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Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1325 -1337 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1331 Cephamycin C (mg/l) DCW (g/l) Fig. 3. Effect of minerals on cephamycin C production by N. lactamdurans NRRL 3802 by SmF have inhibitory effect on the production of cephamycin C. Whereas, removal of NaCl, MgSO 4 , FeSO 4 , Na 2 S 2 O 3 and K 2 HPO 4 decreased the production of cephamycin C suggesting them to play an important role in the production of cephamycin C. Calcium has been identified to be inhibitory to the biosynthesis of cephamycin C in resting cells of N. lactamdurans (21). Therefore, calcium probably does not directly interfere with the biosynthetic enzymes for either cephamycin C or efrotomycin. Inhibition is more likely to be caused by calcium interfering with an enzyme or cellular process, which is generally the case in secondary metabolism (22). MgSO 4 is a cofactor for the enzyme ACV synthetase, and FeSO 4 for isopenicillin N synthase (IPN synthase) and deacetoxycephalosporin C synthase (DAOC synthase). Hence they are essentially required by these enzymes for the biosynthesis of cephamycin C. Sulphur from sodium thiosulphate gets incorporated into cysteine by reduction, and ultimately into â- lactam products. Phosphate, despite not being incorporated directly into the â-lactam nucleus, affects secondary metabolism in â-lactam producing organisms. They can independently inhibit enzyme action, regulate protein expression and limit cell growth. Optimization of fermentation medium using statistical methods Optimization using the L 16 -orthogonal array: Taguchi design was used to determine most significant factors which affect the production of cephamycin C. The 8 factors selected were glycerol, yeast extract, L-glutamic acid, K 2 HPO 4 , NaCl, MgSO 4 , FeSO 4 and Na 2 S 2 O 3 . The concentrations of the medium components were selected based on the production medium composition that was optimized using one factor at a time. Table 4 represents the response table for means (larger is better) and for signal to noise ratio obtained with L 16 -orthogonal array. The last two columns in the Table 4 document the delta Sequential optimization of cephamycin C
Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1325 -1337 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1332 values and ranks for the system. Rank and delta values help to assess which factors have the greatest effect on the response characteristic of interest. Delta measures the size of the effect by taking the difference between the highest and lowest characteristic average for a factor. A higher delta value indicates greater effect of that component. ‘Rank’ orders the factors from the greatest effect (based on the delta values) to the least effect on the response characteristic. The order in which the individual components selected in the present study effect the fermentation process can be ranked as Na 2 S 2 O 3 > yeast extract > K 2 HPO 4 > L-glutamic acid > MgSO 4 > FeSO 4 > glycerol > NaCl suggesting that Na 2 S 2 O 3 had a major effect, and NaCl has the least effect on cephamycin C production by N. lactamdurans. Fig 4 represents the main effects plot for the system. Main effects plot show how each factor affects the response characteristic. A main effect is said to be present when different levels of a factor affect the characteristic differently. MINITAB creates the main effects plot by plotting the characteristic average for each factor level. These averages are the same as those displayed in the response Table 4. A line connects the points for each factor. A horizontal line (parallel to the x-axis) indicates absence of main effect. Each level of the factor affects the characteristic in the same way and the characteristic average is the same across all factor levels. A main effect is present when the line is not horizontal (not parallel to the x-axis). Different levels of the factor affect the characteristic differently. The greater the difference in the vertical position of the plotted points (the more the line is not parallel to the x- axis), the greater is the magnitude of the main effect. In the present study it was seen that among the 8 variables at 2 levels, one level increased the mean compared to the second level. This difference is a main effect. Variables like glycerol, NaCl and MgSO 4 showed greater mean at level one whereas yeast extract, L-glutamic acid, K 2 HPO 4 , FeSO 4 and Na 2 S 2 O 3 showed greater mean at level two. Glycerol Yeast Extract L-Glutamicacid KZHPO 4 NaCl MgSO 4 FeSO 4 Na2SO3 Mean cephamycin C (mg/L) Fig. 4. Main effects plot for means Lalit D. Kagliwal et al
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Page 175: (December 7-9, 2011) Venue: Karunya
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