APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1995, p. 2654–2658 Vol. 61, No. 7
0099-2240/95/$04.0010
Copy igh q1995, Ame ican Socie y o Mic obiology
Con e sion o Chlo obiphenyls in o Phenylhexadienoa es and
Benzoa es by he Enzymes o he Uppe Pa hway o
Polychlo obiphenyl Deg ada ion Encoded by he
bph Locus o Pseudomonas sp. S ain LB400
MICHAEL SEEGER,
1,2
KENNETH N. TIMMIS,
1
AND BERND HOFER
1
*
Depa men o Mic obiology, Gesellscha u¨ Bio echnologische Fo schung-Na ional Resea ch
Cen e o Bio echnology, D-38124 B aunschweig, Ge many,
1
and Depa amen o de
Bioquı´mica, Facul ad de Medicina, Uni e sidad de Chile, San iago, Chile
2
Recei ed 30 Janua y 1995/Accep ed 1 May 1995
Me abolism o 21 chlo obiphenyls by he enzymes o he uppe biphenyl ca abolic pa hway encoded by he
bph locus o Pseudomonas sp. s ain LB400 was in es iga ed by using ecombinan s ains ha bo ing gene
casse es con aining bphABC o bphABCD. The enzymes o he uppe pa hway we e gene ally able o me abolize
mono- and dichlo ina ed biphenyls bu only pa ially ans o m mos ichlo ina ed congene s in es iga ed: 14
o 15 mono- and dichlo ina ed and 2 o 6 ichlo ina ed congene s we e con e ed in o benzoa es. All mono-
and a leas 8 o 12 dichlo ina ed congene s we e a acked by he bphA-encoded biphenyl dioxygenase i ually
exclusi ely a o ho and me a ca bons. This enzyme exhibi ed a high deg ee o selec i i y o he a oma ic ing
o be a acked, wi h he o de o ing p e e ence being non- > o ho->me a->pa a-subs i u ed o mono- and
dichlo ina ed congene s. The in luence o he chlo ine subs i u ion pa e n o he me abolized ing on benzoa e
o ma ion esembled i s in luence on he eac i i y o ini ial dioxygena ion, sugges ing ha he a e o benzoa e
o ma ion may equen ly be de e mined by he a e o ini ial a ack. The abso p ion spec a o phenylhexa-
dienoa es o med co ela ed wi h he p esence o absence o a chlo ine subs i uen a an o ho posi ion.
Con amina ion o he biosphe e wi h syn he ic o ganic com-
pounds ha a e pe sis en and oxic is o inc easing conce n (1,
3, 24). One such ype o chemical ha has been ex ensi ely
used in pas decades is polychlo ina ed biphenyl (PCB). The e
exis a o al o 209 dis inc PCB congene s which di e in
numbe and posi ion o he chlo ines. A ypical comme cial
PCB mix u e, such as A ochlo , Clophen, o Kanechlo , con-
ains be ween 60 and 80 di e en congene s (7).
A p omising app oach o PCB decon amina ion o soils and
sedimen s is bio emedia ion in ol ing he combined ac ion o
anae obic and ae obic mic oo ganisms. Anae obic conso ia
ha a e able o pa ially dehalogena e highly chlo ina ed con-
gene s ha e been desc ibed (1, 19), whe eas a numbe o ae -
obic bac e ia ha a e capable o oxida i ely a acking he ing
sys em o mode a ely chlo ina ed PCBs ha e been isola ed (2,
6, 12). Al hough he spec a o PCBs ha can be ans o med
by a numbe o ae obes ha e been cha ac e ized (4–6, 11, 17),
li le is known abou he inal me aboli es o med om indi-
idual congene s. Mo eo e , in mos cases obse ed ans o -
ma ions could no unequi ocally be asc ibed o speci ic en-
zymes, because he syn hesis o isoenzymes by he o iginal
isola es canno be excluded.
Gene cloning is a con enien app oach o deal wi h such
ques ions. Since genes encoding enzymes o any pa o a
pa hway can be in oduced in o a ecipien de oid o ca aly ic
ac i i ies owa ds he subs a es and incapable o u he ans-
o ma ion o he p oduc s, cha ac e iza ion o indi idual enzy-
ma ic ac i i ies and iden i ica ion o me aboli es p oduced by
hem a e conside ably acili a ed.
