Pareto plot of the PBD showing the significant nutrient factors affecting the yield of the enzyme _L-glutaminase.

<p>The nutrient factors were ranked in order from highly significant (top) value to the least significant. The nutrient factors greater than the alpha value 0.05 were considered as significant.</p

Estimated effects and coefficients from the Plackett Burman design.

<p>The table shows the regression analysis of the nutrient factors with their corresponding <i>P</i>-value. R Square 99.99%, R Square predicted 98.42%, R Square adjusted 99.88%, Significant values *, and non-significant values ^.</p

Comparison of enzyme yield before and after chemical treatment.

<p>Scatter plot showing the yield of enzyme in IU/mL compared between 30 samples of chemically treated strains to that of 30 sets of non-chemically-treated adapted and UV-induced strains. The yield can be seen to increase slightly towards the upper end of the chemically treated strains compared to the control.</p

Analysis of variance of the quadratic model.

<p>The table shows the outcome of ANOVA for the quadratic model. DF -Degrees of Freedom, Seq SS- sequential sum of squares and Adj Ms-Adjusted Mean square.</p

Estimated regression coefficients in the optimization of nutrient factors by central composite design.

<p>The table shows the regression analysis of the nutrient factors with their corresponding <i>P</i>-value. R Square 94.38%, R Square predicted 83.89%, R Square adjusted 90.97%, # denotes significant values, and ^ denotes non-significant values.</p

Enhancement of _L-glutaminase production by adaptive mutation of <i>Av</i>.

<p>The isolated strains of <i>Av</i> were repeatedly sub-cultured every three days in SMA medium for 60 days to ensure adaptive mutation. The yield of the enzyme in the mutated strains was significantly higher when compared with the native strain. The data are shown as means ± SEM of three independent observations (* P<0.05, **P<0.01 and ***P<0.001).</p

Glutaminase gene sequences of wild and mutated organisms of <i>Aeromonas veronii</i>.

<p></p><p></p><p>a)</p><p>Alignment of the gene sequences from 1 to 441 nucleotide bases of wild and mutated organisms of <i>Aeromonas veronii</i>.</p><p></p><p>b)</p><p>Alignment of the gene sequences from 442 to 915 nucleotide bases of wild with 442 to 918 nucleotide bases of mutated organisms of <i>Aeromonas veronii</i>.</p><p></p><p></p><p></p> <p>Alignment of the gene sequences from 1 to 441 nucleotide bases of wild and mutated organisms of <i>Aeromonas veronii</i>.</p> <p>Alignment of the gene sequences from 442 to 915 nucleotide bases of wild with 442 to 918 nucleotide bases of mutated organisms of <i>Aeromonas veronii</i>.</p> <p>The sequences were aligned for similarities by T-Coffee program. Glutaminase_Wil—Glutaminase gene sequence of wild organism, Glutaminase_mut- Glutaminase gene sequence of mutated organism and cons—highly conserved regions of the sequence.</p

Full factorial central composite design matrix showing predicted and real values in the optimization of _L-glutaminase yield with the screened nutrient factors.

<p>E.A = Enzyme activity IU/mL, P.E.A = Predicted Enzyme Activity IU/mL. Corresponding five levels of the actual values were _L-glutamine (1, 3.5, 6, 8.5 & 11 g/L), Lactose (0.5, 2, 3.5, 5& 6.5 g/L), Glucose (0.5, 2, 3.5, 5 & 6.5 g/L), Peptone (1, 3, 5, 7 & 9 g/L) and Fructose (1, 2.5, 4, 5.5 & 7 g/L).</p

Enhancement of the production of _L-glutaminase, an anticancer enzyme, from <i>Aeromonas veronii</i> by adaptive and induced mutation techniques

<div><p>Microbial anti-cancer enzymes have been proven to be effective and economical agents for cancer treatment. <i>Aeromonas veronii</i> has been identified as a microorganism with the potential to produce _L-glutaminase, an anticancer agent effective against acute lymphocytic leukaemia. In this study, a selective medium of <i>Aeromonas veronii</i> was used to culture the microorganism. Strain improvement was done by adaptive and induced mutational techniques. A selective minimal agar media was incorporated for the growth of the strain which further supports adaptive mutation. Strains were also UV-irradiated and successively treated with N-methyl-N'-nitro-N-nitrosoguanidine to find a resilient strain capable of producing _L-glutaminase efficiently. The Plackett-Burman design and central composite designs were used to screen and optimize additional carbon and nitrogen sources. Adaptive mutation resulted in promising yield improvements compared to native strain (<i>P</i><0.001). The mean yield of 30 treated colonies from the induced mutation was significantly increased compared to the non-induced strain (<i>P</i>< 0.001). The economically feasible statistical designs were found to reinforce each other in order to maximize the yield of the enzyme. The interactions of nutrient factors were understood from the 3D response surface plots. The model was found to be a perfect fit in terms of maximizing enzyme yield, with the productivity improving at every stage to a fourfold output of enzyme (591.11 ±7.97 IU/mL) compared to the native strain (135±3.51 IU/mL).</p></div

Two-way ANOVA results of strain, time and strain and time interaction.

<p>Two-way ANOVA results of strain, time and strain and time interaction.</p