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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 |