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Cloning and expression of phenylalanine ammonia-lyase gene of Rhodosporidium toruloides in Escherichia coli

红冬孢酵母苯丙氨酸解氨酶基因pal在大肠杆菌中的克隆与表达



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ChineseJournalofBioprocessEngineering
Vol.10No.5
Sep.2012
doi:10.3969/j.issn.1672-3678.2012.05.012
NOPH
:2012-04-15
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Ã
E.coli
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:1672-3678(2012)05-0061-06
Cloningandexpressionofphenylalanineammonialyasegeneof
RhodosporidiumtoruloidesinEscherichiacoli
HOUWenting,ZHANGLiang,GUZhenghua,DINGChongyang,SHIGuiyang
(NationalEngineeringLaboratoryforCerealFermentationTechnology,KeyLaboratoryofIndustrial
BiotechnologyoftheMinistryofEducation,JiangnanUniversity,Wuxi214122,China)
Abstract:ThecDNAfragmentofpalgenewasamplifiedfromRhodosporidiumtoruloidesbyRTPCR.Nu
cleotidesequencingidentifiedthegeneof99% similaritywithreportedpalgene.UsingpET28a(+)with
T7promoterasavector,therecombinantplasmidpET28a(+)palwasconstructedandthenexpressedin
E.coliBL21(DE3).Afteroptimization,thespecificactivityofPALreached4299U/g.Theconversion
rateoftranscinnamicacidwas4852% andtheconcentrationofLphenylalaninewas173g/LItwas
shownthatthegenepalfromR.toruloideswashighlyexpressed.
Keywords:phenylalanineammonialyase(PAL);reversetranscription;recombinantexpression
  
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22 

pal
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PCR。
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2200bp
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ŸÏ„
pal
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ž€‘

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yÚÞ
2。
,­‡ˆp
1—pMD18Tpal/EcoRI;M—λDNA/PstImarker
f
1 
¾¿À]­Ä
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Fig.1 AgargelelectrophoresispaternsofRNA
extractedfromR.toruloides
¡
pMD18 T pal,
¤
EcoRI
d&F€

žýN
2200bp
20=

yÚÞ
3。
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:RT
PCR
6m2?X¿„
GenBank
cm¬2™š›

cDNA
¿€5µè
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ùúN2”“€
5µè
99%。
1—palgene;M—λDNA/PstImarker
f
2 
¾¿À]­
pal

RT PCR
ëV;fl
Fig.2 ElectrophoresisanalysisofRTPCRproductof
palgenefromR.toruloides
1—pMD18 T pal/EcoRI;M—λDNA/PstImarker
f
3 
«´¼·
pMD18 T pal
Ζ9Ùfl
Fig.3 Electrophoresisanalysisofenzymatic
resultofpMD18Tpal
36 
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4—pET 28a(+) pal/EcoRI+HindII;M—λDNA/PstImarker
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4 
«´¼·
pET 28a(+) pal^
Ζ§¾
Fig.4 Enzymeanalysisofrecombinant
plasmidpET28a(+)pal
24 PAL
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EcoliBL21(DE3)
e^œ”
  
J
E.coliBL21(DE3)
çè-Û

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(DE3) PAL
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

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)ò
³

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2—pET 28a(+) pal/EcoliBL21(DE3);
M—Marker
f
5 SDSPAGE
ØÙ«´t
PAL
œ”
Fig.5 SDSPAGEanalysisofPALexpressionlevels
  
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P
85×104
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OD600ý08` a´lIPTG;s
D´µè
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activityofPAL
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IPTG
D´µè
005、01、03、05、07、09
P
11mmol/L,25℃
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6h
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7 IPTG
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Fig.7 EfectofIPTGconcentrationon
specificactivityofPAL
  
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µµ)2
IPTG
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Fig.8 Efectoftemperatureonspecific
activityofPAL
254 IPTG
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P
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¤

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f
9 IPTG
ŒŽ#Z«´tšÎÖ^_`
Fig.9 EfcetofIPTGaddingtimeon
specificactivityofPAL
  
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IžY¶S2ÅÓg;íWi
j
。1992
$
,Orum
y
[14]
²3â2
pal
Ižðl…
+,
pKK223 3

EcoliSG1611
c…

©
ª;f5
PAL
_`2”“p

é_åîè
01~02
U/g。
+ÅÓIg¯
,1994
$
Faulkner
y
[15]
þ¤

ōüý1˜PŸ ¡Sc%0…+,

J
pPGK:REP2
èzº*
E.coliTG1
c…
pal
I
ž

°_åè
20U/g。2008
$
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y
[8]
þ¤2J
tac
P
PLPR_Ùzº*2p¡ EcoliJM109cd
…;žF_,m)
35U/g,
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RNA
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T7
Üzº*2
pET28a
…
+,

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4299U/g。
26 
«´t„븡¦§
  
ÖÛGžE¼–µž2.[\dž³¼
–

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IPTG
D´µè
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25℃,
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6h
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R5©Tº
ž2

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173g/L,
ØðQ‡ˆ
Sã;©ªœWX2éå

f
10 
ÇàÈÉ÷^¸¡a
Fig.10 Conversionrateoftranscinnamicacid
3 
x
 
)
  
@µRGÔµž2
pal
Iž„
NCBI¯
m¬2
™š›1˜
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99%,
56 
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pal
Ÿ ¡Sc2–¢„…
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pal
Ižñß/±m2F_

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…Ú¡WPªð…™š›1˜
pal,^
HQ…Ú¡þ¤uG2\ç

.GH[
\ᱪz

ž³[\thGžE•–Œ89
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