Isolation and Characterization of Highly Active Uricase from Alcaligenes spp. Strain UR1

Isolate code	Source of strains	Motility		Sporulation	Gram test	Uricase activity (mm)
Isolate code	Source of strains	Gram stain	KOH test	Sporulation	Gram test	Uricase activity (mm)
UR1	Soil	–	+	+	–	75
UR2	Soil	–	+	+	–	74
UR3	Poultry wastes	–	+	+	–	73
UR4	Poultry wastes	–	+	+	–	70
UR5	Poultry wastes	–	+	+	–	70
UR6	Soil	–	+	+	–	70
UR7	Soil	–	+	+	–	70
UR8	Poultry wastes	–	+	+	–	70
UR9	Poultry wastes	–	+	+	–	70
UR10	Poultry wastes	–	+	+	–	70
UR11	Poultry wastes	–	+	+	–	70
UR12	Poultry wastes	–	+	+	–	70
UR13	Poultry wastes	–	+	+	–	68
UR14	Poultry wastes	–	+	+	–	68
UR15	Soil	–	+	+	–	67
UR16	Poultry wastes	–	+	+	–	66
UR17	Poultry wastes	–	+	+	–	65
UR18	Poultry wastes	–	+	+	–	65
UR19	Poultry wastes	–	+	+	–	65
UR20	Poultry wastes	–	+	+	–	65
UR21	Poultry wastes	–	+	+	–	65
UR22	Poultry wastes	–	+	+	–	65
UR23	Poultry wastes	–	+	+	–	65
UR24	Soil	–	+	+	–	62
UR25	Poultry wastes	–	+	+	–	60
UR26	Poultry wastes	–	+	+	–	60
UR27	Poultry wastes	–	+	+	–	60
UR28	Soil	–	+	+	–	60
UR29	Poultry wastes	–	+	+	–	60
UR30	Poultry wastes	–	+	+	–	60
UR31	Poultry wastes	–	+	+	–	60
UR32	Poultry wastes	–	+	+	–	60
UR33	Poultry wastes	–	+	+	–	60
UR34	Poultry wastes	–	+	+	–	60
UR35	Poultry wastes	–	+	+	–	60
UR36	Poultry wastes	–	+	+	–	60
UR37	Poultry wastes	–	+	+	–	60
UR38	Poultry wastes	–	+	+	–	55
UR39	Poultry wastes	–	+	+	–	55
UR40	Poultry wastes	–	+	+	–	55
UR41	Poultry wastes	–	+	+	–	55
UR42	Poultry wastes	–	+	+	–	55
UR43	Poultry wastes	–	+	+	–	50
UR44	Poultry wastes	–	+	+	–	50
UR45	Poultry wastes	–	+	+	–	50
UR46	Poultry wastes	–	+	+	–	50
UR47	Poultry wastes	–	+	+	–	50
UR48	Poultry wastes	–	+	+	–	45
UR49	Soil	–	+	+	–	45
UR50	Poultry wastes	–	+	+	–	45
UR51	Soil	–	+	+	–	40
UR52	Soil	–	+	+	–	40

1 Uric acid degrading bacteria were enriched in uric acid-supplemented (0.8%) enrichment broth followed by incubation for 48 h at 37°C under shaking conditions. Uric acid turbidity gradually disappeared during fermentation. Then, dilutions of enrichment cultures were inoculated on uric acid-supplemented (0.3%) BT agar plates where all the isolates formed clear zones.

Table II

Morphological and partial characterization of uricase-producing isolates.

Characteristics	Most promising uricase-producing isolates
Characteristics	UR1	UR2	UR5	UR6	UR10
Colony morphology:
Form	Irregular	Irregular	Irregular	Irregular	Irregular
Color on NA	whitish	Whitish	Whitish	Whitish	Whitish
Pigment	–	–	–	–	–
Elevation	Flat	Flat	Flat	Flat	Flat
Opacity	Opaque	Opaque	Opaque	Opaque	Opaque
Consistency	Dry	Dry	Dry	Dry	Dry
Cell morphology:
Shape	Rods shaped	Rods shaped	Rods shaped	Rods shaped	Rods shaped
Gram stain	–	–	–	–	–
Catalase	+	+	+	+	+
Oxidase	+	+	+	+	+
Sporulation	+	+	+	+	+
Motility	+	+	+	+	+
Carbon source utilization:
Maltose	+\–	+	+	+	+
Dextrose	+	+	+	+	+
Fructose	+	+	+	+	+
D-Glucose	+	+	+	+	+
Sucrose	+	+	+	+	+
Nitrogen source utilization:
Casein	+	+	+	+	+
Yeast extract	+	+	+	+	+
Urea	+	+	+	+	+
Growth on NaCl (%):
0	+	+	+	+	+
1%	+	+	+	+	+
2%	+\–	+\–	+\–	+\–	+\–
5%	–	+\–	+\–	+\–	+\–
10%	–	–	–	–	–
pH optimum:
5	+	+\–	+\–	+\–	+\–
6	+	+\–	+\–	+\–	+\–
7	+	+	+	+	+
8	+	+	+	+	+
9	+\–	+\–	+\–	+\–	+\–
Temperature optimum:
20°C	+\–	+\–	–	+\–	+\–
30°C	+	+	+	+	+
37°C	+	+	+	+	+
40°C	+	+	+	+	+
50°C	–	–	–	–	–
MacConkey agar:	–	–	–	–	–
Blood agar hemolysis	α-hemolytic	α-hemolytic	α-hemolytic	α-hemolytic	α-hemolytic
Enzyme secretion:
Uricase	+	+	+	+	+
Urease	+	+	+	+	+
Protease	–	–	–	–	–
Amylase	–	–	–	–	–
Lipase	–	–	–	–	–
Cellulose	+	+	+	+	+

