Folia Microbiol. 54 (6), 505–508 (2009) http://www.biomed.cas.

cz/mbu/folia/

Modulation of Enzyme Activities of a Lead-Adapted Strain

of Rhizopus arrhizus during Bioaccumulation of Lead
S. BHATTACHARYYAa *, T.K. PALb, A. BASUMAJUMDARa
aDepartment of Chemical Technology, University of Calcutta, Kolkata 700 009, India

e-mail sauryya@hotmail.com
bDepartment of Biotechnology, Bengal Institute of Technology, Kolkata 700 150, India

e-mail tapankpal@gmail.com

Received 13 January 2009

Revised version 24 June 2009

ABSTRACT. Activity of some enzymes of a Pb-adapted strain of Rhizopus arrhizus augmented signifi-
cantly during bioaccumulation of Pb from a chemically defined medium. The mycelium of a Pb-adapted strain
exposed to 1 μg/mL Pb in a defined medium for 10 d at 28 ± 2 °C exhibited, relative to a wild-type strain,
increased activities of antioxidant enzymes, i.e. SOD, CAT and GPX and of enzymes like AP and PPO in-
volved in defense against pathogens. Another response is a significant increase in the cellular thiol content
after 4 d. The responses to Pb exposure thus include an increase in the antioxidant and anti-pathogen defense,
and an increased metal-chelating ability.

Abbreviations
AP acid phosphatase PPO polyphenol oxidase
CAT catalase SOD superoxide dismutase
GPX glutathione peroxidase WT wild-type (strain)
NBT nitroblue tetrazolium

Rhizopus arrhizus, a common filamentous fungus (class Zygomycetes, family Mucoraceae), was
found to remove a few toxic heavy metals in a metabolism-dependent manner from aqueous culture medium
in laboratory conditions and the capacity of metal uptake, especially Pb, could be increased when the fungus
was adapted to higher concentrations of the metal (Bhattacharyya et al. 2002). Modulation of the physiologi-
cal conditions of fungi due to stress has been reported, particularly in reference to antioxidant defense sys-
tems (Li et al. 2008). The presence of toxic heavy metals also produces stress that ultimately affects antioxi-
dant enzyme activities of a fungus (Todorova et al. 2008).
This paper evaluates the enzyme activities of a Pb-adapted strain of R. arrhizus growing in the pre-
sence of Pb, which could be useful for identifying the metabolic and molecular effects caused by Pb. The en-
zymes include antioxidant enzymes, such as SOD, CAT, GPX, and also enzymes active in fungal self-defense,
such as AP and PPO. The consequent cellular responses would contribute to the understanding of the dif-
ferent mechanisms involved in fungal resistance to heavy metal stress.

MATERIAL AND METHODS

Chemicals used (AR grade) were purchased from Merck (India) or SRL (India) except for 5,5´-di-
thiobis(2-nitrobenzoic acid) (DTNB) and standard enzymes, which were purchased from Aldrich and Sigma.
The culture media were prepared using double-distilled water.
A high concentration of Pb-adapted R. arrhizus strain M1 was developed and maintained as descri-
bed previously (Bhattacharyya et al. 2002; Chattopadhyay et al. 1999). The WT strain was obtained from
Bioengineering Division, Department of Chemical Engineering, University of Calcutta. The strain was main-
tained on solid potato-dextrose agar slants at 4 °C and grown at 30 °C for 5 d prior to inoculation. Inoculum
was prepared by washing spores with double-distilled water. Flasks were inoculated with spore suspension
having 4.5 × 107 spores per mL. The WT strain was grown in synthetic culture medium containing 100 mg/mL
Pb (as Pb(NO3)2) for 7 d at 28 ± 2 °C; pH of the medium was 4.5. Twenty productive variants were isolated

*Address for correspondence: 87/46, A.K. Mukherjee Road, Kolkata 700 090, India.
506 S. BHATTACHARYYA et al. Vol. 54

