Investigation of the media pH effects on the amount of protein secretion of Burkholderia mallei by UV-VIS spectroscopy

Glanders occurs primarily in equines, but other species,including humans, may become incidental hosts [1]. Thcausal agent of glanders is the bacterium Burkholderia mallei, a small, Gram negative, non-motile, encapsulated,facultative intracellular rod [2,3]. The disease presents in three main forms: pulmonary, nasal and cutaneous [4]. The malination test is used to identify the disease. Because of the low concentration of these malein-derived proteins, optimization of existing methods was carried out.One of the factors affecting the secretion of these proteins is pH bacterial culture. Media were prepared, adjusted to the desired pH with HCl or NaOH and sterilized in theautoclave. Before using, the pH of the broth was determined electrometrically. Burkholderia mallei after 72 days incubation in 37° C the broth culture with difference pH as: 6.4, 6.8, 7, 7.2 was steamed at 100 for 1 hour ,sterility-tested, centrifuged at 10000g and the supernatant filtered and stored aseptically at 4 C. this filtrate served as crude mallein and was further processed for separation of malleo-proteins. After incubation, by observing the growth of the bacteria and activating the bacteria in order to obtain the most suitable culture conditions, the Lurie method was used to protein assay by using the UV spectrometer. The Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques. The method combines the reactions of copper ions with the peptide bonds under alkaline conditions with the oxidationf aromatic protein residues. The Lowry method is based on the reaction of Cu+, produced by the oxidation of peptide bonds, with Folin-Ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid in the Folin-Ciocalteu reaction).Experiments have shown that cysteine is also reactive to the reagent. Therefore, cysteine residues in protein probably also contribute to the absorbance seen in the Lowry assay [4]. The result of this reaction is an intense blue molecule known as hetero polymolybdenum Blue [5]. The concentration of the reduced Folin reagent (heteropoly molybdenum Blue) is measured by absorbance at 660 nm [6]. As a result, the total concentration of protein in the sample can be deduced from the concentration of tryptophan and tyrosine residues that reduce the Folin-Ciocalteu reagent. With regard to read absorption, It was found that the highest growth was observed in pH 7 and This pH has the highest protein content. So the sample with the pH 7 is suitable for the production of PPD Mallein.