46

The Open Biotechnology Journal, 2009, 3, 46-49

Open Access

Screening of Xanthine Oxidase Producing Microorganisms Using
Nitroblue Tetrazolium Based Colorimetric Assay Method
Amit Agarwal and U.C. Banerjee*
Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research, Sec-67, SAS Nagar-160067, Punjab, India
Abstract: Xanthine oxidase is a highly versatile and ubiquitous complex molybdoflavoprotein, which controls the rate
limiting step of purine catabolism pathway. Microbial xanthine oxidase can be used to address a number of questions
presently not feasible with the eukaryotic enzymes. In the present study, a high-throughput microtitre plate-based colorimetric assay for xanthine oxidase producing microorganism was developed. Superoxides produced by microbial cultures, grown on xanthine rich medium interacts with nitroblue tetrazolium (NBT) solution and produces dark blue color,which facilitates the rapid screening of xanthine oxidase producing microorganisms and could be adapted for quick quantitative assessment and distribution of xanthine oxidase in many heterogeneous microbial communities. The method developed may be utilized for the rapid screening of a variety of xanthine oxidase producing microorganisms from the nature.

Key Words: Xanthine, nitroblue tetrazolium, allopurinol, screening, microbial xanthine oxidase.
INTRODUCTION
Xanthine oxidase is a highly versatile and ubiquitouscomplex molybdopterin, first identified a century ago in milk
[1]. Most of the xanthine metabolizing organisms possess amolybdopterin cofactor-containing enzyme that oxidizes
xanthine to uric acid and transfers electrons to NAD+ (xanthine dehydrogenase) or oxygen (xanthine oxidase) [2]. Although mammalian xanthine oxidase exists originally as adehydrogenase form, but it can be easily converted to oxidase form either irreversibly [3], or reversibly [3-5]. These
different forms of xanthine oxidoreductases are generally
conserved in living organisms, including archaea, bacteria,
fungi, plants, and metazoans [6] and it has been revealed
from different sources that these isoforms are all fundamentally similar with regard to molecular properties, prosthetic
group content and substrate specificity [7]. Xanthine oxidase
is capable of oxidizing varieties of purines and related compounds and physiologically participates in a variety of biochemical reactions including hydroxylation of various
purines, pterines and aromatic heterocycles, as well as aliphatic and aromatic aldehydes and hence plays a significant
role in the detoxification or activation of endogenous compounds and xenobiotics [8].
The standard spectrophotometric assay of xanthine oxidase is based on the oxidation of xanthine/hypoxanthine touric acid [9]; in case of microbial xanthine oxidase assay, it
suffers from the drawback of being slow and less sensitive.
There is a pressing need to develop high throughput assays,*Address correspondence to this author at the Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research, Sec-67, SAS Nagar-160067, Punjab, India; Tel: +91172-2214682-87, Ext.2142; Fax: +91-172-2214692;
E-mail: ucbanerjee@niper.ac.in
1874-0707/09

which would greatly facilitate work on xanthine oxidase/xanthine dehydrogenase (and other enzymes), and specifically would potentiate the use of combinatorial chemical
libraries to screen for novel lead compounds (antigout, antimicrobial, antitumor). We made an attempt to develop microbial xanthine oxidase assay based on the reactivity ofsuperoxides produced during the course of the reaction. The
aerobic oxidation of xanthine/hypoxanthine by xanthine oxidase is associated with the formation of hydrogen peroxideand superoxides [10]. This has prompted us to use the xanthine oxidase system with xanthine/hypoxanthine as substrate as a model of the high throughput screening of microbial xanthine oxidase. The underlying principle [11] wasused to develop the colorimetric assay for screening of xanthine oxidase producing microorganisms from the soil sample.
Xanthine + O2

Uric acid + NBT (yellow)
Superoxides

Formazan(dark blue)

MATERIALS AND METHODS
Xanthine, allopurinol, xanthine oxidase (from bovine
milk; grade II), were purchased from Sigma- Aldrich Inc.,
USA. Nitroblue tetrazolium and all other chemicals wereobtained from Hi-media Inc. Mumbai, India.
Microbial Culture
A 2 x 2 ft. area in the institute’s garden was selected forthe primary enrichment of xanthine utilizing microorganisms
in the soil. A solution of xanthine (2 mmol l-1) was sprayedon the designated soil at a regular time interval and samples
were collected from it. This process of spraying of xanthineand collecting soil samples continued for 4-6 months. A soil
suspension was prepared by adding 10 ml tap water to 1 g
2009 Bentham OpenScreening of Xanthine Oxidase Producing Microorganisms

