xsgÿgx~|`tYtsV_ªVxï|Ät[y^c|

¥¦`KoÃe

xkY`V»t[$¶YkZáVm`V»tÛýªtgxkYyÀtÛ|`tÛª[i g^j[_|`tV L#YkZcmL gjZm

$¨Y$¨YiK[sÿVZcYptªi (YïwÿKsgi`[_wY~wY~}j[YkZcÿY~^YïwYgjZ^c|wÿ[]Vxsgjÿgjx:twn^:tÛwXV_ª[]VZyáV_ª[Xd

£¨p`§(Ð

£¨p·RÆ:Ð´]eÃ

d%' C =EC#C ' F>@#Cü @#@)

' U c\]XV_^j[XdcUL $hgn{t »no[_ªü n^`gcnosÿd{z*°rViKVX[]VZyU r[m`gc|d

!

xsgÿgx~|`tYtsV_ªVxï|°[y

^c|

¥¦`KoÃe

xkY`V»t[$¶YZKVm`V»tÛýªtgxkYyÀtÛ|`twª[i c[vvw[s_tgv áV_ª[XgZ ù YkZ[_wYs `VXViK[_ª[X;td

£¨p`§(Ðe£·RÆ:Ð´]eÃ

' =EC#C1F% `ü*

'/@#%0QXY`g:tQlZsïd '#@#@+z`gnwÿKVZx[_|fhy)~d{zc+°rgcn`YptdU YptAtgn`XYÃd ' `d%I+z

!

EC#Rz* 9 [YZ[Xd1L ! 6gxkxkiKVZd ! "V»t[d I 4"`zYkZcmÿd "#\µÿYg`dÄ\. ! xÃVm`gjZVd{z L#Xgvw[_ªVd

L ° 9

[YkZ[Xd

!

Z[ vwgXs[vY[xy(vwgjXQiKgx[sn`xVXQiK[sÿVZYsVx:tªYkin`xV_wYÃgjZKgv{Z»nosxÃ[Ys]VsYÃyÄtVZy

Xgcª[YkZ`td

Ð´§6Ð:¥¦`§6Ð

¯

Ð

'/C ='/@) #F=; ü ;) *

%'/Rz* 9 [YZ[Xd1L ! 6gxkxkiKVZd 4032 mcn|[Zd ! "V»t[d ! ZáVxkx`V_ªgi vwgjXs[vY[xy(vwgX

tYin`xV_wYgZÄt gjv*Xgcª[YkZ`tVZy(Zenosx[Ys]VsYy)

Ð:¥Rc§#·¬`Ðo¥r`§(Ð ¹

='/@) 1F%#Cü *

(16)

E I `[Z»^j[Xm`dcU0 °áVZÄt[Zd{z* L V_

ù

VXVZy.0 ! gZ[etd0 ÿ[t[»no[Zs[d`sXÛ|`tÛªVx:tÛwXÛnos_n`X[

y[_ª[Xªi(YkZVwYgjZáVZy(X[vYZ[iK[Z» gjv*KvwgXiÀtgv{xkY`V»t[$¶vXgi

¥¦`KoÃe

d ¯

Ã¬°¬c

='/@#@ #F%E@ü#C)

# °U $h[XªiKVZd 9 [»tÛ^XgÄg

ù

d °#[Zcm`d`\\µYxkxkYkxVZyd032 $¨ÿV_ªdc 9 [Ypt;tYkm`dl 32

zÿcYkZy»|Vxgc}`d1L q $hgcn`XZ[d 0 ÿ[ Xgcª[YkZKyV_ªV4^jVZ ù d

È¬pp´]Q¨

=EC#C#C1F% ü *

ERz*f]V3eVdc uXVZÄt;tgZdc °n`x~ªdcqRZVZ»wYÃgut[x[s_wYï}Y~|6YkZ

¥¦`Kokep

xkY`Vet[$

!

iKgx[s_n`xVXy»|cZVi(YsetRtÛnoy»|d

R`

¯

pÐ

'/C =EC#C ' FE@ü)

_wYpt;t[d{z*°rViKVX[dcU °r[m`g|áVZyU c\]XV_^j[Xd{zÄgxkYy»ýþm`V»t!^YgÄsV_ªVx~|`tYtcVZKV3Xg6XªYV_ª[