DNA segmen s in ol ed in PCB deg ada ion ha e been iso-
la ed om a numbe o bac e ia including Pseudomonas sp.
s ain LB400 (9, 17), a mic oo ganism which is pa icula ly
no ewo hy o i s abili y o oxidize a b oad spec um o con-
gene s (4–6, 17). To da e, 10 cis ons o he LB400 bph locus
ha e been cha ac e ized and shown o be in ol ed in he
b eakdown o biphenyls (10, 15, 16). Th ough his wo k, LB400
became he i s s ain o which comple e sequence in o ma-
ion a he DNA as well as a he enzyme le el was a ailable o
he biphenyl uppe pa hway, i.e., he ca abolism o biphenyls o
benzoa es and 2-hyd oxypen a-2,4-dienoa es which is ca alyzed
by ou enzymes, biphenyl-2,3 dioxygenase (BphA), 2,3-dihy-
d o-2,3-dihyd oxybiphenyl-2,3 dehyd ogenase (BphB), 2,3-di-
hyd oxybiphenyl-1,2 dioxygenase (BphC), and 2-hyd oxy-6-
oxo-6-phenylhexa-2,4-dienoa e hyd olase (BphD) (Fig. 1).
We p e iously iden i ied me aboli es p oduced by he ac ion
o BphA and/o BphB and obse ed dioxygenase-media ed
dechlo ina ion o o ho-subs i u ed congene s (21). He e we
in es iga e which o a b oad ange o chlo ina ed biphenyls
(CBs) a e deg aded o 2-hyd oxy-6-oxo-6-phenylhexa-2,4-di-
enoa es (HOPDAs o phenylhexadienoa es) o chlo obenzo-
a es (CBAs).
MATERIALS AND METHODS
Chemicals. PCB congene s (99% pu i y) we e ob ained om Lancas e Syn-
hesis, Whi e Lund, Mo ecambe, England; P omochem, Wesel, Ge many; o
Res ek, Sulzbach, Ge many. CBAs (98% pu i y) we e pu chased om Fluka
AG, Buchs, Swi ze land, o Lancas e Syn hesis.
Bac e ial s ains, plasmids, and cul u e condi ions. The Esche ichia coli
s ains used in his s udy we e DH1 (14), DH5a(13), and BL21(DE3)[pLysS]
(23). The ecombinan plasmids pAIA50 and pAIA74 a e desc ibed in Fig. 2.
Bac e ia we e g own in Lu ia-Be ani medium (20) a 378C. Whe e app op ia e,
chlo amphenicol and/o ampicillin was used o selec ion a concen a ions o 20
and 50 mg/ml, espec i ely.
Recombinan DNA echniques. Plasmids we e cons uc ed by s anda d ecom-
binan DNA echniques simila o published p o ocols (20). T ans o ma ions o
* Co esponding au ho . Mailing add ess: Depa men o Mic obi-
ology, Gesellscha u¨ Bio echnologische Fo schung, Masche ode
Weg 1, D-38124 B aunschweig, Ge many. Phone: (49-531) 6181467.
Fax: (49-531) 6181411.
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E. coli s ains we e ca ied ou ei he by he Hanahan me hod (14) o by
elec opo a ion (8).
Res ing cell assays. E. coli BL21(DE3)[pLysS] ha bo ing ei he pAIA50 o
pAIA74 was g own o an op ical densi y o 0.6 a 600 nm. A e u he incuba-
ion in he p esence o 0.4 mM isop opyl-b-D-galac opy anoside o 30 min a
308C, cells we e ha es ed, washed wi h 50 mM sodium phospha e bu e (pH
7.5), and esuspended in 1/10 olume o he same bu e . Cells we e incuba ed
wi h single PCB congene s (nominal concen a ion, 62 o 125 mM) o 20 o 24
ha 308C on a gy a o y shake .
Moni o ing o HOPDA and CBA o ma ion. Fo ma ion o HOPDAs was
moni o ed by isible spec al scanning o es ing cell assay mix u es wi h a
Beckman DU-70 spec opho ome e . Accumula ion o CBAs was ollowed by
high-pe o mance liquid ch oma og aphy (HPLC). Cell- ee supe na an s o
es ing cell incuba ions (20 ml) we e analyzed on a Beckman liquid ch oma o-
g aph equipped wi h a diode a ay de ec o wi h a SC125/Lich osphe 5-mm
column (Bischo , Leonbe g, Ge many) and an aqueous sol en sys em con ain-
ing 1 ml o 85% o ho-phospho ic acid and 640 ml o me hanol pe li e . CBAs
we e iden i ied and quan i a ed by compa ison wi h au hen ic s anda ds.
