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Strain-screening in biotransformation of d-pseudoephedrine and expression of its bcdh gene

生物转化产d-伪麻黄碱菌株的筛选及其关键酶基因的验证



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ChineseJournalofBioprocessEngineering
Vol.11No.3
May2013
doi:10.3969/j.issn.1672-3678.2013.03.007
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BacilusclausiB0658,
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y
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1283mg/L。
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B.clausiB0658
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bcdh
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‰
pET28a(+)
«†ÛÈ
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:1672-3678(2013)03-0034-06
Strainscreeninginbiotransformationofdpseudoephedrine
andexpressionofitsbcdhgene
CHAIMankun,ZHANGLiang,GUZhenghua,DINGZhongyang,SHIGuiyang
(NationalEngineeringLaboratoryforCerealFermentationTechnology,
KeyLaboratoryofIndustrialBiotechnologyoftheMinistryofEducation,JiangnanUniversity,Wuxi214122,China)
Abstract:Basedontheresultsofblastinggeneandaminoacidsequencesofcarbonylreductasefrom
MorganelamorganiJ8inGenBankandUniprotdatabase,5strainsfortransforming1phenyl2
methylamineacetone(MAK)todpseudoephedrinewerescreenedoutfromtheCICIMCUbyHPLC
detection.ThetransformationprocessfordpseudoephedrineproductionfromBacilusclausiB0658was
investigatedUndertheoptimaltransformationcondition,theyieldofdpseudoephedrinecouldreach
1283mg/L.Moreover,leucinedehydrogenasegene(bcdh)fromB.clausiB0658wasamplifiedA
recombinantplasmidpET28abcdhwasconstructedandtransformedintoE.coliBL21(DE3).The
catalyticfunctionofleucinedehydrogenasefromB.clausiB0658wassubsequentlyverifiedasbcdhwas
expressed,dpseudoephedrine could be detected in the reaction system catalyzed by the
recombinantE.coli.
Keywords:dpseudoephedrine;carbonylreductase;leucinedehydrogenase
  
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10,
NaCl10;pH70~72,01MPa
Å[
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P>‚ï(æ–‡100μg/mLh:9h1!
(Amp)
Ù
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MLDH
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99
BacilusmegateriumB0108
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84
OceanobacilusiheyensisB1109
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77
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XanthoonascampestrisB1070
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1 
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24h
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76g/L,
Îh‚0̈ƒn=ž
1087mg/L。
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17h
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.ŒÀ Ã¥;ƒ8
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+MAK+Glu
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3。
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biotransformation
h+Ç>^cËÎh¬å

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(
005、010、015、020、025
O
030mg/mL
MAK,
|}+‡\§Ìˆ×–‹×*hST

3
@46
4。
‘6

á7
:MAK
文\ 
005
mg/mL
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010mg/mL
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ז½!í@

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Ÿ*+¢+–ús

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020mg/mL
P=ž‚0

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A•|B

Q
020mg/mL
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D
4 
½¾rsc

/0126w;de
Fig.4 EfectsofMAKconcentrationon
dpseudoephedrineproduction
243 pH
§

ÓÎN/זhST
  
ÎI‘Œˆ5‚g²chW/ÛÜ

.e
Qh
pH
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60、
65、70、75、80
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73 
!

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žŸ

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5
łƒ&̈
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5 pH
c

/0126w;de
Fig.5 EfectsofpHonbiotransformation
fordpseudoephedrine
244 
ˆ\§

ÓÎN/זhST
  
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zsc.Œ

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fordpseudoephedrine
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&̈

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cû’ŠßPZ¯iÚh
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mg/L,
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9::;<Š%’;=>f?
251 
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252 
±ªæÛ
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