ªg`gjxcªg+tÛnoy»|wÿ[YkZvx~no[Zs[]gjv#gXm`VZcYÃs]sgji `gn`ZyÄtgjZ ù YkZ[_wYÃs»tgv xY`V»t[]sV_ªVx~|4e[yKVxÃsgjÿgjxï|ÄtªYpt

$¨Yg`sÿcYki $¨Ygÿ»|`t

!

s_ªV]ü!L#Xgcª[YkZ`t L#Xgcª[gi(Ys»td ' =EC#C1F% ;ü *

(17)

>&

$#H

öò ; ¯

oÄ`¢cpc.kÅÅ°Ãc.

Æ:§(`°pÆuÐ

0Qÿ[][vvw[s_ gjvQtgx~}j[Z»#YkZÿcY~^Y~wYgZKgjZwÿ[

Vs_wYï}Y~|ágjv#V4wXVZÄt[etÛª[XYvYsV_wYgZ(X[Vs_wYgZ^`[&K[[Z(iK[_wÿe|cxXg6`VZgÄVª[]VZy 'üXg`VZgjxYptrtÿg6Z

vwgXQYkZsX[V»tYZcm+tgx~}`[Z»{wÿ[XªiKgÄy»|ZVi(Ys]VswY~}Y~wYÃ[»t ! Z(YkZÿcY~^Y~ªgjXw|K[vvw[s_*KV»trt[[Z6vwgjX!wÿ[tgx~}`[Zet

`ü`[ZeªVZgZ[d ü`[Z»ªVZgjZ[]VZy`üÛiK[wÿ»|xü`ü^nªVZgxd(ÿ[X[VetQZg+tYkmZYkvYsVZe [vvw[s_*áV»tRt[[Z(vwgX

`üÛiK[wÿ»|x`[ZeªVZ[düÛiK[wÿ»|x`[ZeªVZ[d`üÛiK[wÿ»|xü`ü`[Z»ªVZgjx`VZy.üÛiK[_wÿe|cxÃüü`[ZeªVZgx&

>&

$#H öòE;

± Ï

0 ÿ[YkZcYïwYVxXV_ª[d}1,ädiK[V»twn{t[y(YkZwÿ[ X[»t[Zs[]gv*`ü`[ZeªVZgZ[d

xgcª[yKVm`VYkZ`tÛ{wÿ[4wÿ[XiKg`y»|ZVi6Ys]Vs_wY~}Y~|ágjv{iK[_wÿe|xXg6YÃgjZV_ª['=V ! FV_ ycYkvvw[X[Z»/Xg`VZgx

wÿ[XiKg`y»|cZVi(Ys]Vs_wY~}YïwY[»t>=V

!

F50Qÿ[]sgZÄtwªVZ»{wÿ[XiKg`y»|ZVi(YÃs]Vs_wY~}Y~|Kgv*`ü`[ZeªVZgZ[][»noVxpt

C);)

>&

$#H öò * Â

eA§6opc.Å.Ã`··Ro

f][sYXg`sVxYkZY~wYVxXV_ª[»t%xgcª[y}`[XAtÛn{t!wÿ[

X[sYXg`sVxcwÿ[XiKg`y»|cZVi(Ys]Vs_wY~}Yï|Kgv{iK[_wÿ»|xXg`VZgÄV_ª[YZwÿ[ X[et[Zs[]gjv*`ü`[Z»ªVZgjZ[0Qÿ[

}`Vx~no[]gjv 'Aý #Ypt mY~}`[ZáV»t!wÿ[]gXycYkZV_ª[4}`Vx~no[]V_ 'AýwV ! ' 0Qÿ[]sgjZ`tÛªVZe wÿ[XiKg`y»|ZVi6Ys]Vs_wY~}Y~|

gv `ü `[Z»ªVZgjZ[][»noVxpt C);)

>&

$#H öò#; Â

eA§6opc.ÅK`¢cþcÀcÃokÅQ·R·R`

î

40Qÿ[YkZÿcY~^Y~wYgZKsgZÄtÛªVZ»ªdc-,äd

gv Xg6`VZgjxE=;zÄsÿ[iK[ ' FQYZwÿ[ X[et[Zs[]gjv*`ü`[Z»ªVZgjZ[d1áV»ty[ª[Xi(YkZ[y(vXgi VX[üxgc gjv:wÿ[