RESULTS
Cons uc ion o ecombinan s ains and desc ip ion o he
assay sys em. Recombinan exp ession ec o s ha bo ing bph
genes downs eam o a phage T7 la e p omo e we e desc ibed
p e iously (16). Plasmid pAIA50 con aining bphABC was gen-
e a ed om one o he ea lie cons uc s, pAIA5, as shown in
Fig. 2A. The cons uc ion o a bphABCD-con aining exp es-
sion ec o comp ised se e al s eps. Fi s ly, bphC and bphD
we e sepa a ely ampli ied by PCR o in oduce es ic ion si es
a hei ends and subsequen ly linked in such a way ha 34 bp
FIG. 1. Uppe pa hway o me abolism o biphenyls as encoded by hebph locus
o Pseudomonas sp. s ain LB400. Me aboli es: 1, biphenyl; 2, 2,3-dihyd o-2,3-dihy-
d oxybiphenyl; 3, 2,3-dihyd oxybiphenyl; 4, 2-hyd oxy-6-oxo-6-phenylhexa-2,4-di-
enoic acid; 5a, benzoic acid; 5b, 2-hyd oxypen a-2,4-dienoic acid. Enzymes: BphA,
biphenyl-2,3 dioxygenase; BphB, 2,3-dihyd o-2,3-dihyd oxybiphenyl-2,3 dehyd oge-
nase; BphC, 2,3-dihyd oxybiphenyl-1,2 dioxygenase; BphD, 2-hyd oxy-6-oxo-6-phe-
nylhexa-2,4-dienoa e hyd olase.
FIG. 2. Cons uc ion o ecombinan plasmids. Di ec ions o sense ansc ip ion o genes a e indica ed by a ows. P-T7, phage T7 gene 10 p omo e ini ia ing
ansc ip ion in he di ec ion indica ed by he a owhead. o i, o igin o eplica ion. bla, gene encoding b-lac amase which media es ampicillin esis ance. (A)
Cons uc ion o pAIA50. A SacI-XhoI agmen was dele ed om pAIA5 o inac i a e bphK. (B) Cons uc ion o pAIA74. Fo de ails, see ex . (C) Sequence o he
a i icial bphCD junc ion. The s op codon o bphC and he s a codon o bphD a e unde lined. The Shine-Dalga no sequence o bphD is o e lined. The newly
in oduced SpeI si e is shown in bold ace wi h he single base-pai exchange indica ed by an as e isk.
VOL. 61, 1995 CONVERSION OF CHLOROBIPHENYLS INTO BENZOATES 2655
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o he bphD leade sequence (wi h a single exchange o c ea e
aSpeI si e) was a ached o he s op codon o bphC (Fig. 2C).
The bphD s a codon was he eby placed in almos he same
posi ion no mally occupied by he bphK s a codon (15). The
esul ing cons uc , pAIA3, is shown in Fig. 2B. An adap e
con aining se e al es ic ion si es was inse ed in o he
HindIII si e o pAIA5 (Fig. 2A) o yield pAIA71 (Fig. 2B).
pAIA3 and pAIA71 we e used o cons uc pAIA74 con aining
bphABCD as shown in Fig. 2B.
pAIA50 o pAIA74 was in oduced in o he E. coli hos
s ain BL21(DE3)[pLysS], which pe mi s induc ion o an-
sc ip ion o cloned genes om phage T7 la e p omo e s (23).
A e induc ion, es ing cells we e incuba ed wi h single PCB
congene s. Assay mix u es we e analyzed di ec ly, ei he by
isible spec oscopy o by HPLC and UV spec oscopy.
Con e sion o chlo ina ed biphenyls in o phenylhexadieno-
a es. Con e sion o all mono- and dichlo ina ed and six i-
chlo ina ed biphenyls in o hei HOPDAs by E. coli cells ex-
p essing bphABC was assessed spec opho ome ically. All o
he mono-, 11 o 12 di-, and 5 o 6 ichlo ina ed congene s
we e me abolized o HOPDAs. No HOPDAs we e de ec ed
o he double o ho-subs i u ed 2,6-diCB o o 2,5,39- iCB
(Table 1). While oxida ion o 2,5,39- iCB by biphenyl dioxy-
genase has been e i ied by gas ch oma og aphy-mass spec-
ome y (22), i is no clea a p esen whe he 2,6-diCB is
a acked by BphA, al hough oxida ion by s ain LB400 has
been desc ibed p e iously (18).