1 + – good growth; – – no growth; +/– – weak growth

Table III

API-20E profile of sugar fermentation and enzyme activity of uricase-producing isolates.

Biochemical test		Uricase-producing isolates					Reference strain
Biochemical test		UR1	UR2	UR5	UR6	UR10	Alcaligenes faecalis DSM13644
1	O-nitrophenyl-b D-galactopyranoside (ONPG)	–	–	–	–	–	–
2	Arginine dihydrolase (ADH)	–	–	–	–	–	–
3	Lysine decarboxylase (LDC)	–	–	–	–	–	–
4	Ornithine decarboxylase (ODC)	–	–	–	–	–	–
5	Citrate (CIT)	+	+	+	+	+	+
6	Hydrogen sulfide (H₂S)	–	–	–	–	–	+
7	Urease (URA)	+	+	+	+	+	–
8	Tryptophan deaminase (TDA)	–	–	–	–	–	+
9	Indole (IND)	–	–	–	–	–	+
10	Voges-Proskauer test (VP)	+	–	+	+	+	+
11	Gelatinase (GEL)	–	–	–	–	–	–
Sugar fermentation:
12	Glucose (GLU)	+	+	+	+	+	+
13	Mannose (MAN)	–	–	–	–	–	–
14	Inositol (INO)	–	–	–	–	–	–
15	Sorbitol (SOR)	–	–	–	–	–	–
16	Rhamnose (RHA)	–	–	–	–	–	–
17	Sucrose (SAC)	+	+	+	+	+	+
18	Melibiose (MEL)	–	–	–	–	–	–
19	Amygdalin (AMY)	–	–	–	–	–	–
20	Arabinose (ARA)	–	–	–	–	–	–

1 + – good growth, – no growth, +/– weak growth

1 + – positive enzyme reaction, – negative enzyme reaction

Table IV

bioMérieux VITEK^®-based biochemical tests of uricase-producing isolates.

