Further investigations showed that the addition of Fe and or Mn, as ions or oxides influenced significantly the
formation and the resulting enzymic properties of urease– tannic acid complexes Gianfreda et al., 1995a,b. The
purpose of the work reported here was to compare the ability of Fe and Mn as ions and oxides, to promote the formation
of complexes of tannic acid and acid phosphatase and to affect their catalytic properties. Acid phosphatase was
chosen as it is one of the most studied enzymes in soil because of its essential role in the phosphorus cycle Speir
and Ross, 1978. The influence of the clay montmorillonite on the enzymic properties of the various complexes was also
investigated.
2. Materials and methods
2.1. Chemicals Acid phosphatase P EC. 3.1.3.2, from potato,
MW , 100 kDa, Type I, 60 U mg
21
was purchased from Boehringer Mannheim, Germany. Proteinase K EC
3.4.21.14, about 20 Anson U g
21
was purchased from Sigma Chemical Co., St Louis, Mo, USA. Tannic acid T
MW 1701.23 was a reagent from Fluka AG. All the other chemicals were reagent grade and were supplied by Analar,
BDH, Ltd Poole, UK.
2.2. Inorganic components An Al-substituted hematite a-Fe
2
O
3
was synthesised and characterised as described by Colombo et al. 1994.
The analysis by X-ray dffraction of a commercial MnO
2
indicated the presence of peaks at 31.4, 24.1, 21.3 and 16.3 nm which are characteristic of pyrolusite g-MnO
2
Brindley and Brown, 1980. A Wyoming montmorillonite M Source Clay Minerals
Repository, University of Missouri-Columbia, USA was ultrasonically dispersed and the fraction
,0.2 mm was sepa- rated by sedimentation Gianfreda et al., 1993. The clay
was Na-saturated with 0.1 M NaCl solution, then washed with deionized distilled water and dialysed until Cl
2
free. 2.3. Phosphatase complexes
Phosphatase–tannic acid complexes P–T were prepared at 30
8C in the presence or absence of different inorganic components. Usually, 1 ml of 0.1 M Na-acetate buffer solu-
tion at pH 5.0 containing 0.02 mg of acid phosphatase, 0.2 mg of tannic acid [TP w:w of 10] either 1 mM
FeCl
3
and MnCl
2
cation Fe
3 1
or Mn
2 1
tannic acid acid molar ratio 0.4 or 10 mg of Fe
2
O
3
and MnO
2
were incu- bated at 30
8C for 1 h. Phosphatase–tannic acid–Fe and –Mn chloride or oxide complexes were also prepared in
the presence of montmorillonite at the ratio tannic acid montmorillonite TM w:w of 0.1. After incubation
the residual activity of the suspensions were assayed before centrifugation at 10,000g for 30 min. The pellets,
consisting of the insoluble complexes, were collected, washed twice with 1 ml acetate buffer at pH 5.0 and
resuspended in an equal volume of buffer. The activities of the insoluble complexes, supernatant fractions and
washings were measured.
The insoluble complexes were usually used immediately after preparation but, if necessary, were stored at 10
8C and pH 5.0 and their residual activity periodically measured.
When the enzymatic activity had declined by more than 20 with respect to that of the initial preparation, new
complexes were prepared.
2.4. Phosphatase assay The activity of free and immobilised phosphatase was
assayed with
1 ml of 6 mM p-nitrophenylphosphate
pNPP in 0.1 M Na-acetate buffer at pH 5.0 and 30 8C.
After 20 min incubation 1 M NaOH was added and the concentration of p-nitrophenol was determined by measur-
ing the adsorbance at 405 nm with a spectrophotometer. To avoid interference by turbidity, the samples were centri-
fuged at 3000g for 15 min prior to measurement.
One enzymatic unit was defined as the mmol of p-nitro- phenol produced by 1 ml of free or immobilised enzyme in
1 min at 30 8C and at pH 5.0. The specific activity was
expressed as the units measured per mg
21
of protein. The dependence of activity on temperature was obtained
using the range 10–60 8C and the activation energy E
a
was calculated by plotting the log of activities of free and immo-
bilised phosphatase vs. 1T in K according to the Arrhe- nius equation. The value of activation energy was obtained
by a computed linear regression analysis of the experimental data. The enthalpies of activation DH
a
were evaluated as the slopes of the curves obtained by plotting log activityT K
vs. 1T Segel, 1975. 2.5. Kinetic tests
The kinetics of free and immobilised phosphatase were determined by activity assays at 30
8C and pH 5.0 with pNPP substrate concentrations ranging from 0 to 6 mM. The
kinetic parameters V
max
and K
m
were evaluated, according to Michaelis–Menten method Segel, 1975, by a computed
non-linear regression analysis. 2.6. Stability studies
The stability of free and immobilised phosphatase in the presence of proteinase was studied by determining the resi-
dual enzymatic activity after 24 h-exposure to a bacterial protease 500:1, ratio of proteinase K to phosphatase activ-
ity at 37 8C and pH 5.0. Controls were performed in the
absence of proteinase. Enzyme thermal stability was measured at 60
8C over 2 h incubation time. At predetermined time intervals, suitable
volumes of free phosphatase solution or immobilised
M.A. Rao, L. Gianfreda Soil Biology Biochemistry 32 2000 1921–1926 1922
enzyme suspension were withdrawn and the residual activ- ity assayed under standard conditions.
All experiments were carried out in triplicate.
3. Results and discussion
