Growth Na2HPO4 contain significant positive effect on E.coli growth

Growth condition
has been reported as an important factor for increasing the production of a
protein. Hence, the optimization of growth medium
composition has been proposed as a notable way to augment the production of
recombinant protein (Nikerel,
Öner et al. 2006, Vásquez-Bahena,
Vega-Estrada et al. 2006, Baskar,
Dharmendira Kumar et al. 2009). In the present work, the effect of nine different variables was
investigated on E.coli growth and production of LjPAL6 using
fractional factorial design. The level of each variable and the results of the
experiments are provided in Table 5.  Statistical data analysis is presented
in Table
6 for the selected variables demonstrating that three variables including
tryptone, yeast extract and Na2HPO4 contain significant
positive effect on E.coli growth and production of LjPAL6
(P<0.05). The linear regression coefficient (R2) determined for biomass yield and expression level was 0.996 and 0.995, respectively, indicating the validation of the model for fractional factorial design. Different carbon sources including, sucrose and manitol had a positive effect on both of LjPAL6 production and E.coli growth but were not significant. Glucose had a positive and negative influence on E.coli growth and LjPAL6 production, respectively, but was not significant. It has been reported that glucose has a negative effect on the regulatory system of lac operon decreasing the production of protein (Grossman, Kawasaki et al. 1998, Studier 2005). Tryptone and yeast extract as nitrogen sources had a significant positive influence on both of responses while NH4Cl had a positive effect but not significant on responses and among different mineral sources, Na2HPO4 and NaH2PO4 had a significant and not significant positive effect on the responses, respectively, while MgSO4 had a negative influence but not significant on the responses.  It has been observed that nitrogen sources are basic requirements for bacterial growth (Berenjian, Mahanama et al. 2011). The results indicated that nitrogen sources are important factors influencing the biomass yield and production of LjPAL6. According to the results, among different variables tryptone, yeast extract and Na2HPO4 had a significant positive effect (P<0.05) on both of biomass yield and expression level and therefore, these variables were selected for optimization of LjPAL6 production by response surface methodology (RSM). To determine the interaction among effective variables and their optimal levels central composite design (CCD) was applied. The results of CCD experiments are provided in Table7. The experimental design regression analysis for linear, quadratic and interaction terms are presented in Table. 8. The R2 value of 0.93 and 0.91 assessed for relative expression of LjPAL6 and biomass yield, respectively, confirmed the validation of the model. The regressions predicted for the LjPAL6 relative expression (Eq. 1) and biomass yield (OD600) (Eq. 2) are included in the following equations: Y= 6057.46+2158.16 X1+1643.96 X2+1525.85 X3+2935,65 X1X2-1956.35 X2X3+693.44 X12 Y=1.15+0.19 X1+0.19 X2+0.13 X3+0.29 X1X2-0.19 X2X3  The insignificant terms were not considered in the equations. X1, X2 and X3 are the concentrations of tryptone, yeast extract and Na2HPO4, respectively. ANOVA test and the model goodness are given in Table. 9. Low p value shows the model's validity. The results showed that high production of LjPAL6 is associated with increasing in the biomass yield. Similar results have been obtained by other studies that high cell density is led to the increase in production of proteins (Chen, Hwang et al. 1992, Vásquez-Bahena, Vega-Estrada et al. 2006). The surface plots of LjPAL6 production and biomass yield (OD600) are presented in Fig. 10 for each pair of variables which indicate the LjPAL6 production and biomass yield (OD600) are increased by enhancing in concentration of tryptone, yeast extract and Na2HPO4. The optimal values of tryptone, yeast extract and Na2HPO4 were 10, 8.5 and 7.5 g/l, respectively. Under these conditions, the production of LjPAL6 was about three times (83.7 mg/l) more than the basal medium (33.5 mg/l).  

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