X[sYXg`sVxxgc tªÿg(Z6YkZ(vYkmcn`X[#)40Qÿ[txÃg6`[»t gjv:wÿ[YZ»ª[XAt[s_wYkZmKxkYkZ[et K[X[ xgcª[yKVm`VYkZ`tÛ{wÿ[

wÿ[XiKg`y»|cZVi(Ys]Vs_wY~}Yï|Kgv*Xg6`VZgxdÄV! 40Qÿ[YkZeª[XAt[s_wYgZ^`[_&á[[Zwÿ[Z[ xkYkZ['=ÛX C)@ 1FVZy

wÿ[]V^:tsYptAtV][»noVxptü-,

(18)

>&

$#H öò#;

¯

oj§6`cûk``eþÐ

0 ÿ[]Vs_wY~}j[tY~ª[]gv

¥r`KoÃ°p

xY`V»t[$

0Qÿ[]sV_ªVx~|ewYst[XªYkZ['=Az`[X '/C FVZy(ÿcYtÛwYycYkZ['= 6Ypt #FVX[tÿg6Zªgjm`[wÿ[X 6Y~wÿ.0QÿcX ECKVZy

\]xkZ '/C d(ÿYsÿKsgjZ`tÛwY~nª[»t#wÿ[]gc|VZcYÃgjZ(ÿgjxÃ[0QX%'/C .sgZÄtwwY~nª[»tV vxg`gjX ]YZwÿ[]VswY~}`[tªY~ª[

sV_}Y~|áVZy(YptRtªÿg(Z^`gcwÿ(YkZÀtwwYs

ù

tVZy(YkZÀt`Vs[vYkxkxiágÄy[x 19

qrYkmÿ»#ÿ»n`ZycX[yÀtVix[yKVZy

tÛn`[XYki`g:t[yÀtwwXÛnos_n`X[»t gjv*`üÛiK[wÿ»|xü`ü`[Z»ªVZgjxYkZwÿ[]Vs_wYï}`[tYïª[]gjv

¥¦`KoÃe

xkY`V»t[

$ 0Qÿ[]VxsgÿgxYpt!wYkmÿewx~|^jgn`Zy(YkZwÿ[]Vs_wY~}`[tªY~ª[]VZyáX[iKVYkZ`t!wÿ[X[]y»n`XYkZmáwÿ[(ÿgjxÃ[tYkin`xÃV_wYgZ)

,

qrYkmÿ»#ÿen`ZycX[yÀtVix[yÀtwwXÛnos_n`X[»t gjv*`ü`[ZeªVZgZ[40Qÿ[

ù

[_ªgZ[áV»t!wYmÿ»wx~|^`gcn`Zy(YkZwÿ[

Vs_wYï}`[tYïª[]Vx~wÿgcn`mÿKgcwÿ[XsgjZvwgjXiáV_wYgZÄt!wÿVZwÿ[]gc|VZYgZ(ÿgx[ÿ»|ycXgm`[Z^jgjZy[yKgjZ[»t á[X[

Vxptg vwgcn`Zy)40 ÿ[

ù

[_ªgZ[X[iáVYkZ[yáYZwÿ[]VswY~}`[tªY~ª[]y»n`XYkZmáwÿ[(ÿgjxÃ[tYkin`xÃV_wYgZ)÷

qrYkmÿe

ÿ»n`ZycX[yÀtVix[yÀtÛwXwnos_n`X[»tgv*`üÛiK[_wÿe|x`[Z»ªVZ[40 ÿ[X[á[X[Zg.V_wXVswYgjZ^`[_&á[[Zwÿ[]Vxù VZ[

VZy6wÿ[]Vs_wY~}j[tY~ª[dÄVZyKVx~wÿgcn`mÿwÿ[ `üÛiK[_wÿe|cx`[Z»ªVZ[KV»t`gutªY~wYgZ[y(YkZwÿ[4^YkZycYkZcm+tªY~ª[vXgi

wÿ[twªVXÛ gvuwÿ[tYkin`xV_wYgZdY~tÛªVXwª[yK[xgXYkZm(wÿ[tÛn`XªXgn`ZycYkZcm.VZy(vwgn`ZyáYït KV|Kgcn{wÿgcn`mÿ

wÿ[]Vs_wY~}`[tªY~ª[][ZewXVZs[

(19)

OH O

O O

M eOH O

+ C A LB +

M Pr M P PPr

K _I P E P

E MPr E-Pr M E-Pr E-Pr P E PPr

E E

MPr methyl propanoate, M methanol, P propanol, PPr propyl propanoate, E enzyme,

‘ • ’denotes noncovalent bond, ‘-‘ denotes covalent bond.

