full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 5 (3) 1325 -1337 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)<br />
1326<br />
requirement can be manipulated by the<br />
conventional or statistical methods (3). The<br />
conventional method involves changing one<br />
independent variable at a time, while keeping<br />
other variables at a fixed level. Statistical<br />
methods, on the other h<strong>and</strong>, (a) shortlist<br />
significant variables, (b) help in underst<strong>and</strong>ing<br />
the interactions among the variables at different<br />
levels, <strong>and</strong> (c) reduce the total number <strong>of</strong><br />
experiments resulting in saving time <strong>and</strong><br />
resources. Hence these methods are more rapid<br />
<strong>and</strong> reliable (4).<br />
There are very few reports on production<br />
<strong>of</strong> cephamycin C from Nocardia lactamdurans<br />
using submerged fermentation (SmF) (5,6). To<br />
the best <strong>of</strong> our knowledge, no report on<br />
optimization <strong>of</strong> the fermentation media<br />
composition <strong>and</strong> conditions for cephamycin C<br />
production are available, which prompted us to<br />
undertake this study. This paper reports a detailed<br />
study on the optimization <strong>of</strong> cephamycin C<br />
production from Nocardia lactamdurans NRRL<br />
3802 using SmF, initially by the conventional<br />
one factor at-a-time method <strong>and</strong> subsequently by<br />
statistical methods such as Taguchi L 16<br />
-<br />
orthogonal array design <strong>and</strong> central composite<br />
rotatable design. Furthermore, the effects <strong>of</strong><br />
precursors <strong>and</strong> inducer on the production <strong>of</strong><br />
cephamycin C were also undertaken.<br />
Materials <strong>and</strong> Methods<br />
Materials: Nutrient broth, yeast extract, malt<br />
extract, peptone, L-glutamic acid, sucrose,<br />
fructose, lactose, maltose, galactose, myoinositol<br />
<strong>and</strong> 1,3-diaminopropane were purchased<br />
from Himedia Limited, Mumbai, India. Glucose,<br />
glycerol, starch, ammonium chloride, calcium<br />
carbonate, potassium dihydrogen<br />
orthophosphate, dipotassium hydrogen<br />
orthophosphate, sodium chloride, magnesium<br />
sulphate, ferrous sulphate, manganese sulphate,<br />
zinc sulphate, sodium thiosulphate,<br />
orthophosphoric acid, methanol, L-cysteine, L-<br />
valine, DL-methionine, L-lysine hydrochloride,<br />
heptanesulphonic acid sodium salt for HPLC <strong>and</strong><br />
HPLC grade acetonitrile were purchased from<br />
s.d. Fine-Chem Limited, Mumbai, India.<br />
Soybean oil, sesame oil, coconut oil, groundnut<br />
oil <strong>and</strong> mustard oil were procured from local<br />
market <strong>of</strong> Matunga, Mumbai, India. All solvents<br />
used were <strong>of</strong> AR grade unless mentioned.<br />
St<strong>and</strong>ard cephamycin C (authentic sample) was<br />
a gift sample through kind courtesy <strong>of</strong> Merck<br />
Research Laboratories, Rahway, NJ, USA.<br />
A cephamycin C producing strain,<br />
Nocardia lactamdurans NRRL 3802, was a gift<br />
sample from Northern Regional Research<br />
Laboratory, Peoria, Illinois, USA.<br />
Preparation <strong>of</strong> seed inoculums: The strain was<br />
maintained as glycerol stock by adding 1 ml <strong>of</strong><br />
culture solution, prepared by growing 1 ml (1 x<br />
10 9 CFU/ml) <strong>of</strong> the organism in 50 ml NYG<br />
medium [(g/l) nutrient broth 8, yeast extract 2,<br />
glucose 10, MgCl 2<br />
4, pH 6.8) for 48 h (7)], to<br />
preautoclaved 1.5 ml microcentrifuge tube<br />
containing 0.5 ml <strong>of</strong> 50% glycerol solution <strong>and</strong><br />
stored at -20°C. For seed inoculum, the glycerol<br />
stock (1 x 10 9 CFU/ml) prepared earlier was<br />
inoculated into 50 ml <strong>of</strong> sterilized NYG media<br />
<strong>and</strong> kept on orbital shaker at 180 rpm for 48 h.<br />
Fermentative production <strong>of</strong> cephamycin C: The<br />
production medium for cephamycin C contained<br />
(g/l) glucose, 10.0; L-glutamic acid, 4.25; NH 4<br />
Cl,<br />
1.0; CaCO 3<br />
, 0.25; K 2<br />
HPO 4<br />
, 2.0; myo-inositol,<br />
0.2; MgSO 4<br />
.7H 2<br />
O, 0.5; NaCl, 0.5; FeSO 4<br />
.7H 2<br />
O,<br />
0.025; MnSO 4<br />
.H 2<br />
O, 0.005; ZnSO 4<br />
.7H 2<br />
O, 0.01;<br />
para-amino benzoic acid (PABA), 0.0001 <strong>and</strong><br />
Na 2<br />
S 2<br />
O 3<br />
.5H 2<br />
O, 0.36. The pH <strong>of</strong> the production<br />
medium was adjusted to 6.8 ± 0.05 (5). 50 ml <strong>of</strong><br />
production medium was taken in 250 ml<br />
Erlenmeyer flask <strong>and</strong> sterilized by autoclaving<br />
at 121°C for 20 min. After cooling, the media<br />
was inoculated with 1 ml <strong>of</strong> 48 h old seed culture<br />
Lalit D. Kagliwal et al