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Electrochemical enzyme immunoassay for detection of toxic substances
Biocatalysis for the 90s THE EFFECTS OF RIFAMPICIN AND CHLORAMPHENICOL ON PRODUCT AND ENZYME LEVELS OF THE ACID AND SOLVENT PRODUCING PATHWAYS OF ~LOSTR1D1UM ACETOBUTYLICUM (ATCC 824). Richard W. Welch, Sandra W. Clark, George N. Bennett, and Frederick B. Rudoloh. Department of Biochemistry and Cell Biology, P.O. Box 1892, Rice University, Houston, Texas 77251 C. acetobutylicurn is a saccharolytic, spore forming anaerobe which has been used in the industrial production of a number of chemicals including solvents such as butanol and acetone. The organism has two distinct phases during its life cycle. The acidogenic first phase is characterized by vigorous growth and the production of organic acids, such as butyric and acetic acid, and molecular hydrogen. The solventogedic second phase is characterized by a slower growth rate and the production of solvents, mainly butanol, ethanol and acetone. Many of the changes that occur in the switch from acid to solvent production have been characterized and numerous studies on the levels of key enzymes have been performed. As a part of an ongoing study of regulation of acid and solvent production in C. acetobutylicum with the goal of developing genetically modified strains, the effects of rifampicin and chloramphenicol addition at various points in the fermentation were determined on product formation, enzyme activity and enzyme stability in vivo. The in vitro activities of phosphotransbutyrylase, butyrate kinase, CoA transferase, butyraldehyde dehydrogenase and butanol dehydrogenase were assayed over the entire fermentation. The enzymes in the solvent producing pathways were more effected by the addition of rifampicin and chloramphenicol than those in acid producing pathways. The position of butyraldehyde dehydrogenase as a branchpoint enzyme in die use of butyryl-CoA, coupled with the shon half life for butyraldehyde dehydrogenase in vivo indicates that butyraldehyde dehydrogenase may be a key enzyme in controlling the switch from the production of butyrate to the production of butanol.
DIRECT DETERMINATION ViTY OF INNOBILIZED
OF THE CEPHALOSPORIN TRANSFORMING ACTIC E L L S ~ r f T H U S E O F AN E N Z Y M E T H E R N I S T O R
P. ~ereeiner~ A. W e l w a r d o v ~ , E. ~ [ [ c h ~ I k o v ~ , ~ L. K u r l l l o v ~ , V. ~ t e f u c a , + B. D a r ~ i e l s s o n , ~ a n d E.
Welward,~ Berznekov~
E. f
Z r~e~ute o/ Ch~.mfstrp, Slo~a~ Academ~ o/ Sciences, Bma~sla~a, C~ech and ~lo~a;~ Federal Republtc (CSFRD *Reseamch Ir~st~u~e ~or Dm~gs, Slo~ens~ £ u p ~ a , CSFR +Depo~tm~n~ o/ C h e m i c a l a n d B i o c h e m i c a l Engfneerfng. S[ova& TocAnZcaL University. Bratfsla~e. CSFR #Ptu~e a n d A p p l £ e d B ~ o c A e m f s z r y . Chemzc~l Center. Un~ersLty el i~nd. iund. S ~ d e n
T h e E7 Js a E:low caller i m e t r i c iristtHmeht ~. F h e t h e ~ m o m e t r tc s i g n a l ~ a s iderltit:ied bv ilie~.~[i~ o~ HF'tC-.-arla.... i y s i s to be a r i s i n g F r o m t h e ~ c t i v i t y o~: ( , r i o s p e c ] . + i ( : doml!~=l t i n g e n z y m i c s t e p t!l t h e Eel I~. The c,btalr~ed r e s t ! ] ~.; Wet e superveri#ied in t h e s a m e w a y w i t h t h P - ~ d o~ b-a~mtno =,( ~a e ~ i d a s e (DANe, E.C, 1 . 4 . V , ~> i s o ] a ~ P d tI-(.-~mT.~. c e i l s aild imm o b i l i z e d by gel entt ,]pment. T h e thr~r m o m e t ~ ]c Lclnla] v,as l't o p(,rtional t o t h e n u m b e r c:)E c e l l s =~s }~P!l +,~ lo t h e a m o t m t cH erlzvme (DAAD) i m m o b i l i z e d in t h e E] iIi[riic!-t~unn. Fltrthe~more,, g o ( ) o ~ _ o r r e i a t i n n b e t N p , m t~,e ~ h e ~ m o m e t t ~< siHr,al a s s o c i a t e d w i t h c:ephalospo! in t r a n f o r m i n g ,~{:[ivit,; er both t h e LmmeL, i l i z m d c e l l s a n d DAAC} ~r~i;! t h e - h e i a h r o f tr'i~ g]!da~ yl 7-aminac:ephalosporanic a c i d p e a k ir~ t b , HF'!C c h ~ < , m ~ f o ~ r - m W,~s Eo~tnd. T h e b e s t a g r e e m e n t ~,~s. obt~ir,~-.d w h e n t h e ET ~,~ Obef a t e d {,t a F l o w r a t e o F 0 . 8 to l.] m] JTI]~'I, rh[~ ~,~ime ~(:)r£inq
