full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 5 (3) 1325 -1337 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)
1328
combined effect of four independent variables
identified by L 16
-orthogonal array design (A:
Na 2
S 2
O 3
(g/l); B: yeast extract (g/l); C: K 2
HPO 4
(g/l); D: L-glutamic acid (g/l)) on maximum
production of cephamycin C, media optimized
by one factor at-a-time method was used. RSM
using central composite rotatable design (CCRD)
was applied. Each variable in the design was
studied at five different levels, with all variables
taken at a central coded value of zero. The
experiments were designed using the software,
Design Expert Version 6.0.10 version (Stat Ease,
Minneapolis, MN) which gave (factorial portion
2 4 = 16 with 8 star points where á is equal to
square root of k and k is 4) and 24 plus 6 centre
points leading to 30 experiments. The CCRD
design matrix in terms of coded and actual values
of independent variable is given in Table 2.
The second order polynomial coefficients
were calculated using the software package
Design Expert Version 6.0.10 to estimate the
responses of the dependent variable. Response
surface plots were also obtained using Design
Expert Version 6.0.10. The quadratic model
suggested by RSM was validated by using the
optimized medium composition shown in Table
3.
The effect of metabolic precursors on
cephamycin C production: The effects of amino
acids on the production of cephamycin C was
studied by supplementing the medium with
different amino acids viz. L-lysine hydrochloride,
L-valine, L-cysteine and DL-methionine. Here
amino acids were added at 0.5% - 1.5% in RSM
optimized media.
The effect of inducer on cephamycin C
production: Diamines are known to increase the
production of cephamycin C by inducing the
enzymes responsible for conversion of lysine to
á-aminoadipic acid (8,9). 1,3-Diaminopropane
(a three carbon diamine) was used as an inducer
for the production of cephamycin C. It was varied
from 0.4-1.6% v/v to the RSM optimized media
supplemented with amino acids.
Analytical methods
Cephamycin C assay by HPLC: The
fermentation broth was centrifuged at 4000 x g
for 10 min to remove biomass and 1 ml of
supernatant was extracted with 4 ml of methanol
to precipitate the proteins. The precipitated
proteins were removed by centrifugation at 4000
x g for 10 min and the supernatant was filtered
through 0.45 µm membrane filter (Millex ® -HV,
Millipore, USA). Cephamycin C was estimated
by HPLC method reported by Kagliwal et al.
(10). Jasco HPLC system fitted with a reverse
phase column Thermo ODS-2 Hypersil (C 18
octadecylsilane, 250 x 4.6 mm ID) was used. The
mobile phase was 50 mM KH 2
PO 4
with 20 mM
heptanesulphonic acid (HSA) adjusted to pH 4.0
with concentrated phosphoric acid. A 90% 50
mM KH 2
PO 4
with 20 mM HSA (pH 4.0) and 10%
acetonitrile mixture was used for fine resolution
of peaks. 20-ìl fermentation broth (the
centrifuged and deproteinized extract) was
injected and eluted at a flow rate of 0.8 ml/min.
Cephamycin C was detected at 253 nm.
Dry cell weight determination: To determine the
biomass produced, 1 ml of fermentation media
was taken in microcentrifuge tube and
centrifuged at 10,000 x g for 10 min. The
supernatant was decanted and settled biomass
was dried at 60°C for 24 h.
Results and Discussion
Optimization of fermentation medium using
one factor at-a-time method
Effect of carbon sources: During the microbial
fermentations, the carbon source not only acts
as a major constituent for building of cellular
material, but is also used as energy source.
Glycerol supports the production of antibiotics,
and glucose supports the growth of the organism
Lalit D. Kagliwal et al