Microorganisms was as follows: uricase can catalyse the

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Microorganisms was as follows: uricase can catalyse the

Microorganisms
and cultural conditions:
seventy-six of actinomycet strains selected from Iran Biological Research
Center (IBRC) for revived them. All strains were cultured on specific malt-yeast
agar medium (ISP2) consists
of (g L-1): Yeast extract
4g, Malt extract 10g, Glucose monohydrate 4g; distilled water up to 1 L; pH 7. For
plates 20.0 g agar was added and incubated at 28°C for seven days.

 

Primary and
secondary screening of uricase production: The culture Modified
Bennett’s agar medium for screening consists of (g L-1): Uric acid,
3.0 g; glycerol, 2.0 g; Yeast extract 1.0
g; Malt extract 1.0 g; Pepton,
2.0 g; distilled water up to 1 L; pH 7. For plates 20.0 g agar was added (Azab et
al., 2005). Preliminary screening for uricase production was done by
conventional spot inoculation of pure actinomycetes strains on agar medium and
incubated at 30°C for seven days. Uricase production by the microorganisms is indicated by
the appearance of clear zone around the colonies. The strains which forming
bigger clear zones in a shorter time were selected for subsequent screening
under submerged fermentation conditions. Fifty milliliter of fermentation
medium were dispensed in 250 mL Erlenmeyer conical flasks, sterilized and
inoculated. The fermentation media were incubated for at 28°C in a rotatory
incubating shaker (180 rpm). The enzyme production was measured after 7 days.
The strain which showed the most promising result was selected for further
experiments.

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Uricase
assay: The principle of enzyme measurement was as follows: uricase can
catalyse the oxidation of uric acid to form allantoin, carbon dioxide and
hydrogen peroxide which is then analyzed by the oxidative coupling of
4-aminoantipyrine, phenol and peroxidase as chromogens. Uricase activity was
measured by incubating 300 ?L enzyme solution with a mixture of 400 ?L sodium
borate buffer (pH 8.5, 0.1 M) containing 2 mM uric acid, 150 ?L
4-aminoantipyrine (30 mM), 100 ?L phenol (1.5%), 50 ?L peroxidase (15 U mL-1)
at 37°C for 20 min (Suzuki, 1981). The reaction was stopped by addition of (200
?L of 0.1 M potassium cyanide solution. In the blank, the solution of potassium
cyanide was added to the mixture before the addition of the crude enzyme. The
absorbance was measured against the blank in a spectrophotometer at 540 nm. One
unit of uricase enzyme is defined as the amount of enzyme that produces 1 ?mol
of H2O2 per minute under the standard assay conditions.

 

Characterization
of the selected actinomycete isolate: The
actinomycete selected as the best uricase-producing organism was
characterized and identified. Morphological studies were conducted after
growth on ISP
media 2-7 as described by Shirling and Gottlieb (1966); all plates were
incubated at 28°C for 14 days.  Phospholipid types were determined by 2-dimensional
thin-layer chromatography.

Phylogenetic
analysis of 16S rDNA sequence: The preparation
of genomic DNA of the strain was conducted in accordance with the methods
described by Wilson. The PCR amplification reaction was performed in a total
volume of 50 ?l which contained 5 ?l DNA, 1.3 ?l 0.4 mM deoxyribonucleotide
5′-triphosphate (dNTP’s); 4.5 ?l DMSO; 9 ?l PCR buffer, 1.8 ?l 2.5 mM MgCl2 and
0.6 ?l Taq polymerase, 9 ?l of 0.5 mM(each) forward 16s rRNA primer 27f
(5′-AGAGTTTGATCMTGGCTCAG-3′) and reverse 16s rRNA primer 1492 r (5′ GGTTACCTTGTTACGACTT-3′)
and water was added up to 50 ?l. The PCR-apparatus was programmed as follows: 5
min denaturation at 95°C, followed by 35 amplification cycles of 1 min at 95°C,
1 min of annealing at 55°C and 90 sec of extension at 72°C, followed by a 10
min final extension at 72°C. The PCR reaction mixture was then analyzed via
agarose gel electrophoresis. The purification and sequencing of the PCR product
of the previous step was carried out by Korea Bioneer Company. The sequence
received from this company was edited and revised using the ChromasPro
software, and then compared with the sequences recorded in the Eztaxon and
EzBioCloud genomic databases, and their similarities were recorded in different
strains and their phylogeny tree drawn up by MEGA7 software.