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ELECTROCHEMICAL ENZYME IMMUNOASSAY FOR DETECTION OF TOXIC SUBSTANCES. J. P. O ' D a l v . J. Z h a o . a n d R. W. H e n k e n s . Enzyme Technology R e s e a r c h G r o u p , Inc., 7 1 0 W e s t M a i n Street, Durham, North Carolina 27701. Sensors that provide reliable, rapid measurement of toxic substances are needed to solve significant human health and safety problems. We have developed a new biosensor d e s i g n t h a t con~oines t h e a d v a n t a g e s of immunoassay with electrochemical response. The biosensor consists of two major elements: an electrical conducting layer having immobilized enzyme, polyclonal or monoclonal antibodies, and other necessary reagents; and the electronic components used in the signal readout. We have established that this immunosensor measures toxic substances in biological samples. T h e r e s u l t is a n ampezometric immunoassay b a s e d on c o u p l i n g t h e irmmunochemical reaction to the electrode response by using a soluble electrochemically active mediator. The specific question addressed was: Does the system's irmmunochemical detection r e l i a b l y r e s p o n d at s u f f i c i e n t l y low analyte concentrations? We will describe our work in these areas: i) e n z y m e i n z m o b i l i z a t i o n o n c o l l o i d a l g o l d ; 2) c o l l o i d a l gold-enzyme deposition on the electrode s u r f a c e ; 3) mediator-antigen conjugate synthesis; 4) a n t i b o d y incorporation at t h e e l e c t r o d e s u r f a c e ; 5) b i o e l e c t r o d e characterization and optimization; a n d 6) i m m u n o s e n s o r demonstration to detect antigen. Sensors that employ immunochemical detection will have broad applicability to detect/diagnose toxic substances in biological samples such as b l o o d a n d u r i n e a n d in e n v i r o n m e n t a l s a m p l e s s u c h as waste water and drinking water.
524
Enzyme Microb. Technol., 1991, vol. 13, June
t T ~pproach ( ) G # : e r s ~, {:Jibe{ t arx:! < ~ , n , ' " , , L P ~ l l t {:l;~r,e.to ~stlmatl(-,r, o~: k i r 3 e t ~ c i::),.~ ~ m ~ t e l ~: , , , n . , b i ~ r ~ c t t,~[~ c:l (." i) c~.ll~5 F o r a c ~ r + a i r l !/t n s * . ~ 2 ~ m ~ n ( ] : . ( ~ ~-~t -,rid
The',
Struc£ure-Activlty Relationships Generated DV Protein Engineering
ot
Protease
and Lipase
Variants
A.J. P c u l o s e , D. h s t e ! ! , T. G r a y c a r , R. Bott, S. P o w e r , R. A d a m s - S i l v a , o. V a n b i e l i n , B. Hhew, G. G r a y , a n d S. N o r t o n . G e n e n c o r i n t e r n a t i o n a l , S. S a n F r a n c i s c o , C A a n d J. W e l l s , G e n e n t e c h , inc., S. S a n F r a n c i s c o , CA. V a r i a n t s of t w o " a c t i v e s e r i n e - h y d r o l a s e s " , o n e b e l o n g i n g t o t h e protease family and the other to the lipase family, have been g e n e r a t e d b y s i t e s p e c i f i c m u t a g e n e s i s at f u n c t i o n a l l y r e l e v a n t r e g i o n s of t h e s e e n z y m e s . C h a r a c t e r i z a t i o n of t h e s e v a r i a n t s r e v e a l e d t h a t m a n y of t h e s e v a r i a q t s , w i t h s i n g l e a m i n o a c i d s u b s t i t u t i o n s , s h o w e d d r a m a t i c d i f f e r e n c e s in t h e i r s u b s t r a t e specificity, catalytic efficiency and stability.
DIRECT DETERMINATION ViTY OF INNOBILIZED
OF THE CEPHALOSPORIN TRANSFORMING ACTIC E L L S ~ r f T H U S E O F AN E N Z Y M E T H E R N I S T O R
P. ~ereeiner~ A. W e l w a r d o v ~ , E. ~ [ [ c h ~ I k o v ~ , ~ L. K u r l l l o v ~ , V. ~ t e f u c a , + B. D a r ~ i e l s s o n , ~ a n d E.
