Soil Enzymes
E + S ES E+P
Definition of Enzymes
Enzyme (E) – A protein produced by a
cell to act as a catalyst
Substrate (S) – A compound acted upon
by an enzyme
Enzymes are proteins that allow reactions to
proceed at faster rates by reducing the energy of
activation of the reaction:
Substrate
Product
Enzyme +
Substrate Enzyme
Substrate
Sources of Enzymes in Soil
Plants, soil animals, and microorganisms
Enzymes accumulated in soils are present as:
Free enzymes (Exoenzymes)
Endoenzymes (cytoplasm, periplasm)
Kiss et al. 1975
Types of Enzymes
Constitutive enzymes
Used frequently in the cell – always present
Inducible enzymes
In the genetic code – only produced when
needed
State of Soil Enzymes
Enzymes may be destroyed by proteases
Enzymes may be protected by clay or humus
Enzyme-clay and enzyme-organic polymer
complexes resistant to denaturation
Categories of soil enzymes
1. Enzymes associated with living, metabolically active
cells in soil; found in cell’s cytoplasm, bound to cell
wall, or as extracellular enzymes that have been
recently produced by the cell
2. Enzymes associated with viable but non-proliferating
cells (such as spores, etc.)
3. Enzymes that are attached to dead cells or to cell
debris, or which have diffused away from dead/dying
cells that originally produced them.
4. Enzymes that are “permanently” immobilized on soil
clay & humic colloids
-Such enzymes can remain active for long periods of
time
-Such immobilized soil enzymes can arise from either
eukaryotic or prokaryotic cells
Location of Enzymes in Soils
Dead intact
Resting cell (vi)
(i) Intracellular enzymes structure
(ii) Periplasmatic enzymes (V )
(vii)
(iii) Enzymes attached to outer Lysis
surface of cell membranes R estin g stru ctu re D ead intact cell
(iv) Enzymes released during Living cell
(vi)
L iving cell
cell growth and division (iii)
(ii)
(v) Enzymes within non-
proliferating cells (spores, (i)
(I) D ead d isin tegrated Dead
cysts, seeds, endospores)
cell disintegrated
(vii) cell
(vi) Enzymes attached to dead S ecretion
(viii)
cells and cell debris (vii) E n zym Enzyme-substrate
e-su b strate
(vii) Enzymes leaking from intact com p lex
complex
Clay
C lay m inerals
cells or released from lysed E xtracellu lar en zym es
Extracellul minerals
cells ar enzymes
(viii) Enzymes temporarily
associated in enzyme- L iving cells
Living H um ic acids
substrate complexes cell E ntraped enzym es
(ix) Enzymes absorbed to Humic (x) A dsorbed enzym es (ix)
acids
surfaces of clay minerals H u m uHumus-
s-enzym e com p lex C lay-en zymClay-
e com p lex
(x) Enzymes complexed with enzyme enzyme
complex complex
humic colloids
Prepared by my colleague Dr. Klose
according to Burns 1982, Nannipieri 1994
Soil enzyme activity
• Can be used as assay of microbial community
enzyme activities
• Have been used to assay impact of
environmental stresses on soil microbial activity
• Assays of soil enzyme activities sometimes
designed to measure enzyme activities of
enzymes both inside & outside of cells; or only
enzymes that are outside of cells (= soil-bound
enzymes)
Soil enzyme assays…
E+S ES E+P • Are based mostly on the
quantification of the product (P)
+ + released upon adding known
I I amount of substrate (S) to the
soil, and allowing the reaction to
EI + S ESI occur under controlled
temperature, pH
and ionic strength.
E = Enzyme;
S = Substrate; • If an inhibitor (I) (i.e., metal) is
present in the soil, EI and/or ESI
I = Inhibitor ; complexes could be formed in
P= Product. addition to the ES (enzyme-
substrate) complex normally
formed.
