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Importance of Soil Enzymes
1. Release of nutrients in soil bymeans of organic matterdegradation
2. Identification of soils
3. Identification of microbial activity
4. Importance of soil enzymes assensitive indicators of ecologicalchange
ENZYME CLASSIFICATIONENZYME CLASSIFICATION
Oxidoreductases - Oxidation reduction reaction(Dehydrogenase, Catalase, Peroxidase)
Tranferases - The transfer of group of atoms fromdonor to an acceptor molecule.
(Aminotransferases, Rhodonese)
Oxidoreductases - Oxidation reduction reaction(Dehydrogenase, Catalase, Peroxidase)
Tranferases - The transfer of group of atoms fromdonor to an acceptor molecule.
(Aminotransferases, Rhodonese)
Hydrolases - Hydrolitic cleavage of bonds.(Phosphatase, Cellulase, Urease)
Lyases - Cleavage of bonds other than hydrolysisor oxidation.
(Aldolase)
Hydrolases - Hydrolitic cleavage of bonds.(Phosphatase, Cellulase, Urease)
Lyases - Cleavage of bonds other than hydrolysisor oxidation.
(Aldolase)
Isomerases - Isomarization reaction.
Ligases - Formation of bonds by the cleavage ofATP.
(Acetyl-CoA carboxylase)
Isomerases - Isomarization reaction.
Ligases - Formation of bonds by the cleavage ofATP.
(Acetyl-CoA carboxylase)
1.
2.
3.
4.
5.
6.
Kin
ds
of
En
zym
es
Kin
ds
of
En
zym
es
Alw
ays
pre
sentin
nearly
consta
ntam
ounts
ina
cell
(not
affecte
dby
additio
nofany
part
icula
rsubstr
ate
...g
enes
alw
ays
expre
ssed.)
(pyro
phosphata
se)
Alw
ays
pre
sentin
nearly
consta
ntam
ounts
ina
cell
(not
affecte
dby
additio
nofany
part
icula
rsubstr
ate
...g
enes
alw
ays
expre
ssed.)
(pyro
phosphata
se)
Pre
sentonly
intr
ace
am
ounts
or
notatall,
butquic
kly
incre
ases
inconcentr
ation
when
its
substr
ate
ispre
sent.
(Am
idase)
Pre
sentonly
intr
ace
am
ounts
or
notatall,
butquic
kly
incre
ases
inconcentr
ation
when
its
substr
ate
ispre
sent.
(Am
idase)
Both
enzym
es
are
pre
sentin
the
soil.
Both
enzym
es
are
pre
sentin
the
soil.
Inducib
le
Constitu
tive
ORIGIN OF SOIL ENZYMESORIGIN OF SOIL ENZYMES
STATE OF ENZYMES IN SOILSSTATE OF ENZYMES IN SOILS
1. Role of Clays1. Role of Clays
1. Microorganisms -Living and dead
1. Microorganisms -Living and dead
2. Plant Roots and Plant Residues2. Plant Roots and Plant Residues
3. Soil Animals3. Soil Animals
3. Role of Clay - Organic MatterComplexes
3. Role of Clay - Organic MatterComplexes
2. Role of Organic Matter2. Role of Organic Matter
+
STATE OF ENZYMES IN SOILSTATE OF ENZYMES IN SOIL
Role of ClaysRole of Clays
Role of Organic Matter
Role of O.M. - Clay ComplexRole of O.M. - Clay Complex
a. Most activity associated with clays.
b. Increases resistance to proteolysis andmicrobial attack
c. Increases the temperature of inactivation.
a. Most activity associated with clays.
b. Increases resistance to proteolysis andmicrobial attack
c. Increases the temperature of inactivation.
a. Humus material provides stability to soilnitrogen compounds
b. Enzymes attached to insoluble organicmatrices exhibit pH and temperaturechanges.
c. Inability to purify soil enzymes free of
a. Humus material provides stability to soilnitrogen compounds
b. Enzymes attached to insoluble organicmatrices exhibit pH and temperaturechanges.
c. Inability to purify soil enzymes free of
a. Lignin + bentonite ( clay ) protect enzymesagainst proteolitic attack, but not bentonitealone.
b. Enzymes are bound to organic matter whichis then bound to clay.
a. Lignin + bentonite ( clay ) protect enzymesagainst proteolitic attack, but not bentonitealone.
b. Enzymes are bound to organic matter whichis then bound to clay.
soil organic matter ( bound to O.M. )soil organic matter ( bound to O.M. )
Schematic representation of methods ofimmobilizing enzymes.
