d(GC) - Association of Biotechnology and Pharmacy
d(GC) - Association of Biotechnology and Pharmacy
d(GC) - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 6 (2) 190-195 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)<br />
Pradesh) including one sample from petrol pump<br />
where soil was mixed with petroleum products.<br />
Screening <strong>of</strong> bacterial isolates: All soil samples<br />
were made semi-dried in the laboratory using<br />
autoclaved water. The 10 2 to 10 7 times dilutions<br />
were made from the suspension <strong>of</strong> different soil<br />
samples. Serially diluted soil samples were plated<br />
on Luria Broth (LB) nutrient agar plates <strong>and</strong><br />
incubated at 37°C for 24 hours. The LB medium<br />
was comprised <strong>of</strong> bacto-tryptone, 10gm; yeast<br />
extract, 5gm; sodium chloride, 10gm; agar, 15gm<br />
per litre <strong>and</strong> adjusted to pH 10 using 0.1N sodium<br />
hydroxide. Few distinct colonies with distinct<br />
morphology were isolated <strong>and</strong> transferred to<br />
Petri-plates having Emersion medium (yeast<br />
extract, 5 gm; peptone, 5 gm; K 2 HPO 4 , 1 gm;<br />
MgSO 4 . 7H 2 O, 0.2 gm per litre <strong>and</strong> pH adjusted<br />
to 9 with 1N NaOH) <strong>and</strong> incubated at 37°C for<br />
24 hours. The Emersion medium was<br />
supplemented with 1% xylan in order to isolate<br />
xylan utilizing bacteria.<br />
Screening for xylanase activity: The colonies<br />
obtained were screened for xylanase activity<br />
using congo red dye method (16). A set <strong>of</strong> replica<br />
plates was prepared <strong>and</strong> incubated at 37°C for<br />
24 hours. Thereafter, one <strong>of</strong> the replica plate was<br />
flooded with 0.5% congo red dye <strong>and</strong><br />
subsequently with 1 mM solution <strong>of</strong> sodium<br />
chloride. A clear zone around the colony was<br />
considered as indication <strong>of</strong> xylanase activity. The<br />
corresponding colonies from other replica plate<br />
were inoculated individually in Emersion medium<br />
broth, allowed to grow overnight at 37°C at 200<br />
rpm in an orbital shaker. The xylanase activity<br />
was analyzed in the medium.<br />
Morphological, biochemical <strong>and</strong> physiological<br />
studies <strong>of</strong> the bacteria: Gram staining was<br />
done using Kit from Hi-media. After staining,<br />
slides were observed in a phase contrast<br />
microscope. For biochemical <strong>and</strong> physiological<br />
studies <strong>and</strong> identification <strong>of</strong> the bacteria, the<br />
culture was sent to Microbial Type Culture<br />
Collection <strong>and</strong> Gene Bank, Institute <strong>of</strong> Microbial<br />
Technology, Ch<strong>and</strong>igarh.<br />
Meeta Sharma <strong>and</strong> Anil Kumar<br />
191<br />
Production <strong>of</strong> xylanase: The isolated bacteria<br />
was maintained on Emerson medium slants by<br />
routine sub-culturing every month <strong>and</strong> used for<br />
the production <strong>of</strong> xylanase. The growth from 24<br />
hours old Emerson slant was scrapped into<br />
sterile Emerson medium contained in a 250 ml<br />
capacity Erlenmeyer flask <strong>and</strong> allowed to grow<br />
at 37 o C on an orbital shaker with a speed <strong>of</strong> 200<br />
rpm. A 2 ml aliquot <strong>of</strong> this suspension was used<br />
as an inoculum for 100 ml medium. The flasks<br />
were incubated at 37 o C for 48 hours with a speed<br />
<strong>of</strong> 200 rpm. Thereafter, the bacterial cells were<br />
harvested by centrifugation <strong>of</strong> the broth at 10,000<br />
x g for 30 minutes at 0 to 4°C in a cooling<br />
centrifuge. The supernatant contained most <strong>of</strong><br />
the xylanase activity.<br />
Growth time optimization for xylanase<br />
production: Samples from growing broth were<br />
drawn at 4 hours intervals, centrifuged at 10,000<br />
x g for 15 minutes in the cold condition (0 to 4°C).<br />
The resulting supernatants were analyzed for<br />
xylanase activity.<br />
Enzyme assay: Xylanase enzyme was assayed<br />
by measuring the release <strong>of</strong> the reducing sugars<br />
from birch wood xylan following the dinitrosalicylic<br />
acid (DNS) method (17). The D- xylose was used<br />
as st<strong>and</strong>ard during the colorimetric estimation.<br />
One unit <strong>of</strong> xylanase activity was taken as the<br />
amount <strong>of</strong> the enzyme required to release one<br />
micromole <strong>of</strong> the reducing sugar equivalent to<br />
one micromole <strong>of</strong> xylose per minute at 50 o C<br />
under the conditions <strong>of</strong> the enzyme assay.<br />
Optimization <strong>of</strong> culture conditions for<br />
xylanase production: For optimization <strong>of</strong> culture<br />
conditions, pHs ranging from pH 8.0 to 11.0 with<br />
a difference <strong>of</strong> 0.1 unit were maintained in the<br />
culture medium. The xylanase activity <strong>and</strong><br />
bacterial growth was measured. The bacterial<br />
growth was measured using absorbance at 600<br />
nm. The time <strong>of</strong> growth was also optimized in<br />
the range <strong>of</strong> 12 to 96 hours by withdrawing the<br />
aliquots aseptically at 12 hours intervals <strong>and</strong><br />
analyzed for the xylanase activity. For