Soil enzyme assays…
• To assay the activity of an immobilized soil
enzyme, a known amount of soil can be
combined with a known concentration of
substrate, & rate of product formation resulting
from enzymatic reaction can be determined
• Such assays must try to separate/differentiate
extracellular enzyme activity from enzyme activity
associated with living organisms
To help achieve such separation/differentiation:
1. Chemicals (such as toluene) which inhibit
microbial proliferation can be added to soil
2. Duration period of enzyme assay is kept short,
to reduce chance of an increase in microbial
numbers
3. Attempts are made to keep microbial cells in
sample intact. Is not desirable to have cells in
sample become leaky, so that cytoplasmic
enzymes leak out into surrounding soil
Enyzme Classification
• Oxidoreductases
Oxidation reduction reactions
• Transferases
Transfer of a group from one molecule to another
• Hydrolases
Hydrolysis reactions
• Isomerases
Rearrangement reactions
• Lyases
Removal or addition of groups to form a double
bond
• Ligases
Attaching groups to a molecule using the
hydrolysis of ATP as the source of energy.
Selected enzyme reactions
Carbon mineralization:
-Glucosidase catalyzes the final limiting step
of
HO
cellulose
HOH C
2
O degradation HOH C 2
O
HO O-R HO
+ H2O
HOH 2C OH HO OH + R-OH
O -glucosidase HOH 2C OH
HO O
HO O-R HO
+ H2O
p-nitrophenyl--D-glucoside
OH -glucoside
HO
p-nitrophenol
OH + R-OH
OH
p-nitrophenyl--D-glucoside -glucoside p-nitrophenol
R= NO 2
R= NO 2
Selected enzyme reactions
Nitrogen mineralization
Aminization Ammonification Nitrification
RNH2 NH4+ NO2- NO3- + energy
i.e.=Proteins
Amino acid mineralization in soils
(begins by the release of amino acids
from organic matter)
Amino acid Ammonification Nitrification
Amidohydrolases
Arylamidase Amino acid NH4+ NO2- NO3- + E
activity:
activity (RNH2)
e.g.,
L-Glutaminase activity
L-Asparaginase activity
L-Aspartase activity
Hypothetical structure of humic acid
(Stevenson)
Suga
r
Peptide
Arylamidase Activity
NH2
O
NHCCHCH2CH(CH3)2
-Naphthylamine
NH2 Arylamidase
+ H2O
+
HOOC
CHCH2CH(CH3)2
L-Leucine -naphthylamide NH2
(Substrate for assay)
Leucine
L-Asparaginase activity
COOH COOH
+ H 2O L-Asparaginase
HC NH2 HC NH2 + NH3.
CH2 CH2
CO COOH
NH2
L-Aspartase activity
COOK COOK
L-Aspartase
HC NH2 + H2O CH + NH3.
CH2 CH
COOH COOH
L-Glutaminase activity
COOH COOH
H C NH2 H C NH2 + NH3.
+ H 2O L-Glutaminase
CH2 CH2
CH2 CH2
CO COOH
NH2
Amidase activity
Amidase
RCONH2 + H 2O NH3 + RCOOH
-Glucosiminidase Activity in Soils
HO
O HO
O O
HO
NH
O-R + H 2O
-Glucosaminidase HO
HO OH + R-OH
NH
C=O
C=O
CH3
CH3
p-Nitrophenyl-N-acetyl- -D- glucosaminide N -acetyl- -D-glucosaminide p -nitrophenol
(Substrate for assay)
R= NO2
Urease activity
O
Urease
NH2C NH2 CO2 + 2NH3
H2O
Selected enzyme reactions
Phosphorus mineralization
O O
Acid or alkaline
RO P O + H 2O phosphatase HO P O + ROH
O O
OH OH
O P OR1 + H 2O Phosphodiesterase O P OH + R1OH
OR2 OR2
Selected enzyme reactions
Sulfur mineralization
HOH 2C
O HOH 2C
HO O
HO O-R HO
+ H2O
_
+ H2O Arylsulfatase HO ROH + H
OH +
+
SO4 2-.
+ R-OH
ROSO3 OH
OH
Arylsulfatase is believed to be partly
p-nitrophenyl--D-glucoside responsible
-glucoside for S
p-nitrophenol
cycling in soils as it is involved in the mineralization of
organic sulfur compounds to inorganic forms (SO 42-) for plant
uptake (Tabatabai, 1994).