( Weetall, 1975 )
Schematic representation of methods ofimmobilizing enzymes.
( Weetall, 1975 )
Adsorption Entrapment
Microencapsulation Ion exchange
Adsorption and cross-linkingAdsorption and cross-linking
Covalent attachmentCovalent attachment
Cross-linking
Copolymerization
Enzyme
Enzyme
Enzyme
Enzyme
Enzyme
Enzyme
Enzyme
Enzyme
Membrane
Carrier
Carrier
Carrier
Polymer
Polymer
RRRR RR R+ + ++ + + +
vvvvvvvvvv
vvvvvvvvvv
(HD
TM
A-
hexad
ecylt
rim
eth
yla
mm
on
ium
bro
mid
e-
serv
es
as
acati
on
exch
an
ge
su
pp
ort
.)
E
E-
E-
E
vvvvvvvvvv
vvvvvvvvvv
vvvvvvvvvv
vvvvvvvvvv
vvvv
vvvv
vv
vvvvvvv
vvvvvvvvv
vvvvvvvvv
vvvvvvvvv
vvvvvvvvv
vvvvvvvvv
vvvvvvvvv
vvvvvvvvv
vvvvvvvvvvv
vvvvvvvvv
vvvvvvvvv
vvvvvvvvv
+
+N
+N
+N
+N
+N
+N
+N
+N
+N
+N
+N
+N N
N
N
N
N
N
N
0.9
nm
Am
od
elfo
rb
ind
ing
ure
ase
toh
yd
rop
ho
bic
HD
TM
Asm
ecti
te.
Th
ed
ark
sit
eo
fth
een
zym
es
are
hyd
rop
ho
bic
are
as.
QUANTITATIVE ASSAY OF ENZYMATIC ACTIVITY *QUANTITATIVE ASSAY OF ENZYMATIC ACTIVITY *
1. The overall stoichiometry of the reaction catalysed.1. The overall stoichiometry of the reaction catalysed.
Things we must know.Things we must know.
4. Its optimum pH .4. Its optimum pH .
* Usually measure enzyme activity at substrate concentrationsabove saturation level, where the reaction rate is at a maximum.
* Usually measure enzyme activity at substrate concentrationsabove saturation level, where the reaction rate is at a maximum.
5. A temperature zone in which it is stable and has
high activity.
5. A temperature zone in which it is stable and has
high activity.
6. A simple analytical procedure to measure the
disappearance of substrate or the appearance
of product.
6. A simple analytical procedure to measure the
disappearance of substrate or the appearance
of product.
2. Whether the enzyme requires the addition of
cofactors such as metal ions or coenzymes.
2. Whether the enzyme requires the addition of
cofactors such as metal ions or coenzymes.
3. Its dependence on substrate and cofactor
concentrations .
3. Its dependence on substrate and cofactor
concentrations .
SOIL STORAGE
Methodology
Air Dry
Heat Treatment
Enzyme concentration declines in theabsence of renewed synthesis.
Once dry, enzyme activity is maintainedat the same level for a long time -good for comparative studies.
Soil protects against heat and cold extremes.To inactivate an enzyme in soil requires alonger time and a higher temperature thanenzymes in solution.