Welward,~ Berznekov~
E. f
Z r~e~ute o/ Ch~.mfstrp, Slo~a~ Academ~ o/ Sciences, Bma~sla~a, C~ech and ~lo~a;~ Federal Republtc (CSFRD *Reseamch Ir~st~u~e ~or Dm~gs, Slo~ens~ £ u p ~ a , CSFR +Depo~tm~n~ o/ C h e m i c a l a n d B i o c h e m i c a l Engfneerfng. S[ova& TocAnZcaL University. Bratfsla~e. CSFR #Ptu~e a n d A p p l £ e d B ~ o c A e m f s z r y . Chemzc~l Center. Un~ersLty el i~nd. iund. S ~ d e n
T h e E7 Js a E:low caller i m e t r i c iristtHmeht ~
ra]
metl~od
=-
ELECTROCHEMICAL ENZYME IMMUNOASSAY FOR DETECTION OF TOXIC SUBSTANCES. J. P. O ' D a l v . J. Z h a o . a n d R. W. H e n k e n s . Enzyme Technology R e s e a r c h G r o u p , Inc., 7 1 0 W e s t M a i n Street, Durham, North Carolina 27701. Sensors that provide reliable, rapid measurement of toxic substances are needed to solve significant human health and safety problems. We have developed a new biosensor d e s i g n t h a t con~oines t h e a d v a n t a g e s of immunoassay with electrochemical response. The biosensor consists of two major elements: an electrical conducting layer having immobilized enzyme, polyclonal or monoclonal antibodies, and other necessary reagents; and the electronic components used in the signal readout. We have established that this immunosensor measures toxic substances in biological samples. T h e r e s u l t is a n ampezometric immunoassay b a s e d on c o u p l i n g t h e irmmunochemical reaction to the electrode response by using a soluble electrochemically active mediator. The specific question addressed was: Does the system's irmmunochemical detection r e l i a b l y r e s p o n d at s u f f i c i e n t l y low analyte concentrations? We will describe our work in these areas: i) e n z y m e i n z m o b i l i z a t i o n o n c o l l o i d a l g o l d ; 2) c o l l o i d a l gold-enzyme deposition on the electrode s u r f a c e ; 3) mediator-antigen conjugate synthesis; 4) a n t i b o d y incorporation at t h e e l e c t r o d e s u r f a c e ; 5) b i o e l e c t r o d e characterization and optimization; a n d 6) i m m u n o s e n s o r demonstration to detect antigen. Sensors that employ immunochemical detection will have broad applicability to detect/diagnose toxic substances in biological samples such as b l o o d a n d u r i n e a n d in e n v i r o n m e n t a l s a m p l e s s u c h as waste water and drinking water.
524
Enzyme Microb. Technol., 1991, vol. 13, June
t T ~pproach ( ) G # : e r s ~, {:Jibe{ t arx:! < ~ , n , ' " , , L P ~ l l t {:l;~r,e.to ~stlmatl(-,r, o~: k i r 3 e t ~ c i::),.~ ~ m ~ t e l ~: , , , n . , b i ~ r ~ c t t,~[~ c:l (." i) c~.ll~5 F o r a c ~ r + a i r l !/t n s * . ~ 2 ~ m ~ n ( ] : . ( ~ ~-~t -,rid
The',
Struc£ure-Activlty Relationships Generated DV Protein Engineering
ot
Protease
and Lipase
Variants
A.J. P c u l o s e , D. h s t e ! ! , T. G r a y c a r , R. Bott, S. P o w e r , R. A d a m s - S i l v a , o. V a n b i e l i n , B. Hhew, G. G r a y , a n d S. N o r t o n . G e n e n c o r i n t e r n a t i o n a l , S. S a n F r a n c i s c o , C A a n d J. W e l l s , G e n e n t e c h , inc., S. S a n F r a n c i s c o , CA. V a r i a n t s of t w o " a c t i v e s e r i n e - h y d r o l a s e s " , o n e b e l o n g i n g t o t h e protease family and the other to the lipase family, have been g e n e r a t e d b y s i t e s p e c i f i c m u t a g e n e s i s at f u n c t i o n a l l y r e l e v a n t r e g i o n s of t h e s e e n z y m e s . C h a r a c t e r i z a t i o n of t h e s e v a r i a n t s r e v e a l e d t h a t m a n y of t h e s e v a r i a q t s , w i t h s i n g l e a m i n o a c i d s u b s t i t u t i o n s , s h o w e d d r a m a t i c d i f f e r e n c e s in t h e i r s u b s t r a t e specificity, catalytic efficiency and stability.