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)
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requirement can be manipulated by the
conventional or statistical methods (3). The
conventional method involves changing one
independent variable at a time, while keeping
other variables at a fixed level. Statistical
methods, on the other hand, (a) shortlist
significant variables, (b) help in understanding
the interactions among the variables at different
levels, and (c) reduce the total number of
experiments resulting in saving time and
resources. Hence these methods are more rapid
and reliable (4).
There are very few reports on production
of cephamycin C from Nocardia lactamdurans
using submerged fermentation (SmF) (5,6). To
the best of our knowledge, no report on
optimization of the fermentation media
composition and conditions for cephamycin C
production are available, which prompted us to
undertake this study. This paper reports a detailed
study on the optimization of cephamycin C
production from Nocardia lactamdurans NRRL
3802 using SmF, initially by the conventional
one factor at-a-time method and subsequently by
statistical methods such as Taguchi L 16
-
orthogonal array design and central composite
rotatable design. Furthermore, the effects of
precursors and inducer on the production of
cephamycin C were also undertaken.
Materials and Methods
Materials: Nutrient broth, yeast extract, malt
extract, peptone, L-glutamic acid, sucrose,
fructose, lactose, maltose, galactose, myoinositol
and 1,3-diaminopropane were purchased
from Himedia Limited, Mumbai, India. Glucose,
glycerol, starch, ammonium chloride, calcium
carbonate, potassium dihydrogen
orthophosphate, dipotassium hydrogen
orthophosphate, sodium chloride, magnesium
sulphate, ferrous sulphate, manganese sulphate,
zinc sulphate, sodium thiosulphate,
orthophosphoric acid, methanol, L-cysteine, L-
valine, DL-methionine, L-lysine hydrochloride,
heptanesulphonic acid sodium salt for HPLC and
HPLC grade acetonitrile were purchased from
s.d. Fine-Chem Limited, Mumbai, India.
Soybean oil, sesame oil, coconut oil, groundnut
oil and mustard oil were procured from local
market of Matunga, Mumbai, India. All solvents
used were of AR grade unless mentioned.
Standard cephamycin C (authentic sample) was
a gift sample through kind courtesy of Merck
Research Laboratories, Rahway, NJ, USA.
A cephamycin C producing strain,
Nocardia lactamdurans NRRL 3802, was a gift
sample from Northern Regional Research
Laboratory, Peoria, Illinois, USA.
Preparation of seed inoculums: The strain was
maintained as glycerol stock by adding 1 ml of
culture solution, prepared by growing 1 ml (1 x
10 9 CFU/ml) of the organism in 50 ml NYG
medium [(g/l) nutrient broth 8, yeast extract 2,
glucose 10, MgCl 2
4, pH 6.8) for 48 h (7)], to
preautoclaved 1.5 ml microcentrifuge tube
containing 0.5 ml of 50% glycerol solution and
stored at -20°C. For seed inoculum, the glycerol
stock (1 x 10 9 CFU/ml) prepared earlier was
inoculated into 50 ml of sterilized NYG media
and kept on orbital shaker at 180 rpm for 48 h.
Fermentative production of cephamycin C: The
production medium for cephamycin C contained
(g/l) glucose, 10.0; L-glutamic acid, 4.25; NH 4
Cl,
1.0; CaCO 3
, 0.25; K 2
HPO 4
, 2.0; myo-inositol,
0.2; MgSO 4
.7H 2
O, 0.5; NaCl, 0.5; FeSO 4
.7H 2
O,
0.025; MnSO 4
.H 2
O, 0.005; ZnSO 4
.7H 2
O, 0.01;
para-amino benzoic acid (PABA), 0.0001 and
Na 2
S 2
O 3
.5H 2
O, 0.36. The pH of the production
medium was adjusted to 6.8 ± 0.05 (5). 50 ml of
production medium was taken in 250 ml
Erlenmeyer flask and sterilized by autoclaving
at 121°C for 20 min. After cooling, the media
was inoculated with 1 ml of 48 h old seed culture
Lalit D. Kagliwal et al