Initia
lV
elo
city
Initia
lV
elo
city
Initia
lV
elo
city
Initia
lV
elo
city
Enzyme
Substrate
First order (substrate dependent)First order (substrate dependent)
Zero order(substrate independent)
Zero order(substrate independent)
A
B
MICHAELIS - MENTEN ASSUMPTIONMICHAELIS - MENTEN ASSUMPTION
1. The rate of an enzyme catalyzed reaction changesfrom first order to zero order kinetics.
1. The rate of an enzyme catalyzed reaction changesfrom first order to zero order kinetics.
4. Enzyme total concentration defined as free and incomplex state.
4. Enzyme total concentration defined as free and incomplex state.
6. V max when ES complex reaches a maximumsaturation (no free enzyme).
6. V max when ES complex reaches a maximumsaturation (no free enzyme).
5. Initial rate limiting parameter is the decompositionof the enzyme / substrate (ES) complex from theproduct k .
5. Initial rate limiting parameter is the decompositionof the enzyme / substrate (ES) complex from theproduct k .
3. A steady state equilibrium between the rate offormation and the rate of degradation of ES israpidly achieved.
3. A steady state equilibrium between the rate offormation and the rate of degradation of ES israpidly achieved.
TE = E + ESE = E + ES
v ~ ESv ~ ES
2. Enzyme (E) reversibly binds with substrate (S) to forman intermidiate (ES) complex which then breaks downto form product (P). Each reaction is described by aspecific rate constant: k , k , k .
2. Enzyme (E) reversibly binds with substrate (S) to forman intermidiate (ES) complex which then breaks downto form product (P). Each reaction is described by aspecific rate constant: k , k , k .21 3
3
k (E - ES)(S) = k (ES) + k (ES)k (E - ES)(S) = k (ES) + k (ES)
DERIVATION OF THE MICHAELIS - MENTEN EQUATIONDERIVATION OF THE MICHAELIS - MENTEN EQUATION
v = k (ES)v = k (ES)2
k1 k2
k -1
maxv = k (E )v = k (E )2 T
d (ES)d (ES)
d (ES)d (ES)
(ES)
= k (ES) + k (ES)= k (ES) + k (ES)2
2
2
1
1
-1
-1
-1
1m
1
but E = (E - ES)but E = (E - ES)(E )(S)(E )(S)
(E - ES)(S)(E - ES)(S)
T
T
T
T
f f= k= k
= k= k
(S)(E - ES) k + k(S)(E - ES) k + k
E + S ES E + PE + S ES E + P
dt
dt
1. Rate of formation of ES1. Rate of formation of ES
2. Rate of breakdown of ES2. Rate of breakdown of ES
3. Setting the rates equal to each other3. Setting the rates equal to each other
4. Rearranging equation 34. Rearranging equation 3
= =k k
-
v = v (S)v = v (S)
v vs. v / S = Eadie-Hofstee plot
S / v vs. S = Hanes-Woolf plot
v vs. v / S = Eadie-Hofstee plot
S / v vs. S = Hanes-Woolf plot
k (ES)k (ES)
(ES)
2
2 2
2
2
(E )(S)(E )(S)T
(E )(S)(E )(S)T
T
+ (S)+ (S)
+ (S)+ (S)
k (ES) = vk (ES) = v
k (E ) = vk (E ) = v
6. Multiply by k6. Multiply by k
7. But7. But
8. Lineweaver - Burk transformation8. Lineweaver - Burk transformation
Michaelis - Menten EquationMichaelis - Menten Equation
5. Rearranging again5. Rearranging again
K + (S)K + (S)
m
max
max
m
K
mK
=
= k
max=
1 K 1 11 K 1 1m
v v v+max(S)( )( )
SOIL ENZYMOLOGY
Methodology
Problems
No way to separate extracellular fromintracellular activity
Presence of recently secreted freeenzymes accumulated in soil.
Separation between chemical andbiological catalysis.
Storage and treatment of soils greatlyaffects enzymatic activity.
Toluene
Disadvantages
Advantages
Ideal - Inhibit microbial activity without celllysis or extracellular enzyme inhibition.
Stops synthesis of enzymes by living cells.
Prevents assimilation of products of enzymaticreactions. (Important to study individual reactions.)
Acts as a plasmolytic agent, releasing cellcontents and intracellular enzymes.