The 19 congene s ha we e ans o med o HOPDAs may
be di ided in o h ee classes, acco ding o he a e o con e -
sion. The kine ics o con e sion o one ep esen a i e o each
class is shown in Fig. 3. Class 3 congene s, which a e subs i-
u ed in bo h ings a he me a o pa a posi ion (3,39-, 3,49-, and
4,49-diCB), we e con e ed slowly. Rema kably, hei mo e
highly chlo ina ed analogs con aining an addi ional chlo ine a
an o ho posi ion (2,3,39-, 2,5,49-, and 2,4,49- iCB) we e con-
e ed a mode a e a es (class 2 congene s). The esidual
congene s (class 1) we e quickly and p obably quan i a i ely
con e ed in o HOPDAs. Al hough mola ex inc ion coe i-
cien s o he HOPDAs a e no known, comple eness o con-
e sion can be in e ed in all bu wo cases om quan i a i e
p oduc ion o he co esponding benzoa es (see Table 2, be-
low).
Abso p ion maxima o he HOPDAs o med ell in o wo
dis inc wa eleng h egions lying ei he be ween 430 and 440
nm (as o he unsubs i u ed HOPDA) o be ween 390 and 400
nm (Table 1). We ound a s ic co ela ion be ween hese
alues and he p esence (low l
max
) o absence (high l
max
)o a
chlo ine subs i uen a an o ho posi ion in he HOPDA, as
deduced om iden i ica ion o he co esponding CBAs (Table
2). We assume ha he o ho chlo ine p e en s a coplana
con o ma ion o he pelec on sys ems o he a oma ic ing
and he alipha ic side chain, he eby leading o a hypsoch omic
shi o l
max
.
While l
max
alues gene ally emained s able, signi ican
shi s owa ds sho e wa eleng hs we e obse ed du ing he
incuba ions o 3,39-diCB and 2,3,39- iCB, sugges ing a u he
eac ion o he ini ially o med compounds.
Con e sion o CBs in o chlo ina ed benzoa es. Con e sion
o CBs in o CBAs by bphABCD-ha bo ing E. coli cells was
ou inely analyzed a e o e nigh incuba ions (20 o 24 h).
Kine ics wi h some class 1 (Table 1) mono- and dichlo ina ed
congene s had indica ed ha o ma ion o benzoa es eached
comple ion wi hin his ime (da a no shown). CBAs we e
iden i ied by HPLC and UV spec um by compa ison wi h
au hen ic s anda ds. The esul s a e summa ized in Table 2.
Wi h he excep ion o 4,49-diCB, all o he mono- and di-
chlo ina ed congene s ha we e deg aded o HOPDAs we e
also ca abolized o benzoa es. Howe e , o he i e ichlo i-
na ed CBs con e ed in o HOPDAs, only wo we e me abo-
lized in o CBAs. Thus, 15 o 21 congene s es ed we e de-
g aded o CBAs. In 11 cases, CBAs we e ob ained in i ually
quan i a i e amoun s.
Iden i ica ion o he CBAs p oduced pe mi ed us o ace
back which o he biphenyl ings was a acked by he ini ial
dioxygenase. All mono- and dichlo ina ed CBs which we e
con e ed in o CBAs and we e subs i u ed in only one ing
we e ound o be oxidized i ually exclusi ely in he nonhalo-
FIG. 3. Time cou se o con e sion o CBs in o HOPDAs by es ing E. coli
cells ha bo ing bphABC. Abso p ions we e measu ed a he maxima o he
indi idual HOPDAs (Table 1). HOPDAs we e o med by deg ada ion o 4-CB
(ci cles), 3,39-diCB (squa es), and 2,3,39- iCB ( iangles).
TABLE 1. Fo ma ion o HOPDAs om a ious CBs by
E. coli cells con aining bphABC
CB
HOPDA
Abso p ion
a
(l
max
[nm]) Ra e o
o ma ion
b
2-
c
393 621
3-
c
436 621
4-
c
437 621
2,3-
d
392 621
2,4-
d
392 621
2,5-
d
392 621
2,6-
d
ND
3,4-
d
440 621
3,5-
c
439 621
2,29-
c
392 621
2,39-
d
435 621
2,49-
d
438 621
3,39-
d
430 65
e
3
3,49-
d
432 653
4,49-
d
430 610 3
2,3,5-
d
393 621
2,3,39-
d
400 65
2
2,4,49-
d
437 622
2,5,29-
d
392 621
2,5,39-
d
ND
2,5,49-
d
394 622
a
ND, no de ec ed.
b
See ex o de ails.
c
20-h incuba ion; 62 mM CB.
d
24-h incuba ion; 125 mM CB.
e
Measu ed a e 2 h; shi ed o 410 65 nm du ing he incuba ion.
