Microbiology Research - Academic Journals

Enzyme assay
Triplicate samples of field moist soil (1 g) were treated with toluene
(0.5 ml) in Universal bottles closed with screw caps and left for 15
min. The enzyme reaction was initiated by addition of Tris HCl
buffer (3 ml, 0.2 M, pH 8.3), L-cysteine (5 mm, 1 ml) and pyridoxal
phosphate, 0.2 mM, 1 ml). The contents of the bottles were
thoroughly mixed and the bottles were incubated at 37°C. After 2 h,
the enzyme reaction was stopped by addition of trichloroacetic acid
(TCA, 10% w/v). Two sets of controls were included a) where no
cysteine was added and b) where the enzyme reaction was
stopped immediately. The soils suspensions were filtered through
Whatman No.1 filter paper and CDA activity was measured by the
determining the concentration of pyruvate as follows:
Determination of pyruvate
To the filtrate (0.5 ml) was added TCA, a 0.3 ml (of 50% w/v
solution), distilled water (2.2 ml) and 2,4-dintrophenylhydrazine (1
ml, of a 1% solution in 2 M HCl); mixed and left for 10 min at room
temperature. Sodium hydroxide (5 ml of a 2.5 N solution) was then
added, and after 10 min incubation at room temperature, the
reddish brown colour formed was measured at 445 nm. The
pyruvate concentration was then determined by reference to a
standard curve c ranging from 0-0.1 µmoles pyruvate (Case, 1932).
Development of the CDA assay
By using the basic assay described above, and varying one
parameter at a time, the optimum conditions for the assay of CDA in
this soil was determined. The following were determined: a) the
optimum amount of soil; the reaction mixture was incubated for 2 h
at 37°C with one of the following, 0, 0.5, 1.0, 2.0, 3.0, 4.0 g of soil;
b) period of incubation; the reaction mixture was incubated at 37°C
for 0, 2, 4, 8, 15, 25 hours; c) substrate concentration, L-Cysteine
concentration of 0, 0.4, 0.8, 1.0, 1.5, 2.0, 3.0, and 4.0 mM; d) effect
of temperature on CDA, reaction mixtures were incubated for 2 h at
10, 20, 25, 40, 50, 60, and 80°C; e) effect of pH- on CDA was
assayed using Tris-HCL buffer over the following pH range, 7.0 ,7.5
,8.0 , 8.5, 9.0, 10.0.
Determination of pyridoxal phosphate in soil
Soil (10 g) was shaken for a period of 1 h with Tris-HCl buffer (0.2
M pH 8.3, 100 ml) and then filtered through a Whatman No. 1 filter
paper. The concentration of pyridoxal phosphate in the filtrate was
then determined by the following method (Wada and Snell, 1961).
Phenlyhyrdazine hydrochloride (0.2 ml, 2 % w/v dissolved in 10 N
H2SO4) was added to 3.8 ml of soil filtrate. The mixture was then
heated at 60°C for 20 min. and then allowed to stand at room
temperature for 10 min, and the intensity of the colour formed was
read at 410 nm.
RESULTS AND DISCUSSION
Linearity was observed between CDA and a) the amount
of soil used (0 - to 0.75 g), b) length of incubation (0 - to
3.5 h.) and c) the concentration of L-cysteine (0.5- to 1.8
mM), (Figures 1a, b and c); showing that the assay
Alharbi et al. 5087
method employed measures the hydrolysis of L-cysteine
and that the measured enzyme activity was not limited by
any of the parameters employed.
The optimum temperature for CDA in this soil was 60°C
(Figure 2a) which is higher than that reported by
Fromageot (1951) for the enzyme in bacteria and
mammals. Soil enzymes generally show a higher optimum
temperature than is observed for pure enzymes, or
when enzyme activity is measured from cells; this is
because soil enzymes are immobilized onto clays and
humus particles (Burns, 1979; Sarkar et al., 1989) .The
optimum pH for CDA in soils was pH 8.5 (Figure 2b). This
pH optimum is the same as that found for Salmonella
typhimirium (Guarneros and Ortega, 1970), but
somewhat higher than that found in other bacteria
(Fromageot, 1951); again because of soil immobilization
soils; enzymes often show broader and higher pH
maximum than enzyme activities measured in other
systems. Table 1 shows and important property of CDA,
namely that pyridoxal phosphate is needed in order for it
to exhibit its maximum activity. Stimulation of CDA by
pyridoxal phosphate was also reported for this enzyme
from Proteus morganii (Kallio, 1951) and E.coli
(Delwiche, 1951).
Pyridoxal phosphate was not detected in this
agricultural soil [1:10 w/v soil 0.2 M Tris-HCL buffer (pH
8.3), extracted by shaking for 1 h], showed that either
pyridoxal phosphate is not extracted by the method, or
more probably that it is not present in detectable concentrations
in this soil. The lack of pyridoxal phosphate in this
soil means that CDA could not function in this soil (and
presumably in most other soils also) at its maximum
activity because of the lack of a necessary cofactor,
namely pyridoxal phosphate. Burns (1979) emphasised
that cytoplasmic enzymes from animals, plants and
microorganism, which rely upon co-factors, electron
transport chains or multi enzyme complexes will not
operate in soils unless such cofactors are present. CDA
provides an excellent example of an enzyme which is
present is soil, probably bound to humus and clay
particles which, because of a lack of necessary cofactors
cannot function in vivo. Activity of the enzyme can
however, be measured in vitro when the necessary
cofactors (in this case pyridoxal phosphate) is added.
The present study therefore illustrates the important point
that just because an enzyme can be assayed in a soil it
does not necessarily mean that it functions in the
environment. Enzymes such as cellulase (Benefield,
1971), urease (Bremner and Mulvaney, 1978) and ophenol
oxidase (Wainwright, 1979), which do not require
cofactors would probably exhibit maximum activity under
environmental conditions. The important conclusion
which can be derived from this study is that although
certain soil enzymes (like CDA) can be assayed in the
laboratory where all cofactors are provided, this does not
mean that they will function in the environment and play a
role in mineral cycling.