Carbon source utilization, enzymatic activities and resistance		Conc. (mg)	Uricase isolates/identification results
		Conc. (mg)	UR1	UR2	UR5	UR6	UR10
1	Ala-Phe-Pro-arylamidase (APPA)	0.0384	–	–	–	–	–
2	Adonitol (ADO)	0.1875	–	–	–	–	–
3	L-Pyrrolydonyl-arylamidase (PyrA)	0.018	–	–	–	–	–
4	L-Arabitol (lARL)	0.3	–	–	–	–	–
5	D-Cellobiose (dCEL)	0.3	–	–	–	–	–
6	β-Galactosidase (BGAL)	0.036	–	–	–	–	–
7	Production (H2S)	0.0024	–	–	–	–	–
8	β-N-acetyl-glucosaminidase (BNAG)	0.0408	–	–	–	–	–
9	Glutamyl arylamidase pNA (AGLTp)	0.0324	–	–	–	–	–
10	D-Glucose (dGLU)	0.3	+	+	+	+	+
11	γ-Glutamyl-transferase (GGT)	0.0228	–	–	–	–	–
12	Fermentation/glucose (OFF)	0.45	+	+	+	+	+
13	β-Glucosidase (BGLU)	0.036	–	–	–	–	–
14	D-Maltose (dMAL)	0.3	–	–	–	–	–
15	D-Mannitol (dMAN)	0.1875	–	–	–	–	–
16	D-Mannose (dMNE)	0.3	–	–	–	–	–
17	β-Xylosidase (BXYL)	0.0324	–	–	–	–	–
18	β-Alanine arylamidase pNA (BAlap)	0.0174	–	–	–	–	–
19	L-Proline arylamidase (ProA)	0.0234	+	+	+	+	+
20	Lipase (LIP)	0.0192	–	–	–	–	–
21	Palationse (PLE)	0.3	–	–	–	–	–
22	Tyrosine arylamidase (TyrA)	0.0276	+	+	+	+	+
23	Urease (URE)	0.15	+	+	–	+	+
24	D-Sorbitol (dSOR)	0.1875	–	–	–	–	–
25	Saccharose/sucrose (SAC)	0.3	–	–	–	–	–
26	D-Tagatose (dTAG)	0.3	–	–	–	–	–
27	D-Trehalose (dTRE)	0.3	–	–	–	–	–
28	Citrate (sodium) (CIT)	0.054	–	–	+	–	–
29	Malonate (MNT)	0.15	–	–	+	–	–
30	5-Keto-D-gluconate (5KG)	0.3	–	–	–	–	–
31	L-Lactate alkalinisation (ILATk)	0.15	+	+	+	+	+
32	α-Glucosidase (AGLU)	0.036	–	–	–	–	–
33	Succinate alkalinisation (SUCT)	0.15	–	–	+	+	–
34	β-N-acetyl-galactosaminidase (NAGA)	0.0306	–	–	–	–	–
35	α-Galactosidase (AGAL)	0.036	–	–	–	–	–
36	Phosphatase (PHOS)	0.0504	–	–	–	–	–
37	Glycine arylamidase (GlyA)	0.012	–	–	–	–	–
38	Ornithine decarboxylase (ODC)	0.3	–	–	–	–	–
39	Lysine decarboxylase (LDC)	0.15	–	–	–	–	–
40	L-Histidine assimilation (IHISa)	0.087	-\+	–	–	+	+\-
41	Coumarate (CMT)	0.126	+	–	–	–	–
42	β-Glucoronidase (BGUR)	0.0378	–	–	–	–	–
43	O/129 Resistance (comp.vibrio.) (O129R)	0.0105	–	–	–	–	–
44	Glu-Gly-Arg-arylamidase (GGAA)	0.0576	–	–	–	–	–
45	L-malate assimilation (IMLTa)	0.042	-\+	+\-	+	–	–
46	Ellman (ELLM)	0.03	-\+	-\+	–	+	–
Identification and similarity according to VITEK^® data base			Pseudomonas oleovorans (95%)	Pseudomonas oleovorans (95%)	Alcaligenes faecalis (97%)	Acinetobacter lwoffii (92%)	Acinetobacter lwoffii (96%)

1 + – good growth, – no growth, +/– weak growth; + – positive enzyme reaction, – negative enzyme reaction

Table V

Antibiotic susceptibility of uricase-producing promising isolates.

Antibiotics	Antibiotics/Disc potency	Uricase-producing isolates
Antibiotics	Antibiotics/Disc potency	UR1	UR2	UR5	UR6	UR10
Amikacin	30 μg (AK)	R	R	S	R	S
Gentamicin	10 μg (GM)	S	S	S	S	R
Tetracycline	30 μg (T)	S	S	S	S	S
Aztreonam	30 μg (ATM)	R	R	R	R	R
Ciprofloxacin	5 μg (CIP)	S	S	R	S	S
Cefotaxime	30 μg (Cf)	S	S	R	R	R
Ceftazidime	30 μg (CAZ)	S	R	S	R	S
Cefepime	30 μg (CPM)	R	R	S	R	R
Imipenem	10 μg (IMI)	S	S	S	S	S
Piperacillin	100 μg (PRL)	S	S	R	R	S
Trimethoprim/sulfamethoxazole	25 μg (TS)	S	S	R	S	R
Chloramphenicol	30 μg (C)	S	S	S	S	S

1 R – resistant; S – sensitive

Table VI

16S rRNA gene sequence-based molecular identification of uricase-producing promising strains.