Measu ed a e 1 h; shi ed o 370 65 nm du ing he incuba ion.
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gena ed ing. In hose cases o dichlo ina ed congene s pos-
sessing an o ho-monochlo ina ed ing, his ing was he sole
a ge o oxida ion. Also wi h 3,49-, 2,3,5-, o 2,5,29-subs i u ed
congene s, only one ype o CBA was ound (Table 2), indi-
ca ing a ack o he me a-, non-, o o ho-chlo ina ed ing.
Howe e , as u no e in o CBAs was incomple e in hese
cases, addi ional oxida ion o he o he ing canno be uled
ou .
Fo h ee ichlo ina ed congene s, no CBAs we e de ec ed,
bu HOPDAs we e o med. Fo hese CBs, he obse ed
co ela ion be ween l
max
and o ho chlo ine subs i u ion o
HOPDAs pe mi ed p edic ion as o which ing was oxidized.
The l
max
alues obse ed sugges ha 2,3,39- and 2,5,49- iCB
we e oxidized in he monochlo ina ed ings yielding o ho-
subs i u ed HOPDAs, whe eas 2,4,49- iCB was oxidized in he
dichlo ina ed ing yielding a pa a-chlo ina ed HOPDA. These
conclusions a e consis en wi h esul s ob ained o he oxida-
ion o 2,3,39- and 2,4,49- iCB by s ain LB400 i sel (4). Again,
howe e , addi ional oxida ion o he o he ing no leading o
HOPDA o ma ion canno be uled ou .
DISCUSSION
The esul s ob ained indica e he loca ion o he ini ial oxi-
da i e a ack and a co ela ion be ween chlo ine subs i u ion
pa e n and deg adabili y o CB congene s speci ied by he bph
gene clus e o Pseudomonas sp. s ain LB400. The bphA-
encoded biphenyl dioxygenase o s ain LB400 is able o oxi-
dize no only he o ho and me a ca bons bu also he me a and
pa a ca bons (18, 22). Gi en he enzyma ic epe oi e encoded
by he cloned bph genes, he ecombinan s ain can p oduce
benzoa e only i he ini ial oxida ion is di ec ed owa ds he
o ho and me a posi ions. Thus, he pe cen age o benzoa e
o ma ion gi es a lowe limi alue o o ho-me a oxida ion by
biphenyl dioxygenase. Ou esul s demons a e ha all mono-
and a leas 8 o 12 dichlo ina ed congene s a e a acked i -
ually exclusi ely in he o ho-me a mode. 2,3,5- and 2,5,29-
iCB a e a leas pa ly oxidized a hese posi ions. In e es -
ingly, a ack a o ho and me a posi ions is no necessa ily
di ec ed owa ds unchlo ina ed ca bons. Wi h 2,29- and 2,49-
diCB, i ually quan i a i e dioxygena ion a posi ions 2 and 3,
leading o elimina ion o hyd ochlo ic acid, was obse ed (21).
I CBAs a e ob ained om an asymme ic congene in quan-
i a i e yield, hei pe cen age o o ma ion e lec s he a io o
oxida ion o he wo ings by biphenyl dioxygenase. The i u-
ally quan i a i e o ma ion o only one CBA om asymme i-
cal congene s demons a es a high deg ee o p e e ence o he
enzyme o one o he wo a oma ic ings. The ypes o CBAs
p oduced indica e ha he eac i i y o he biphenyl ings o-
wa ds BphA-ca alyzed oxida ion dec eases acco ding o he
ollowing o de : non- .o ho-.me a-subs i u ed. Fu he -
mo e, he exclusi e o ma ion o 4-CBA om 3,49-diCB sug-
ges s ha a me a-chlo ina ed ing is mo e easily a acked han
apa a-chlo ina ed one. This is consis en wi h he obse a ion
o poo e HOPDA o ma ion om 4,49-diCB han om 3,39-
diCB.