R= aryl group
R= NO 2
Substrate for assay= p-nitrophenyl sulfate
Immobilized soil enzyme…
Proteins/enzymes that bind to clay minerals
- Are not as catalytically active as enzymes that are
free in aqueous solution
- But are more stable to some environmental stresses
such as temperature extremes; digestion by
proteases; etc. than enzymes that are free in
aqueous solution
Immobilized soil enzyme…
Binding of enzymes to soil surfaces (esp.
clay & humic materials) may take place
via:
- Ionic interactions
- Covalent bonds
- Hydrogen bonding
- Entrapment of enzyme by soil colloids
- Other mechanisms
Enzymes associated with clay minerals
Immobilized soil enzyme…
• Immobilized soil enzymes can bring about very rapid
(‘immediate”) changes in substrate molecules which
are added to soil
• In such cases, little or no lag period may be noted
before enzyme activity becomes apparent
• Compare this to cell-associated enzymes, which often
need to be induced, so that minutes to hours often
pass before cells produce a certain enzyme, causing a
measurable effect on a specific substrate
EX.: Work of Donald Kaufman
• -Revealed accelerated degradation of
some pesticides in some soils of
midwestern USA
• -Research indicated that some soils can
become “conditioned” to degrade
pesticides more & more rapidly, especially
if same pesticide treatment is used year
after year
2 groups of pesticides were affected by this:
1) Carbofuran insecticides 2) Thiocarbamate herbicides
(tradename Furadan) (tradename Eradicane; etc.)
which are carbamates
• Kaufman’s work suggested this phenomenon may
be due to accumulation of immobilized soil
enzymes which are able to degrade the pesticides
• Such immobilized soil enzymes would
accumulate/build up in soil, in addition to enzymes
of living microbes which degrade the pesticides
R.G. Burns hypothesis on ecological role(s) of soil
enzymes originating from microbes:
• -Suggests that these enzymes serve as an existing
“enzyme shell” in the soil surrounding the microbe
which produces the enzyme
• -1) Immobilized soil enzyme would immediately
respond to available substrate & transform it so that it
is available for cell uptake. NOTE that there would no
need to wait for enzyme induction within the cell
before substrate could be acted on
• 2) Immobilized soil enzymes may cleave off small
fragments (“inducer” molecules) from large polymeric
substrates in soil. The inducer molecule would then be
able to enter the nearby cell, & trigger the synthesis of
inducible extracellular enzymes in large amounts
Chemical changes occurring in soil may be due to:
• 1) living eukaryotic & prokaryotic cells
• 2) immobilized enzymes on clay & organic matter
surfaces
• 3) strictly abiologic chemical reactions
• -In doing experiments, sometimes necessary to
differentiate between these 3; at least differentiate
between (3) & the biologic causes
• Sterile soil controls are useful in many
soil biology/soil biochemistry/soil
microbiology studies to evaluate
whether phenomena are due to
biology or other causes
• To sterilize soil, may use autoclaving;
gamma irradiation; etc.
• Some potential biotechnological importance
of immobilized soil enzymes
• Release of such enzymes into soil
environment would involve few or no
restrictions, unlike extensive restrictions
place don release into environment of
genetically modified/engineered
microorganisms (GMOs)
Immobilized enzymes in biotechnology:
• -Some processes in biotechnology make use of
enzymes that are immobilized on solid supports
• -This mimics/is similar to soil enzymes that are
bound to soil surfaces
References
Burns, R.G. (1982). Enzyme activity in soil: Location and a possible role in
microbial ecology. Soil Biol. Biochem. 14: 423-427.
Kiss, S., Dracan-Bularda, M., and Radulescu, D. (1975). Biological
significance of enzymes accumulated in soil. Adv. Agron. 27: 25-87
Nannipieri, P. (1994) The potential use of soil enzymes as indicators of soil
productivity, sustainability, and pollution. In: Soil biota: management in
sustainable farming systems.
Tabatabai, M. A. (1994). Soil enzymes. In “Methods of Soil Analysis” (R.
W. Weaver, J. S. Angle, and P. S. Bottomley, eds.). Part 2.
Microbiological and biochemical properties. p. 775-833. SSSA Book
Series No. 5, Soil Sci. Soc. Am., Madison, WI.