The Open Biotechnology Journal, 2009, Volume 3soil sample. It was vortexed and 1 ml supernatant was used
as an inoculum in 100 ml minimal salt medium (MSM) containing xanthine (2 mmol l-1) and incubated at 30 ºC in anorbital shaker (200 rpm) for 4 -6 days. Enriched samples
were streaked on selective plates (containing xanthine) in
MSM agar. Positive isolates were selected on the basis of
growth on the selective plates and organisms obtained in thiswaywere purified and maintained on nutrient agar plates(0.5 % peptone, 0.15 % yeast extract, 0.5 % beef extract.
0.5 % sodium chloride, agar 1.5 %, pH 7.6).
Detection of Microbial Xanthine Oxidase Using the Nitroblue Tetrazolium Based Assay
This method was developed by modifying the polyacrylamide gel activity staining method used by Ozer et al. [11]for xanthine oxidase. Microbial culture, grown on xanthine
as a sole source of carbon and nitrogen; was centrifuged andthe pellet was suspended in Tris-HCl buffer (50 mmol l-1, pH
7.6). Each reaction contained 150 μl microbial cell (concentration varied from 25 to 150 mg/ml), lysed with NaOH
(1 mol l-1); 150 μl NBT solution containing 50 mmol l-1 TrisHCl, pH 7.6, 2 mmol l-1 xanthine and 0.50 mmol l-1 nitroblue
tetrazolium. The reaction mixture was incubated for 5 minutes at ambient temperature, centrifuged and absorbance ofsupernatant was measured by microplate readers (Emax,
Molecular Devices, California) at 575 nm. This was used atest sample. Three control experiments were set with this

1

2

3

47

assay. First control consists of 150 μl NBT solution and 150
μl NaOH (1 mol l-1) which is termed as negative control.
This was carried out to see the interaction between NBT and
xanthine in alkaline conditions. Second control experiment
was set in which 150 μl microbial culture was incubated withallopurinol (50 mmol l-1) for 5 minutes at ambient temperature and to this mixture, 150 μl NBT solution was added andagain incubated for 5 minutes at ambient temperature, centrifuged and absorbance of the supernatant was measured by
microplate readers (Emax, Molecular Devices, California) at
575 nm. This control experiment determined the superoxides
formed by the action of enzymes other than xanthine oxidase
present in the microbial cultures. The third control experiment is the positive control which was set up with commercially available xanthine oxidase (0.02 units) from bovine
milk (grade II), keeping all the reaction parameters same.
RESULTSA large number of microorganisms exhibits xanthine
dehydrogenase/xanthine oxidase activity [7, 12], and, insome cases, enzyme activity can be induced by growing the
microorganism in a medium with xanthine/hypoxanthine assole carbon and nitrogen source. Microbial cultures whichshowed positive growth on the nutrient agar plates, containing xanthine as a sole source of carbon and nitrogen; reacts
with NBT solution and give dark blue color. As assumed, nocolor was developed in the negative control (Fig. 1), while

4

5

6

7

Lane 1

Lane 2

Lane 3

Lane 4

Fig. (1). Microtitre plate-based colorimetric assay of xanthine oxidase producing microorganisms.
Lane 1, Showing the blue colour of formazan formed due to the interaction of the super oxides with NBT. well 1: negative control, well 2: 25
mg/ml, well 3: 50 mg/ml, well 4: 75 mg/ml, well 5: 100 mg/ml, well 6: 150 mg/ml cell mass well 7: xanthine oxidase from bovine milk,
grade II (0.02 units), as positive control; Lane 2, Showing the same reactions in the presence of allopurinol (50 mmol l-1), standard inhibitorof xanthine oxidase. Lane 3-4, Showing the microorganisms not having xanthine oxidase as no effect of allopurinol is appearing on enzyme
activity.