by serial-dilution plate method and analyzed for their Pb accumulation potential. The productive strain,
R. arrhizus M1 showed maximum bioaccumulation capacity and was selected for further experiments.
Composition of the synthetic culture media (in g/L): sucrose 50, NH4NO3 2, K2HPO4 2, MgSO4·7H2O
0.5, FeSO4·7H2O 0.5, MnSO4·7H2O 0.1 (Basumajumdar et al. 2004). Sucrose was separately sterilized and
added aseptically to fermentation medium. Both the adapted and the WT strains were cultured in media con-
taining 1 μg/mL of Pb (as Pb(NO3)2) for 10 d at 28 ± 2 °C at pH 4.5. Mycelia were separated after 3–10 d;
activity of antioxidant enzymes (viz. SOD, CAT and GPX), AP, PPO, and the thiol content were estimated
as described below.
Activity of extracellular AP, PPO and thiol content were measured in cell-free culture media, SOD
activity by the NBT reduction method (Tsekova and Todorova 2002). CAT activity was assayed at 37 °C
(Isobe et al. 2006) using H2O2 as substrate, GPX activity by NADPH oxidation (monitored at 340 nm) (Na-
gai et al. 2002). AP activity was determined spectrophotometrically (Gonnety et al. 2006) using 4-nitrophe-
nyl phosphate as substrate, PPO activity was measured according to Toralles et al. (2005) using pyrocate-
chol as substrate. Thiol content was determined after incubating mycelial fraction or cell-free extract with
DTNB (Liszewska et al. 2001). Protein content was measured by the Lowry method.
All data were analyzed by one-way ANOVA
followed by Tukey’s post-hoc test for multiple com-
parisons of the treatment means. The value of p ≤
0.01 was taken as the criterion for statistically signifi-
cant difference. Calculations were done with the soft-
ware Prism 4.0 (GraphPad Software).

RESULTS

The Pb-adapted R. arrhizus strain M1 showed

distinct improvement in enzyme profile during growth
in the presence of Pb. Activities of SOD and CAT in
mycelia of this strain were significantly enhanced (p <
0.001 in both cases) after 5 d (Fig. 1 top and middle),
up to 10 d. Significantly improved activity of GPX
(p < 0.001) in the mycelia of the adapted strain was
observed after 3 d (Fig. 1 bottom) up to 10 d. Activi-
ties of the enzymes of this strain remained practically
constant for the rest of the incubation. On the other
hand, antioxidant enzyme activities in the mycelia of
the WT strain remained unaltered throughout the whole
period. When the two strains were grown in the absence
of Pb for 10 d, no significant difference was observed
in the enzymic activities.
The adapted strain showed significantly enhan-
ced mycelial and extracellular AP and PPO activity
(p < 0.001 in both cases) (Fig. 2 top and middle).
Improvement in mycelial and extracellular thiol con-
tent was also significant (p < 0.001), the improvement
being observed after 4 d (Fig. 2 bottom) till the end of
Fig. 1. Activity (U/mg protein) of SOD (top), CAT (mid-
incubation.
dle), and GPX (bottom) in the mycelia of Pb-adapted
(circles) and WT (squares) strains of R. arrhizus during
bioaccumulation of Pb from a medium containing 1 μg/mL DISCUSSION
of Pb; **p < 0.001 compared to WT.
Enhanced activities of antioxidant enzymes of
R. arrhizus M1 may indicate a role of these enzymes
in cellular adaptation to environmental stress (Tsekova and Todorova 2002). The presence of toxic heavy
metals in the culture medium could produce such stress (Guelfi et al. 2003). There is evidence that even
essential trace metals, such as Cu and Zn, can produce oxidative stress which, in turn, induces overproduc-
tion of SOD and CAT in fungi (Azevedo et al. 2007). This stress further produces oxygen-free radicals that
are generally counteracted by SOD, CAT and GPX. The clear increase in SOD, CAT and GPX activities in
2009 ENZYME ACTIVITIES OF A LEAD-ADAPTED STRAIN OF R. arrhizus 507

the mycelium of R. arrhizus M1 (in comparison to WT strain) during growth in the presence of Pb indicates
that adaptation to Pb might induce changes that made the variant capable of producing more enzymes to
combat oxidative stress. Increase in CAT and GPX activities also indicated that H2O2 produced inside the
cell was efficiently counteracted.

Fig. 2. Activity of mycelial (rhombs and squares) and extracellular

(triangles and circles) AP (U/mg protein; top), PPO (U/mg protein;
middle), and thiol content (nmol/mg protein; bottom) of Pb-adapted
(rhombs and triangles) and WT (squares and circles) strains of R. arrhi-
zus during bioaccumulation of Pb from a medium containing 1 μg/mL
of Pb; **p < 0.001 compared to WT.

mycelial AP extracellular AP
Pb-adapted rhombs triangles
WT squares circles

AP and PPO are two of the many enzymes produced by filamentous fungi having relevant biotech-
nological applications. Both enzymes have roles in the defense against pathogens (Mayer 2006). The increa-
sed enzyme activities during growth of R. arrhizus M1 in the presence of Pb suggest a way to produce strains
potentially useful for enhanced production of commercially important enzymes in liquid culture media.
An improvement in thiol content of R. arrhizus strain M1 in the presence of Pb points to a me-
chanism for removal of the toxic Pb. Toxic Cd can induce the synthesis of glutathione and sulfur containing
amino acids (Guelfi et al. 2003) which are good metal chelators. Pb might also be inducing the production of
sulfur containing compounds in R. arrhizus, which was greater in the Pb-adapted strain. Our data suggest
that the cellular responses of a fungal strain toward toxic heavy metal induced stress can be made more
efficient if the strain is adapted previously to high concentration of these metals.

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