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 .

Sinigrin + myrosinase --> glucose + SO42- + isothiocyanates

Myrosinase Activity in Soil

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.

E + S ES E + PK K

K

1 3

2

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 )