48 The Open Biotechnology Journal, 2009, Volume 3

Agarwal and Banerjee

the microbial cultures which were treated with allopurinol;standard inhibitor of xanthine oxidase, showed the formationof light blue colour (Fig. 1) and the corresponding decreasein absorbance (Fig. 2A). In case of microorganisms, lacking
xanthine oxidase activity there is almost no or negligible
difference in the absorbance of the sample without allopurinol and the sample containing allopurinol (Fig. 2B). Tenstrains were screened for xanthine oxidase activity and it has
been observed that it varied from good to moderate to negligible amount (Table 1).
0.8

Absorbance at 575 nm

Absence of allopurinol
0.6

Presence of allopurinol

0.4

0.2

0.0

Table 1.

0

20

40

60

80

100

120

140

Xanthine Oxidase Activity Shown by Different Microorganism Grown on Xanthine Riched Medium

160

Strain Number

Xanthine Oxidase Activity

1

++

2

++

3

+

4

-

5

++

6

-

7

++

8

++

0.15

9

+

0.10

10

++

Cell mass (mg/ml)
Fig. (2a). Oxidation of NBT by xanthine oxidase producing microorganisms in the absence and presence of allopurinol (All the reactions were replicated thrice with SEM ± 0.10 and ± 0.05, respectively).
0.35

Absence of allopurinol

0.30

Absorbance at 575 nm

increased absorbance indicats the increased amount of formazan formed due to the excess of superoxides produced
during the reduction of the oxygen by microbial xanthineoxidase. However, when allopurinol was added to the reaction mixture, color intensity decreased due to the decrease in
the production of superoxide radicals; as allopurinol inhibited the xanthine oxidase activity and hence free radical formation. Appearance of slight blue color can be attributed tothe production of superoxides by other internal factors likemitochondrial electron chain and many autoxidation reactions. In case of microorganism lacking xanthine oxidase
activity no change in color intensity has been observed in theabsence and presence of allopurinol (Fig. 1), indicating thatthese microorganisms do not have the xanthine oxidase activity. In comparison to previous assays [13]; this assay system is simple and rapid for determining the xanthine oxidase
activity in a large number of microorganisms. This highthroughput colorimetric assay method can increase the efficiency of screening libraries of microorganisms for xanthineoxidase activity and also can be exploited for the screeningof xanthine oxidase inhibitors.

0.25
0.20

Presence of allopurinol++ Good xanthine oxidase activity; + moderate xanthine oxidase activity; - no or
negligible xanthine oxidase activity.

0.05
0.00

CONCLUSION
0

20

40

60

80

100

120

140

160

[Cell mass] mg/ml

Fig. (2b). Oxidation of NBT by microorganisms deficient in xanthine oxidase activity in the absence and presence of allopurinol(All the reactions were replicated thrice with SEM ± 0.13 and ±0.06, respectively).

DISCUSSION
Dark blue color appears due to the formation of formazan, formed as a result of interaction of the NBT and superoxides produced during the course of the oxidation of xanthine to uric acid by xanthine oxidase. It is evident from Fig.
(2A) that with the increase in the cell mass concentration, theThe xanthine oxidase/dehydrogenase system has been
extensively studied with the bovine milk oxidase andchicken and rat dehydrogenase and has become the standardsof comparison for xanthine oxidizing enzymes isolated fromother sources [13, 14]. A more detailed understanding of therelative functional role of the redox cofactor (molybdopterin,
two iron-sulfur centers, and FAD) found in these enzymes[4] will require the use of site-directed mutants and theircharacterization. Microbial xanthine oxidase can be exploited to address a number of questions presently not possible with the eukaryotic enzymes, such as (i) defects in thecorresponding gene leading to hyperuricemia/gout, xanthinuria and various other related disorders; (ii) to know the functional roles of amino acid residues implicated in the catalytic

Screening of Xanthine Oxidase Producing Microorganisms

The Open Biotechnology Journal, 2009, Volume 3

mechanism of the enzyme (iii) to fully explore the complicated process of biosynthesis of functional form of the enzyme; (iv) to throw the light on the cofactor functional roles
and electron transfer pathways in this oxidoreductase class of
enzymes. These preliminary studies although precludes thedetection of allozyme or isozyme multiplicity, but this assay
method can pave a way in achieving the above mentioned
tasks.

[5]

[6]
[7]

ACKNOWLEDGEMENTS[8]

AA gratefully acknowledges the Department of Biotechnology, Government of India, for providing the junior research fellowship to carry out the work.

[9]
[10]

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Accepted: April 29, 2009

© Agarwal and Banerjee; Licensee Bentham Open.
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