Final Report of Partner 5 Moscow State University

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Final Report of Partner 5 Moscow State
University
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1. Mediator research
O2 or H2O2 Ered ? Eox Eox S ? Ered
S? S? F ? S Fox
dP/dt kcat Eo S / (Km S)
(1)
dP/dt EoSkcat/Km kF PF
(2)
P kcat/Km SoEo 1 exp( -kcat Eot /Km
- kFFt)/ (kcat/Km) Eo kFF
(3)
Plim(F) kcat/Km SoEo /(kcat/Km) Eo
kFF
(4)
(5)
Plim(0) So
(6)
Plim(0)/Plim(F)
Plim(0)/Plim(F) 1 kFKm F/kcat Eo
(7)
plot Plim(0)/Plim(F) - F
(8)
(9)
kcatEo/Km
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Fig.1. Effect of phenol on ABTS oxidation
catalyzed by tobacco peroxidase (pH 4.5). a
(left) kinetic curves and b (right) linear plot
used to calculate the rate constant for the
non-enzymatic interaction of phenol and ABTS.

Fig. 2. Effect of o-vanillyl alcohol on the rate
of peroxidase (left) and laccase (right)
catalyzed ABTS oxidation.
Fig. 3. Dependence of lag-period on the
concentration of added o-vanillyl alcohol for
tobacco peroxidase and laccase at same initial
rate of ABTS oxidation in the absence of the
second substrate.
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Table 1. Kinetic parameters for
peroxidase-catalyzed oxidation of ruthenium
complexes at pH 4.5.
bpy bi pyridine HRP horseradish peroxidase
phpy phenylpyridine SPP sweet potato
peroxidase phen- phenanthroline RPTP royal palm
tree peroxidase topy- tosyl pyridine Me2bpy
dimethylbipyridine
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2. Enzyme research Production of wild-type
recombinant tobacco peroxidase
5'-CATATGCAATTAAGTGCAACATTTTATGATACC-3' and
5'-GTCGACGAA-TTCTTAATTAACCCTCTT-3
Table 2. Production and purification of
recombinant wild-type tobacco peroxidase (1.8 L
cultural medium).
Calculated as the ratio of the active enzyme to
the total peroxidase apoprotein solubilized from
inclusion bodies.
Fig. 5. Purification of recombinant TOP
Fig.6. Absorbance spectra
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Fig. 7. Kinetic properties of recombinant enzyme
in the reaction of ABTS oxidation
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1. Ile37Met (active, yield 7) I37M_fwd 5 GGT
GCT AAA ATT ATG CGT CTT CAT TTC 3 I37M_rev 5
GAA ATG AAG ACG CAT AAT TTT AGC ACC 3 2.
Glu141Phe (active OK, yield gt30) E141F_fwd 5
CCT AGC CCC TTT TTC ACA CTT GCT GTA
3 E141F_rev 5 TAC AGC AAG TGT GAA AAA GGG
GCT AGG 3 3. Ala162Tyr (traces of
activity) A162Y_fwd 5 ACT GAT CTT GTT TAT CTA
TCA GGT GCA 3 A162Y_rev 5 TGC ACC TGA TAG
ATA AAC AAG ATC AGT 3 4. HIS (Ala67PheHis)
(low activity, unstable against ammonium sulfate
precipitation) HIS_fwd 5 CAA ACT GAG AAA GCT
TTC CAC CCT GCT AAT GTA GGT 3 HIS_rev 5
ACC TAC ATT AGC AGG GTG GAA AGC TTT CTC AGT TTG
3 5. Leu157Trp (active, yield 7) L157W_fwd 5'-
GGGGATGGATTGGACTGATCTTG -3' L157W_rv 5'-
ATCAGTCCAATCCATCCCCTTAT -3' 6. Thr158Trp (active,
yield 7) T158W_fwd 5'- GATGGATTTATGGGATCTTGTTG
-3' T158W_rv 5'- CAACAAGATCCCATAAATCCAT -3' 7.
Asn116Trp (active, yield 7) Q116W_fwd 5'-
TCCGTCGTGGTGGGTACTTTTTG -3' Q116W_rv 5'-
CAAAAAGTACCCACCACGACGGA -3' 8. Thr151Trp (active,
yield 7) T151W_fwd 5'- CACAATTCTGGAATAAGGGGATG
-3' T151W_rv 5'- CCCCTTATTCCAGAATTGTGGTAT -3' 9.
Ala162Trp (no activity at all) A162W_fwd 5
ACT GAT CTT GTT TGG CTA TCA GGT GCA
3 A162W_rev 5 TGC ACC TGA TAG CCA AAC AAG
ATC AGT 3
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Glu141Phe mutant of tobacco peroxidase
Fig.8. SDS-PAGE of WT and Glu141Phe mutant of
recTOP 1, mutant protein solubilized in 6 M
urea 2, mutant enzyme before purification 3,
mutant enzyme after gel-filtration 4 and 5, WT
enzyme after first and second gel-filtration,
respectively.
Table 3. Production of Glu141Phe mutant of recTOP
from 0.6 L cultural medium.
