full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 7 (1) 518-526 January 2013, ISSN 0973-8916 (Print), 2230-7303 (Online)
519
albidoflavus (7), Streptomyces strains S3, S4 and
K8 (8), Streptomyces gulbargensis (9),
Streptomyces noursei MTCC 10469 (10),
Streptomyces ABR2 (11). Among the various
sources of L-asparaginase, actinomycetes are
the least studied organisms. In the present study
an attempt was made for the first time on the
production and characterization of an extracellular
L-asparaginase, under submerged
fermentation from estuarine Actinomycetes
species.
Materials and Methods
Sample collection: A total of five estuarine
sediments samples were collected for the
isolation of marine Actinomycetes. The five
sediment samples were collected at a depth of 1
to 10 centimetres at 2.5 to 9 km off shores
Parawada coast of Bay of Bengal. The texture
of the samples colour was sandy with brown to
blackish in colour. The estuarine sediment
samples were collected and stored in sterilized
bags.
Media: The media used to isolate Actinomycetes
from the estuarine samples are; Potassium
tellurite agar medium: Potassium tellurite: 0.1gm,
Peptone: 5.0gm, Yeast extract: 2.5gm, Agar:
20gm, D.W: 1lit, pH: 6.4 and Starch- casein agar
medium: Soluble starch: 10gm, Casein: 0.3gm,
KNO 3
: 2gm, NaCl: 10gm, K 2
HPO 4
: 2gm,
MgSO 4
.7H 2
O: 0.05gm, CaCO 3
: 0.02gm,
FeSO 4
.7H 2
O: 0.01gm, Agar: 20gm, D.W: 1.0lit,
pH: 7.0.
Isolation and screening: Different Estuarine
sediments samples were serially diluted in sterile
distilled water under aseptic conditions.
Actinomycetes were isolated from estuarine
sediments by plating them on Starch casein agar
media with different dilutions and Incubated at
30°C for 5 th day and up to three weeks.
Rifampicin (12) (2.5 μg/ml) and Cyclohexamide
(50 μg/ml) added to medium to inhibit bacterial
and fungal contamination. After 5 th day to three
weeks, suspected Actinomycete colonies were
identified based on their morphological
characteristics. The selected Actinomycetes are
shown in Table 1. The isolated Actinomycetes
were maintained as slant culture on yeast extract
malt extract agar medium.
Table 1. Distribution of Actinomycetes from
estuarine sediments samples.
Sample No. of No. of No. of Batch
No. colonies Actino active
mycetes isolates
isolated
PW-01 22 7 6 Batch PW-A
PW-02 14 5 2
PW-03 66 3 1 Batch PW-B
PW-04 6 5 2
(PW-Parawada, PW-A, PW-F- Batch number)
Screening of estuarine isolates for L-
asparaginase production by rapid plate
assay: The estuarine isolates were screened for
L-asparaginase activity using the method of
Gulati et al., (13). The medium used were
modified M-9 (composition in g/l: KH 2
PO 4
- 2.0,
L-asparagine -6.0, MgSO 4
.7H 2
O - 1.0,
CaCl 2
.2H 2
O - 1.0, glucose - 3.0, and agar - 20.0)
supplemented with phenol red as a pH indicator.
L-asparaginase activity was identified by
formation of a pink zone around colonies.
L-asparaginase production by submerged
fermentation: Different mediums (Table 2) were
used for the production of L-asparaginase from
Positive estuarine Actinomycetes was carried out
by submerged fermentation.
Determination of enzyme activity: The crude
enzyme was prepared by cooling centrifuge at
14,000 rpm for 20 min. The supernatant was
taken as the crude enzyme. The enzyme activity
was determined according to the method of
Imada et al. (14), where the liberated ammonia
to the action of enzyme was estimated using
Nessler’s reagent. A reaction mixture containing
0.5 ml of 0.04 M L-asparagine, 0.5 ml of 0.5 M
buffer, 0.5 ml of an enzyme preparation, and
distilled water to a total volume of 2.0 ml
incubated at 37°C for 30 min. The reaction
stopped by adding 0.5 ml of 1.5 M trichloroacetic
Rao et al