(20)

0 20 40 60 80 100 120

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Thermodynamic activity of added organic component

e n z y m a ti c a c ti v it y ( % )

2-pentanone 3-pentanone

2-methylpentane 3-methylpentane

2-methyl-2-pentanol 3-methyl-3-pentanol

2-methyl-2-butanol (results from [25])

(21)

0 0.02 0.04 0.06 0.08 0.1

0 0.05 0.1 0.15 0.2 0.25 0.3

a _MP v i (µ m o l. m in -1 .m g -1 )

aP=0,02 aP=0,03 aP=0,06 aP=0,1 aP=0,15

(22)

-20 -10 0 10 20 30 40 50 60 70 80

-15 -10 -5 0 5 10 15 20

1/a _MP 1 /v i (µ m o l -1 .m in .m g )

aP=0,02 aP=0,03 aP=0,06 aP=0,1 aP=0,15

-1 0 1 2 3 4 5

-0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25

a _P s lo p e 1 /a M P

-20 -10 0 10 20 30 40 50 60 70 80

-15 -10 -5 0 5 10 15 20

1/a _MP 1 /v i (µ m o l -1 .m in .m g )

aP=0,02 aP=0,03 aP=0,06 aP=0,1 aP=0,15

(23)

Solvent as a competitive inhibitor for Candida antarctica lipase B

HAL Id: hal-00329734

https://hal.archives-ouvertes.fr/hal-00329734

Submitted on 13 Oct 2008

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Solvent as a competitive inhibitor for Candida antarctica lipase B

Marianne Graber, Romain Irague, Eric Rosenfeld, Sylvain Lamare, Linda Franson, Karl Hult

To cite this version:

Marianne Graber, Romain Irague, Eric Rosenfeld, Sylvain Lamare, Linda Franson, et al.. Solvent as

a competitive inhibitor for Candida antarctica lipase B. Biochimica et Biophysica Acta Proteins and

Proteomics, Elsevier, 2007, 1774, pp.1052-1057. �hal-00329734�

OH O

O O

M eOH O

+ C A LB +

M Pr M P PPr

K I P E P

E MPr E-Pr M E-Pr E-Pr P E PPr

E E

MPr methyl propanoate, M methanol, P propanol, PPr propyl propanoate, E enzyme,

‘ • ’denotes noncovalent bond, ‘-‘ denotes covalent bond.

0 20 40 60 80 100 120

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Thermodynamic activity of added organic component

e n z y m a ti c a c ti v it y ( % )

2-pentanone 3-pentanone

2-methylpentane 3-methylpentane

2-methyl-2-pentanol 3-methyl-3-pentanol

2-methyl-2-butanol (results from [25])

0 0.02 0.04 0.06 0.08 0.1

0 0.05 0.1 0.15 0.2 0.25 0.3

a MP v i (µ m o l. m in -1 .m g -1 )

aP=0,02 aP=0,03 aP=0,06 aP=0,1 aP=0,15

-20 -10 0 10 20 30 40 50 60 70 80

-15 -10 -5 0 5 10 15 20

1/a MP 1 /v i (µ m o l -1 .m in .m g )

aP=0,02 aP=0,03 aP=0,06 aP=0,1 aP=0,15

-1 0 1 2 3 4 5

-0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25

a P s lo p e 1 /a M P

-20 -10 0 10 20 30 40 50 60 70 80

-15 -10 -5 0 5 10 15 20

1/a MP 1 /v i (µ m o l -1 .m in .m g )

aP=0,02 aP=0,03 aP=0,06 aP=0,1 aP=0,15

K _I P E P

a _MP v i (µ m o l. m in -1 .m g -1 )

1/a _MP 1 /v i (µ m o l -1 .m in .m g )

a _P s lo p e 1 /a M P

1/a _MP 1 /v i (µ m o l -1 .m in .m g )