d(GC) - Association of Biotechnology and Pharmacy

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