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?able 4. Kinetic parameters of ABTs oxidation
catalyzed by recombinant forms of TOP
Table 5. Substrate specificity of recTOP forms
compared to recHRP
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The main results achieved are as follows
(1) We developed a quantitative method to
determine mediator effectiveness with respect to
a particular substrate, i.e. the value of the
rate constant for non-enzymatic interaction
between a mediator radical and a target
substrate (2) A requirement for a prospective
mediator is formulated as the lower limit of the
rate constant for the interaction of oxidized
redox mediator with a target substrate to be
above 1000 M-1s-1 (3) A number of ruthenium
complexes satisfying this condition with respect
to phenolic substrates has been synthesized and
characterized (4) Recombinant tobacco peroxidase
and its mutants mimicking horseradish peroxidase,
soybean peroxidase, and lignin peroxidase have
been constructed, produced and characterized. (5)
The most promising in terms of activity,
stability and production yield and a starting
point for the construction of biocatalyst stable
in solutions with extremal pH is Glu141Phe mutant
of tobacco enzyme which preserves all unique
properties of the wild-type and native tobacco
enzyme.
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PhD thesis defended 1 Mareeva E.A. (2002)
Isolation and properties of tobacco anionic
peroxidase from transgenic plants. (Supervised
by Prof. V.Tishkov Dr.I.Gazaryan) Diploma
Thesis defended 1 Hushpulian D.M. (2003)
Production and comparative characterization of
recombinant tobacco peroxidase and its Glu141Phe
mutant (Supervised by Prof. V.Tishkov
Dr.I.Sakharov). Publications Papers 1.
Alpeeva IS, Soukharev VS, Alexandrova L, Shilova
NV, Bovin NV, Csoregi E, Ryabov AD, Sakharov IY.
(2003) Cyclometalated ruthenium(II) complexes as
efficient redox mediators in peroxidase
catalysis. J Biol Inorg Chem. 8(6),683-8. 2.
Hushpulian D.M., Fechina V.A., Kazakov S.V.,
Sakharov I.Yu., and Gazaryan I.G. (2003) Non
enzymatic interaction of reaction products and
substrates in the course of peroxidase catalysis.
Biochemistry-Moscow 9 (in press) 3. Hushpulian
D.M., Savitski P.A., Rojkova A.M., Chubar T.A.,
Fechina V.A., Sakharov I.Yu., Lagrimini L.M.,
Tishkov V.I., Gazaryan I.G. (2003) Expression and
refolding of tobacco anionic peroxidase from
E.coli inclusion bodies. Biochemistry-Moscow 11
(in press) In collaboration with partner 4 4.
Shleev S.V., Khan I.G., Gazaryan I.G., Morozova
O.V., Yaropolov A.I. (2003) Novel laccase redox
mediators spectral, electrochemical and kinetic
properties. Applied Biochemistry and
Biotechnology 2003 (in press) Conference
presentations International Symposium
Biocatalysis 2002, Moscow, Russia, June 23-27,
2002. Khushpulian, D.M., Fechina, V.A., Kazakov,
S.V., Gazaryan, I.G. Non-enzymatic mechanism of
peroxidase catalysed substrate-substrate
co-oxidation. Savitski, P.A., Rozhkova, A.M.,
Chubar, T.A., Fechina, V.A., Gazaryan I.G.
Tobacco peroxidase cloning in E.coli and
refolding to yield active enzyme. VI
International Symposium on Peroxidases, Murcia,
Spain (July 3-7, 2002). Alpeeva I., Alexandrova
L, Ryabov A.D., and Sakharov I. Cyclometalated
Ruthenium(II) Complexes As Efficient Redox
Mediators in Peroxidase Catalysis 226th American
Chemical Society National Meeting, New York, NY,
September 7-11, 2003. Gazaryan I.G., Savitski
P.A., Rojkova A.M., Hushpulian D.M., Chubar T.A.,
Fechina V.A., Sakharov I.Yu., Tishkov V.I.,
Lagrimini L.M. (2003) Expression and refolding of
recombinant tobacco peroxidase from E.coli
inclusion bodies. Biochemistry-US 42(28), p. 8632.
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Team members 1. Genetic engineering group Dr.
A.Rojkova, research scientist (MSU)
Dr.P.Savitskii, research scientist (INBI RAN)
Prof.V.Tishkov (MSU) 2. Microbiology E.Karpenko,
engineer (MSU) 3. Organic synthesis Prof. P.
Khokhlov (VNII Phytopathology) 4. Enzyme
production and kinetic analysis Dr.T.Chubar,
research scientist (MSU) V.Fechina, research
scientist (INBI RAN) D.Hushpulian, junior
research scientist (MSU) Dr.E.Mareeva, research
scientist (MSU) 5. Stopped-flow measurements
(while visiting the laboratory of Prof.
Thorneley, subconstractor to P2, 3 weeks March
2002) V.Schedrina, 2nd year PhD student
(MSU) S.Smirnov, 3rd year PhD student (MSU)
Team leaders Drs. Irina Gazaryan and Ivan
Sakharov