Destroys dehydrogenase activity.
APPLICATION OF SOIL ENZYMESAPPLICATION OF SOIL ENZYMES
* Correlation with soil fertility.
*Correlation with microbial activity.
*Correlation with biochemical cycling ofvarious elements in soil ( C, N, S ).
Degree of pollution ( heavy metals, SO ).
* Correlation with soil fertility.
*Correlation with microbial activity.
*Correlation with biochemical cycling ofvarious elements in soil ( C, N, S ).
Degree of pollution ( heavy metals, SO ).
To assess the successional stage of anecosystem.
Forensic purposes.
Rapid degradation of pesticides.
Disease studies.
To assess the successional stage of anecosystem.
Forensic purposes.
Rapid degradation of pesticides.
Disease studies.
2
2
1
3
4
5
6
7
8
* Correlation not good because the source of enzymes varies, andcomplexes with O.M., and clay limits substrate atack by theenzyme.
Enzyme activity in soil fluctuates with environment.
* Correlation not good because the source of enzymes varies, andcomplexes with O.M., and clay limits substrate atack by theenzyme.
Enzyme activity in soil fluctuates with environment.
Correlation matrix (r-values) between soil enzyme activities, viable plate counts, respiration,biomass, and soil properties
Frankenberger, Jr., W.T. and W.A. Dick. 1983. Relationships between enzyme activities and microbial growth and activity indices insoil. Soil Sci. Soc. Am. J. 47:945-951.
Imm
obili
zatio
n of
enz
ymes
on
pret
reat
ed c
lays
and
soi
ls.
(Sar
kare
t. al
., 19
89)
LACC
ASE
TRYR
OSI
NASE
ACID
PHO
SPHA
TASE
B-D-
GLU
COSI
DASE
020406080100
LACC
ASE
TRYR
OSI
NASE
ACID
PHO
SPHA
TASE
B-D-
GLU
COSI
DASE
BEN
TONI
TEK
AO
LINI
TESA
NDY
LOA
M S
OIL
SILT
LO
AM
SO
IL
Phosphatase activity in acid and alkaline soil ( Eivazi and Tabatabai, 1977 )
pH of Buffer
4 6 8 10 12 14
Pho
spha
tase
Act
ivity
( ug
p-ni
trop
heno
l rel
ease
d / g
soi
l / h
)
0
50
100
150
200
250
Webster ( pH 5.8 )Nicollet ( pH 6.1)
Ida ( pH 8.0 )Harps ( pH 7.8 )
Acid Soils
Alkaline Soils
Conversion of 1-aminocyclopropane-1-carboxylic acid ( ACC)to ethylene in air-dried soils.
( Frankenberger and Phelan, 1985 )
Conversion of 1-aminocyclopropane-1-carboxylic acid ( ACC)to ethylene in air-dried soils.
( Frankenberger and Phelan, 1985 )
1
10
20
30
40
50
0 3 4 5 6 72
24
Time of Incubation ( days)Time of Incubation ( days)
CH
Rele
ased
(m
mo
l/kg
so
il)
CH
Rele
ased
(m
mo
l/kg
so
il)
Pico soilKitchen Creek SoilAltamont Soil
Pico soilKitchen Creek SoilAltamont Soil
0
5
15
20
10
20
-75
-150
0
75
15105
Days after FloodingDays after Flooding
Redox
Enzyme
Re
do
xP
ote
ntia
l(m
v)
Re
do
xP
ote
ntia
l(m
v)
De
hyd
rog
en
ase
Activity
(m
gtr
iph
en
ylfo
rma
za
n/
10
gso
il/
24
h)
De
hyd
rog
en
ase
Activity
(m
gtr
iph
en
ylfo
rma
za
n/1
0g
so
il/2
4h
)
Relationship between degydrogenase activityand redox potential in flooded soils.
Relationship between degydrogenase activityand redox potential in flooded soils.
( Chendrayan and Sethunathan, 1980 )( Chendrayan and Sethunathan, 1980 )