 

Cloning
uricase gene: The urate oxidase coding sequence (960pb) was obtained by PCR
using primers Uricase-F (5?-
TATACACATATGCTSGGMCAGAACCAGTAC-3?) and Uricase-R
(5? -TTGTTACTCGAGGAGGTTGGTSAKGTC-

-3?)
which were designed based on the                              uricase gene sequence (GeneBank gi:                 ). Primer Uricase-F contained
Nde1 site (underlined) and the translation initiation codon,
whereas Uricase-R incorporated an engineered XhoI site.
Histidine-tag sequence was added to the downstream and upstream of urate
oxidase gene before start and stop codon.  For the polymerase
chain reaction (PCR) amplification, the reaction conditions were 95 ?C for 1 min, 53 ?C for 20 s, and 72 ?C for 60 s for a total of 30 cycles. The PCR product was ligated
into the pET28a expression vector (fig1) to form the recombinant plasmid pET28aU,
which was transformed into E. coli BL21 (DL3). The positive clones were picked,
sequenced, and double digested using Nde1 and XhoI. The resulting construct was transformed into E. coli BL21
(DL3), and the clones were screened for expression.

 

fig1;
pET28a expression vector

 

Expression
of Uricase Enzyme: A single E. coli colony
transformed with pET28aU was inoculated in 6ml Luria-Bertolin (LB) medium
containing 50 ?g/ml
kanamycin incubated at 37?C with shaking
(180-200 rpm) overnight. Five milliliters of this preculture were transferred
to 50ml of LB medium in a 250mL shake flask. The culture was grown under the
same condition until OD600 reached
0.6 when IPTG was added to a final concentration of 0.1mM. One milliliter
samples were collected at different intervals and cell pellet was
resuspended in 50 ?L
of SDS-PAGE ( on 12%
polyacrylamide gel) sample buffer
and boiled for 5 minutes prior to gel electrophoresis analysis. Typically, 1–5 ?g total protein
were loaded in each lane.

 

Western
Blotting: Western blot
analysis of the expressed protein was performed using diluted (1:4,000)
anti-histidine-tag antibodies. Then the membrane was visualized by incubation
with tetramethylbenzidine (TMB) substrate solution at room temperature.

 

Recombinant
Enzyme Purification: In order to purify and analyze the
recombinant protein, one bacterial clone was induced with 0.1mM IPTG in 100mL
LB at 17?C with shaking (180-200 rpm) overnight. The cells were
harvested by centrifugation at 7000 rpm for 8 minutes at 4?C. Cell pellet was resuspended in 2mL lysis buffer (5mM imidazole, 50mM Tris-HCL
pH 8.8, 0.3M NaCl) and lysed by
sonication (3 cycles of 10 seconds of pulses at 0.5% amplitude—59W). The
suspension was centrifuged at 7000 rpm for 7 minutes and in both soluble and
insoluble forms loaded into a Ni-NTA column with a volume of 1mL resin
preequilibrated in lysis buffer. The column was washed 3 times with 1mL
wash buffer (20mM imidazole, 50mM NaH2PO4 pH
8.0, 0.3M NaCl) and 3 times with 1mL elution buffer
(0.8M imidazole, 50mM NaH2PO4 pH 8.0, 0.3MNaCl). All fractions were
stored at 4?C for further analysis. Protein fractions were analyzed by SDS-PAGE, and stained with
Coomassie Blue. The
enzymatic activity of purified uricase was determined spectrophotometrically by
monitoring the increase of hydrogen peroxid in absorbance at 540 nm as
described previously.

 

  Determination
of Protein Content: The protein concentration of the purified uricase was
measured using a Bradford protein assay kit (Fermentas, USA) according to the
manufacturer’s instructions. The protein content was calculated from the
standard curve. The GenBank accession number for the sequence reported in this
paper is………………

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