Ehab R. El-Helow

Application of the response surface methodology for optimizing the activity of an aprE-driven gene expression system in Bacillus subtilis

Abstract The major targets for improvement of recombinant expression systems in microbial cells are gene dosage, transcriptional control machinery and, to some extent, translation. Here we show that optimization of fermentation conditions by applying statistically designed, multifactorial experiments offers an additional method for potential enhancement of gene expression systems. A chromosomally encoded fusion between the Bacillus subtilis aprE regulatory region and the E. coli lacZ gene carried by the B. subtilis host cells was used. The 2 × SG sporulation medium was used as a basal medium. Among the 11 fermentation factors we examined, the most significant variables influencing β-galactosidase expression were statistically elucidated for optimization and included peptone, MgSO4 · 7H2O, and KCl. The optimum concentrations of these variables were predicted by using a second-order polynomial model fitted to the results obtained by applying the Box-Behnken design, a response surface method. Calculated optimum concentrations were predicted to confer a maximum yield of 2,423.5 β-galactosidase specific activity units. A verification experiment performed under optimal conditions yielded 96% of the predicted specific activity value with an increase by a factor of almost 5 compared with the results obtained under basal conditions.

Lichenase production by catabolite repression-resistant Bacillus subtilis mutants: optimization and formulation of an agro-industrial by-product medium

Abstract Among four isogenic catabolite repression-resistant Bacillus subtilis mutants grown on four different media, the strain crsA47 and the formula MII were chosen. Selection was supported by analyzing the results using the two-way Anova test. Supplementation of the medium with pectin resulted in a 1.33-fold increase in enzyme specific activity. A 2 n factorial experiment was then applied to elucidate medium components that significantly affect enzyme formation. Significant factors were simultaneously optimized using the steepest ascent method. Optimized medium (tryptone, 10; yeast extract, 10; pectin, 2; and NaCl, 2 g l −1 ) yielded a crude filtrate with lichenase activity of 114 U ml −1 and a specific activity of 20 U mg −1 dry protein within 24 h of cultivation. Catabolite repression of lichenase synthesis was further limited by growing the crsA47 mutant on complex raw materials. The enzyme was maximally produced (123 U ml −1 , 38 U mg −1 protein) by the cofermentation of cotton seed meal (36 g l −1 ) and lemon peel (9 g l −1 ).

Biodegradation of Chlorpyrifos by a Newly Isolated Bacillus subtilis Strain, Y242

ABSTRACT A bacterial strain Y242 isolated from agricultural wastewater was found to be highly effective in degrading chlorpyrifos. On the basis of morphology, physiological characteristics, biochemical tests, and phylogenetic analysis of 16S rRNA sequence, the isolate was identified as Bacillus subtilis. The efficiency of the B. subtilis Y242 isolate as a chlorpyrifos degrader was examined under different culture conditions formulated according to the Plackett-Burman experimental design. It was observed that B. subtilis Y242 was able to utilize chlorpyrifos as a sole carbon and energy source and grows on a medium containing concentration up to 150 mg/L. A growth medium formulated based on the results of the Plackett-Burman experiment and supplied with 150 mg/L chlorpyrifos recorded 95.12% pesticide decomposition within 48 h. Degradation study of chlorpyrifos by B. subtilis Y242 was examined by gas chromatography–mass spectrometer (GC-MS) and high-performance liquid chromatography (HPLC). These results suggest that B. subtilis Y242 will be potentially useful in the cleanup of contaminated pesticide waste in the environment.

Production of α-mannanase by B. subtilis from agro-industrial by-products: screening and optimization

We have demonstrated that a mixture of wheat bran (35 g l-1), as a main substrate, and palm seed powder (10 g l-1), as a co-substrate, is appropriate for β-mannanase production by Bacillus subtilis. A 2n factorial experimental design was employed as a primary step for medium optimization. The enzyme activity titters obtained at the optimized growth condition were equivalent to about 319% of the β-mannanse activity and 114% of the specific activity levels reached by a galactomannan-based culture.

Microbial control of the cotton leafworm Spodoptera littoralis (Boisd.) by Egyptian Bacillus thuringiensis isolates

Four local Bacillus thuringiensis (Bt) isolates that had been serologically identified as Bt var. kurstaki (Btk2, Btk3, and Btk66) and Bt var. mexicanensis (Btm27), in addition to two reference strains (4D20 and 4AC1), were laboratory assayed as microbial control agents against the Egyptian cotton leafworm Spodoptera littoralis (Boisd.). Polymerase chain reaction (PCR) amplification analysis revealed that each of the six experimental strains carries, at least, a cry1 type gene which expresses a protein toxin active against lepidopterous insects. Additionally, PCR amplification results demonstrated that 4D20 and Btk66 contain the Lepidoptera- and Diptera-active cry2 type gene and that Btk66 contains Coleoptera-active cry7 and cry8 genes. Among the six strains, Btk66 and Btm27 were the most promising microbial control agents against S. littoralis. The present findings were the first to report that Btm27 (classified as B. thuringiensis var. mexicanensis) is a very potent microbial control agent against S. littoralis-tested larvae. For more characterization of these two isolates, the sspO gene was investigated as a molecular chronometer. The DNA sequencing results proved that Btk66 and Btm27 carry sspO open reading frames with identical nucleotide sequences, suggesting a strong phylogenetic relationship between the two strains.

Economic production of baker's yeast using a new Saccharomyces cerevisiae isolate

A total of eight cultivable purified Egyptian yeast phenotypes were isolated from different sources, including fruits, juices and paste, and were compared to a bakers yeast factory reference strain. Genotypic characterization of the most promising new isolate (RO1) confirmed its identification as a Saccharomyces cerevisiae strain. In a shake-flask experiment, the Plackett–Burman multi-factorial design was applied to identify factors that considerably affect the RO1 growth rate. Together with the components of the factory molasses-based medium, six other culture factors, hypothesized to affect yeast biomass production, were examined as independent variables. The calculated main effect results and P-values suggested that by increasing the level of molasses, diammonium phosphate and inoculum size, compared to the factory settings, and by supplementing the medium with yeast extract, calcium pantothenate (vitamin B5) and trace elements, the RO1 biomass production was improved. Application of the predicted near-optimum fermentation conditions with scaling up the culture medium to 22 L in a 40 L airlift bioreactor resulted in 93 g L−1 biomass production, which represented approximately a 1.5-fold increase, when compared to the reference culture condition. Moreover, the dough raising test indicated that the newly isolated yeast strain RO1 caused a 1.75-fold increase in the fermentative power, when compared to the factory reference strain.