Importance of Soil Enzymes
1. Release of nutrients in soil by
means of organic matter
2. Identification of soils
3. Identification of microbial activity
4. Importance of soil enzymes as
sensitive indicators of ecological
1. Oxidoreductases - Oxidation reduction reaction
(Dehydrogenase, Catalase, Peroxidase)
2. Tranferases - The transfer of group of atoms from
donor to an acceptor molecule.
3. Hydrolases - Hydrolitic cleavage of bonds.
(Phosphatase, Cellulase, Urease)
4. Lyases - Cleavage of bonds other than hydrolysis
5. Isomerases - Isomarization reaction.
6. Ligases - Formation of bonds by the cleavage of
Kinds of Enzymes
Always present in nearly constant amounts in a cell (not
affected by addition of any particular substrate...genes
Present only in trace amounts or not at all, but quickly
increases in concentration when its substrate is present.
Both enzymes are present in the soil.
ORIGIN OF SOIL ENZYMES
1. Microorganisms -
Living and dead
2. Plant Roots and Plant Residues
3. Soil Animals
STATE OF ENZYMES IN SOILS
1. Role of Clays
2. Role of Organic Matter
3. Role of Clay - Organic Matter
STATE OF ENZYMES IN SOIL
Role of Clays
a. Most activity associated with clays.
b. Increases resistance to proteolysis and
c. Increases the temperature of inactivation.
Role of Organic Matter
a. Humus material provides stability to soil
b. Enzymes attached to insoluble organic
matrices exhibit pH and temperature
c. Inability to purify soil enzymes free of
soil organic matter ( bound to O.M. )
Role of O.M. - Clay Complex
a. Lignin + bentonite ( clay ) protect enzymes
against proteolitic attack, but not bentonite
b. Enzymes are bound to organic matter which
is then bound to clay.
Microencapsulation Ion exchange
+ ++++ + +
R RRRR R R
Adsorption and cross-linking
Schematic representation of methods of
( Weetall, 1975 )
vv vvv v vN
N+vvvvvvvvvv N+ vvvvvvvv
vv vvv vv v
v vv vv vv v
N N+ N+
vv vvv vv
vv v v vv
vv vvvvN vv vvvN+ vvvN
vvv vv vvv 0.9 nm
E - N vvv vv N+vv v vv N+ vvv
N vvvvvvv N +
vvvvv vvvv N
vvv vv vvvv
A model for binding urease to hydrophobic HDTMA smectite.
The dark site of the enzymes are hydrophobic areas.
(HDTMA - hexadecyltrimethylammonium bromide -
serves as a cation exchange support.)
QUANTITATIVE ASSAY OF ENZYMATIC ACTIVITY *
Things we must know.
1. The overall stoichiometry of the reaction catalysed.
2. Whether the enzyme requires the addition of
cofactors such as metal ions or coenzymes.
3. Its dependence on substrate and cofactor
4. Its optimum pH .
5. A temperature zone in which it is stable and has
6. A simple analytical procedure to measure the
disappearance of substrate or the appearance
* Usually measure enzyme activity at substrate concentrations
above saturation level, where the reaction rate is at a maximum.
Myrosinase Activity in Soil
Sinigrin + myrosinase --> glucose + SO42- + isothiocyanates
Enzyme concentration declines in the
absence of renewed synthesis.
Once dry, enzyme activity is maintained
at 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 a
longer time and a higher temperature than
enzymes in solution.
E+S ES E+P
Initial Velocity A
First order (substrate dependent)
MICHAELIS - MENTEN ASSUMPTION
1. The rate of an enzyme catalyzed reaction changes
from first order to zero order kinetics.
2. Enzyme (E) reversibly binds with substrate (S) to form
an intermidiate (ES) complex which then breaks down
to form product (P). Each reaction is described by a
specific rate constant: k 1, k2 , k 3.
3. A steady state equilibrium between the rate of
formation and the rate of degradation of ES is
4. Enzyme total concentration defined as free and in
E T = E + ES
5. Initial rate limiting parameter is the decomposition
of the enzyme / substrate (ES) complex from the
product k3 .
v ~ ES
6. V max when ES complex reaches a maximum
saturation (no free enzyme).
DERIVATION OF THE MICHAELIS - MENTEN EQUATION
E+S ES E+P
v = k 2(ES) vmax = k 2 (ET )
1. Rate of formation of ES
= k 1 (E f )(S) but E f = (E T - ES)
= k1 (E T - ES)(S)
2. Rate of breakdown of ES
- d (ES) = k (ES) + k (ES)
3. Setting the rates equal to each other
k 1 (E T - ES)(S) = k -1 (ES) + k 2 (ES)
4. Rearranging equation 3
(S)(E T - ES) k -1 + k 2
= = km
5. Rearranging again
(E T )(S)
K m+ (S)
6. Multiply by k 2
k 2 (ES) = k (E T )(S)
K m+ (S)
k 2 (ES) = v
k 2 (E T ) = v max
v = vmax (S)
Michaelis - Menten Equation
K m + (S)
8. Lineweaver - Burk transformation
1 Km 1 1
v = v max ( ) (S)
v vs. v / S = Eadie-Hofstee plot
S / v vs. S = Hanes-Woolf plot
No way to separate extracellular from
Presence of recently secreted free
enzymes accumulated in soil.
Separation between chemical and
Storage and treatment of soils greatly
affects enzymatic activity.
Ideal - Inhibit microbial activity without cell
lysis or extracellular enzyme inhibition.
Stops synthesis of enzymes by living cells.
Prevents assimilation of products of enzymatic
reactions. (Important to study individual reactions.)
Acts as a plasmolytic agent, releasing cell
contents and intracellular enzymes.
Destroys dehydrogenase activity.
APPLICATION OF SOIL ENZYMES
1 * Correlation with soil fertility.
2 *Correlation with microbial activity.
3 *Correlation with biochemical cycling of
various elements in soil ( C, N, S ).
4 Degree of pollution ( heavy metals, SO 2 ).
5 To assess the successional stage of an
6 Forensic purposes.
7 Rapid degradation of pesticides.
8 Disease studies.
* Correlation not good because the source of enzymes varies, and
complexes with O.M., and clay limits substrate atack by the
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 in
soil. Soil Sci. Soc. Am. J. 47:945-951.
Immobilization of enzymes on pretreated clays and soils.
(Sarkar et. al., 1989)
40 SANDY LOAM SOIL
SILT LOAM SOIL
LACCASE TRYROSINASE ACID B-D-
Webster ( pH 5.8 )
Nicollet ( pH 6.1)
( ug p-nitrophenol released / g soil / h )
Ida ( pH 8.0 )
Harps ( pH 7.8 )
4 6 8 10 12 14
pH of Buffer
Phosphatase activity in acid and alkaline soil
( Eivazi and Tabatabai, 1977 )
C 2 H4 Released ( mmol / kg soil )
Kitchen Creek Soil
0 1 2 3 4 5 6 7
Time of Incubation ( days)
Conversion of 1-aminocyclopropane-1-carboxylic acid ( ACC)
to ethylene in air-dried soils.
( Frankenberger and Phelan, 1985 )
( mg triphenylformazan / 10 g soil / 24 h )
Redox Potential (mv)
0 5 10 15 20
Days after Flooding
Relationship between degydrogenase activity
and redox potential in flooded soils.
( Chendrayan and Sethunathan, 1980 )