Isolate code	Blast match	Gene Bank Accession No.	16S rRNA Identity (%)
Strain UR1 (PP563675)	– Alcaligenes faecalis strain NBRC 13111	– NR_113606.1	96.96
	– Alcaligenes ammonioxydans strain HO-1	– NR_180910.1	96.86
	– Alcaligenes aquatilis strain LMG 22996	– NR_104977.1	96.54
	– Alcaligenes endophyticus strain AER10	– NR_156855.1	95.91
	– Alcaligenes pakistanensis strain NCCP-650	– NR_145932.1	95.58
Strain UR2 (PP563676)	– Alcaligenes faecalis strain NBRC 13111	– NR_113606.1	95.40
	– Alcaligenes ammonioxydans strain HO-1	– NR_180910.1	95.19
	– Alcaligenes aquatilis strain LMG 22996	– NR_156855.1	94.97
	– Alcaligenes endophyticus strain AER10	– NR_156855.1	95.15
	– Alcaligenes pakistanensis strain NCCP-650	– NR_145932.1	93.79
Strain UR5 (PP563677)	– Alcaligenes ammonioxydans strain HO-1	– NR_180910.1	97.28
	– Alcaligenes faecalis strain NBRC 13111	– NR_113606.1	97.20
	– Alcaligenes aquatilis strain LMG 22996	– NR_104977.1	96.89
	– Alcaligenes pakistanensis strain NCCP-650	– NR_145932.1	96.11
	– Alcaligenes endophyticus strain AER10	– NR_156855.	95.96
Strain UR6 (PP563678)	– Alcaligenes ammonioxydans strain HO-1 16S	– NR_180910.1	98.77
	– Alcaligenes faecalis strain NBRC 13111 16S	– NR_113606.1	98.56
	– Alcaligenes aquatilis strain LMG 22996	– NR_104977.1	98.15
	– Alcaligenes endophyticus strain AER10	– NR_156855.1	98.15
	– Alcaligenes pakistanensis strain NCCP-650	– NR_145932.1	98.15
Strain UR10 (PP563679)	– Alcaligenes ammonioxydans strain HO-1	– NR_180910.1	98.84
	– Alcaligenes faecalis strain NBRC 13111	– NR_113606.1	98.02
	– Alcaligenes aquatilis strain LMG 22996	– NR_104977.1	97.73
	– Alcaligenes pakistanensis strain NCCP-650	– NR_145932.1	96.98
	– Alcaligenes faecalis subsp. phenolicus	– NR_042830.1	95.87

1 Similarity of uricase-producing isolates and closely related strains of genus Alcaligenes spp.

Phylogenetic tree of uricase-producing isolates (shown in blue) and closely related strains of genus *Alcaligenes*. Bootstrap values out of 100 are mentioned at the nodes. *Escherichia coli* strain ATCC^® 35218^™ (J01859.1) and *Bacillus subtilis* strain: ATCC^® 21331^™ (AB018487.1) served as the out-group.

Effect of incubation period on the growth (OD) and uricase secretion of strain UR1

Effect of temperature on the growth (OD) and uricase activity (U/ml) of strain UR1.

Effect of pH on the growth (OD) and uricase activity (U/ml) of strain UR1.

Impact of carbon and nitrogen sources on strain UR1 growth and uricase activity (U/ml).

Impact of different NaCl (%) concentrations on strain UR1 growth (OD) and uricase activity (U/ml).

Uricase purification from strain UR1 by gel filtration. Uricase activity exhibiting fractions (F7-F12).

Table VII

Gel-filtration-based uricase purification steps from strain UR1.

Purification steps	Volume (ml)	Uricase activity (U/ml)	Total uricase activity (U)	Protein concentration (mg/ml)	Specific activity (U/mg)	Purification (fold)	Yield (%)
Crude enzyme (10 fold concentration)	100	75	7,500	5.4	13.8	1	100
Dialysis after ammonium sulfate precipitation	5	112	560	3.8	29.74	2.13	7.40
Gel filtration (Sephadex)	5	275	1,375	0.3	916	66.73	18.33

SDS-PAGE of the purified uricase from strain UR1.
SDS-denatured samples (20–40 μl) were separated in an SDS-polyacrylamide gel (12%) and stained with Serva blue R. Lane 1: molecular mass standard, Lane 2: protein (10 μg) of purified enzyme.

Effect of temperature on a) uricase activity, and b) stability.

Effect of pH on the uricase activity of strain UR1.

Effect of substrate concentration on uricase activity of strain UR1.

Effect of the metal ions and inhibitors on uricase activity of strain UR1.

References

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Articles in this issue

DOI: https://doi.org/10.33073/pjm-2025-009 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331

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Language: English

Page range: 106 - 129

Submitted on: Dec 25, 2024

Accepted on: Feb 10, 2025

Published on: Mar 26, 2025

Published by: Polish Society of Microbiologists

In partnership with: Paradigm Publishing Services

Publication frequency: 4 issues per year

Keywords:

Uricase,

uricase-producing-bacteria,

therapeutic enzyme,

Alcaligenes spp,

uricase purification,

uricase characterization

Related subjects:

Life sciences,

Microbiology and virology

© 2025 Atheer Alshareef, Mahmoud Z. El-Readi, Leena A. Neyaz, Hussein H. Abulreesh, Ahmad A. Alsaigh, Ashjan F. Khalel, Wafaa A. Alshehri, Khaled Elbanna, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Volume 74 (2025): Issue 1 (March 2025)

Isolation and Characterization of Highly Active Uricase from Alcaligenes spp. Strain UR1

Figures & Tables

Fig. 1.

Table I

Table II

Table III

Table IV

Table V

Table VI

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

Table VII

Fig. 9.

Fig. 10.

Fig. 11.

Fig. 12.

Fig. 13.

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