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

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

Biochemical test		Uricase-producing isolates					Reference strain
		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)	–	–	–	–	–	–

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

Morphological and partial characterization of uricase-producing isolates_

Characteristics	Most promising uricase-producing isolates
	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	+	+	+	+	+

Antibiotic susceptibility of uricase-producing promising isolates_

Antibiotics	Antibiotics/Disc potency	Uricase-producing isolates
		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

Screening and partial characterization of uricase-producing isolates_

Isolate code	Source of strains	Motility		Sporulation	Gram test	Uricase activity (mm)
		Gram stain	KOH test
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

bioMérieux VITEK®-based biochemical tests of uricase-producing isolates_

Carbon source utilization, enzymatic activities and resistance		Conc. (mg)	Uricase isolates/identification results
			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%)

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