Importance of Soil Enzymes

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					Importance of Soil Enzymes

1. Release of nutrients in soil by
 means of organic matter
 degradation

2. Identification of soils

3. Identification of microbial activity

4. Importance of soil enzymes as
sensitive indicators of ecological
change
           ENZYME CLASSIFICATION




1. Oxidoreductases - Oxidation reduction reaction
      (Dehydrogenase, Catalase, Peroxidase)

2. Tranferases - The transfer of group of atoms from
      donor to an acceptor molecule.
         (Aminotransferases, Rhodonese)
3. Hydrolases - Hydrolitic cleavage of bonds.
     (Phosphatase, Cellulase, Urease)

4. Lyases - Cleavage of bonds other than hydrolysis
      or oxidation.
                    (Aldolase)

5. Isomerases - Isomarization reaction.

6. Ligases - Formation of bonds by the cleavage of
      ATP.
              (Acetyl-CoA carboxylase)
                 Kinds of Enzymes




                     Constitutive
Always present in nearly constant amounts in a cell (not
affected by addition of any particular substrate...genes
always expressed.)
                    (pyrophosphatase)

                       Inducible
Present only in trace amounts or not at all, but quickly
increases in concentration when its substrate is present.
                       (Amidase)

          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
       Complexes
                            +
        STATE OF ENZYMES IN SOIL

  Role of Clays

a. Most activity associated with clays.

b. Increases resistance to proteolysis and
   microbial attack

c. Increases the temperature of inactivation.

  Role of Organic Matter

a. Humus material provides stability to soil
   nitrogen compounds

b. Enzymes attached to insoluble organic
   matrices exhibit pH and temperature
   changes.

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
   alone.

b. Enzymes are bound to organic matter which
   is then bound to clay.
    Adsorption                               Entrapment
               Enzyme                                      Enzyme


                                     Polymer
         Carrier

  Microencapsulation                        Ion exchange
                                                            Enzyme
                     Enzyme
                                             + ++++ + +
                                            R RRRR R R




                              Adsorption and cross-linking
Membrane

   Cross-linking                                      Enzyme

                                  Carrier
               Enzyme


                                 Covalent attachment
   Copolymerization                                      Enzyme
Enzyme

                                            Carrier
           Polymer


             Schematic representation of methods of
                  immobilizing enzymes.
                    ( Weetall, 1975 )
                                                 vvv
                          E                   vv
                                    vv vvv               v           vN
       N+vvvvvvvvvv            N+               vvvvvvvv




 v
                                                                                   vvv




 vv
                                                                                vv

       N+                     N+




  vv
                                                         vv
                                                                                 vvv
                     vv vvv          vv                v




    vv
                                   v                          v
                                                                          N+
                                                                              vv

                 v vv vv                 vv                          v




     vv
                                       v                          vv
         N                N+                                              N+
               vv vvv                        vv
             vv v                          v                             vv
                                                                                  E-
                          +
                                                                          vv




              vv vvvvN           vv vvvN+ vvvN
                             vvv      vv    vvv                                  0.9 nm
                          vv        v
                                                                             vvv




           vvvvvvv
          v v
 E - N vvv vv        N+vv       v vv N+ vvv
                                                                               vv vvv




             N                  vvvvvvv N                +
  vv vvvvvvv
                        Nvv
                              vvvvv               vvvv N
                                    vvv   vv vvvv
                                               E+

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

   concentrations .

4. Its optimum pH .

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.
* 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
         SOIL STORAGE
          Methodology


              Air Dry

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.

          Heat Treatment

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.
      K1        K3
E+S        ES        E+P
      K2
   Initial Velocity          A




                      Enzyme



                         B


                                                Zero order
                                           (substrate independent)
Initial Velocity




                        First order (substrate dependent)



                      Substrate
       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
     rapidly achieved.

4.   Enzyme total concentration defined as free and in
     complex state.

                      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
                       k1                k2
             E+S                    ES          E+P
                         k -1

             v = k 2(ES)                 vmax = k 2 (ET )

1.   Rate of formation of ES
        d (ES)
               = k 1 (E f )(S)  but E f = (E T - ES)
           dt
               = k1 (E T - ES)(S)
2.   Rate of breakdown of ES
      - d (ES) = k (ES) + k (ES)
                  -1       2
           dt

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
                (ES)                      k1
5.   Rearranging again
                       (E T )(S)
        (ES) =
                       K m+ (S)
6.   Multiply by k 2

           k 2 (ES) = k (E T )(S)
                       2
                         K m+ (S)
7.   But
           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
                                              max




                          v vs. v / S = Eadie-Hofstee plot
                          S / v vs. S = Hanes-Woolf plot
       SOIL ENZYMOLOGY
           Methodology


             Problems


No way to separate extracellular from
intracellular activity

Presence of recently secreted free
enzymes accumulated in soil.

Separation between chemical and
biological catalysis.

Storage and treatment of soils greatly
affects enzymatic activity.
  Ideal -   Inhibit microbial activity without cell
            lysis or extracellular enzyme inhibition.


                       Toluene

Advantages

   Stops synthesis of enzymes by living cells.

   Prevents assimilation of products of enzymatic
   reactions. (Important to study individual reactions.)

Disadvantages

   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
            ecosystem.


     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.
  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)

100


80


60                                                        BENTONITE
                                                          KAOLINITE
40                                                        SANDY LOAM SOIL
                                                          SILT LOAM SOIL

20


 0
      LACCASE   TRYROSINASE       ACID          B-D-
                              PHOSPHATASE   GLUCOSIDASE
                                                           Acid Soils
                                                           Webster ( pH 5.8 )
                                                           Nicollet ( pH 6.1)
                                             250
( ug p-nitrophenol released / g soil / h )




                                                            Alkaline Soils
                                                           Ida ( pH 8.0 )
                                                           Harps ( pH 7.8 )
                                             200
        Phosphatase Activity




                                             150



                                             100



                                             50



                                               0
                                                     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 )




                                                     Pico soil
                                     50




                                                     Kitchen Creek Soil
                                                     Altamont Soil
                                     40
                                     30
                                     20
                                     10




                                          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 )




                                                                       20                                                 75
                                                                                                                 Enzyme
Dehydrogenase Activity




                                                                                                                                 Redox Potential (mv)
                                                                                         Redox
                                                                       15
                                                                                                                          0

                                                                       10

                                                                                                                          -75
                                                                        5

                                                                                                                          -150

                                                                        0            5           10        15        20

                                                                                           Days after Flooding



                                                                            Relationship between degydrogenase activity
                                                                                and redox potential in flooded soils.
                                                                                ( Chendrayan and Sethunathan, 1980 )