Fo mono- and dichlo ina ed congene s, he pe cen ages o
benzoa e o ma ion obse ed co ela e wi h he chlo ine sub-
s i u ion pa e n o he me abolized ing. Congene s oxidized
in he non- o o ho-chlo ina ed ing yielded benzoa es in i -
ually quan i a i e amoun s. CBs a acked in he me a-chlo i-
na ed ing we e incomple ely con e ed in o CBAs. I he
pa a-chlo ina ed ing was dioxygena ed, no o ma ion o ben-
zoa e was obse ed. This sugges s ha he chlo ine subs i u ion
pa e n o he me abolized ing dec eases he e iciency o
deg ada ion h ough he en i e uppe pa hway in he o de
non- 'o ho-.me a-.pa a-subs i u ed. The ichlo ina ed
congene s assayed yielded simila esul s. Only ichlo ina ed
CBs con aining a non- o mono-o ho-chlo ina ed ing we e
con e ed in o benzoa es. The in luence o he chlo ine sub-
s i u ion pa e n o he a acked ing on benzoa e o ma ion
esembles i s in luence on eac i i y owa ds BphA-ca alyzed
oxida ion (compa e abo e). This sugges s ha he a e o CBA
o ma ion may equen ly be de e mined by he a e o ini ial
a ack.
Da a on CB me aboli es p oduced by he pa en al s ain,
Pseudomonas sp. s ain LB400, a e a he limi ed. Compa ison
o such da a (4) wi h he esul s ob ained wi h he bphABCD-
con aining E. coli s ain indica es ha hey a e simila . In bo h
cases, 3- o 4-CBA, bu no o only ace amoun s o 2-CBA,
was de ec ed as me aboli es o 2,39-o 2,49-diCB. The mono-
chlo ina ed ing o 2,3,39-, bu he dichlo ina ed ing o 2,4,49-
iCB, was ound o be (p edominan ly) a acked. Howe e ,
Beda d and Habe l (4) epo ed o ma ion o 2,3-diCBA (no
quan i a ion gi en) o 4-CBA (13%), espec i ely, om hese
wo congene s, whe eas we de ec ed only he HOPDAs. This
migh be due o an addi ional HOPDA hyd olase p oduced by
s ain LB400, bu o he explana ions such as a highe hyd o-
lase- o-subs a e a io in he expe imen s o hese au ho s can-
no be uled ou . Nei he ecombinan E. coli no s ain LB400
was able o con e 2,5,39- iCB in o an HOPDA. The simila -
i y be ween he da a ob ained wi h he cloned bph genes and
hose wi h he pa en al s ain is consis en wi h he iew ha
he isola ed bph genes speci y he ca abolic capabili y o he
s ain o he con e sion o chlo obiphenyls in o CBAs.
ACKNOWLEDGMENTS
We hank Die ma Piepe o a c i ical eading o he manusc ip .
M.S. was suppo ed by a collabo a i e doc o al ellowship om he
Deu sche Akademische Aus auschdiens (DAAD, Bonn, Ge many)
and by a doc o al ellowship om he Consejo Nacional de Ciencia y
TABLE 2. Fo ma ion o CBAs om a ious CBs by
E. coli cells con aining bphABCD
CB (posi ion[s] o
chlo ine[s])
CBA
Posi ion(s) o
chlo ine(s) Yield (%)
a
2
b
2 95–100
3
c
3 95–100
4
b
4 95–100
2,3
c
2,3 95–100
2,4
c
2,4 95–100
2,5
c
2,5 95–100
2,6
c
ND
3,4
c
3,4 95–100
3,5
b
3,5 95–100
2,29
b
2 95–100
2,39
c
3 95–100
2,49
c
4 95–100
3,39
c
3 20–25
3,49
c
4 25–30
4,49
c
ND
2,3,5
c
2,3,5 40–45
2,3,39
c
ND
2,4,49
c
ND
2,5,29
c
2,5 35–40
2,5,39
c
ND
2,5,49
c
ND
a
ND, no de ec ed.
b
20-h incuba ion, 62 mM CB.
c
24-h incuba ion, 125 mM CB.
VOL. 61, 1995 CONVERSION OF CHLOROBIPHENYLS INTO BENZOATES 2657
on Feb ua y 7, 2017 by GESELLSCHAFT FUR BIOTECHNO-h p://aem.asm.o g/Downloaded om
Tecnologı´a (CONICYT, San iago, Chile). This wo k was suppo ed in
pa by g an s om he Fonds de Chemischen Indus ie.
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