Optimization of Production of the Anticancer Agent L-Methioninasebypseudomonasextremaustralis

International Journal of Pharmaceutical and Phytopharmacological Research (eIJPPR) | December 2019| Volume 9| Issue 6| Page 81-88 Al-Zahrani, SHM,Optimization of Production of the Anticancer Agent L-methioninasebyPseudomonasextremaustralis Optimization of Production of the Anticancer Agent L-methioninase byPseudomonas extremaustralis Al-Zahrani, SHM1, Bughdadi, A2, Alharbi, AA3, Bukhari, KA2* 1Biology department, Faculty of Science, King Abdulaziz University, KSA 2Biology department, Faculty of Science, University of Jeddah, KSA 3 Biology department, Faculty of Science, University of Jazan, KSA. ABSTRACT The present study aim optimization Pseudomonas extremaustralis production of L-methioninase. The bacterial isolate ability for L-methioninase production was tested on a modified mineral salt M9 agar medium, using phenol red as the pH indicator. Result of optimization L-methioninase production, the cultural and nutrition parameters revealed to the maximum amount of L-methioninase produced by P. extremaustralis were 0.192U\min\ml, obtained after 48h at 35°C of incubation in shaking incubator (150rpm) in culture supplemented with L-methionine mixed with L-glutamine (0.15%), in the presence of glycerol (0.3%) as carbon source at pH6. Key Words:L-methioninase, methionine, antitumor agent, anti-cancer, Pseudomonas extremaustralis. eIJPPR 2019; 9(6):81-88 HOW TO CITE THIS ARTICLE:Al-Zahrani, SHM, Bughdadi, A, Alharbi, AA, Bukhari, KA(2019).“Optimization of Production of the Anticancer Agent L-methioninase by Pseudomonas extremaustralis”, International Journal of Pharmaceutical and Phytopharmacological Research, 9(6), pp.81-88. INTRODUCTION Nesslerization method, the highest enzyme production was obtained from a seashell sample, then the optimization of MGL production involves one-step degradation of L- the production conditions for maximum L-methioninase methionine by versatile enzyme L-methionine g-lyase. A production. pyridoxal -phosphate-dependent enzyme catalyzing α,γ - elimination of L-methionine to α-ketobutyrate, MATERIALS AND METHODS methanethiol and ammonia and that were existed in many bacteria such as Pseudomonas putida[1].Tumor freed Bacterial isolate: from efficient methionine synthase and external Pseudomonas extremaustralis obtained from other studies methionine comes from the diet [2]. L-methioninase is a by Al-Zahrani and Bukhari, (10). therapeutic agent against tumors [3, 4]. Methionine reduction has a broad spectrum of antitumor activities [5]. OptimizeL-methioninase production byPseudomonas L-methioninase found in most organisms except extremaustralis mammals. Moreover, found in the cell-free extract [6,7] Effect of incubation conditions: and many bacteria species as intracellular enzyme[8], in A loop full of a 24h culture of the selected bacterial isolate bacteria, intracellular L-methioninlytic enzyme have extra was inoculated with 30ml of a sterile seed medium in a limiting step during the scale production while in fungi test tube and incubated at 35±2°C in a constant incubator was both intracellular and extracellular [9]. for 24h. The optimization of enzyme production was evaluated by varying one factor at a time. Aim: For the best incubation conditions for L-methioninase Bacteria screening of L-methioninase in the Saudi marine production by Pseudomonas extremaustralis, a modified environment, the enzyme production was estimated by the Corresponding author:Bukhari, KA Address:Biology Department, Faculty of Science, University of Jeddah, KSA . E-mail: Kawtharbukhari @ hotmail.com Relevant conflicts of interest/financial disclosures: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Received: 16 July 2019; Revised: 24 November 2019; Accepted: 06 December 2019 ISSN (Online) 2249-6084 (Print) 2250-1029 www.eijppr.com International Journal of Pharmaceutical and Phytopharmacological Research (eIJPPR) | December 2019| Volume 9| Issue 6| Page 81-88 AAl-Zahrani, SHM,Optimization of Production of the Anticancer Agent L-methioninasebyPseudomonasextremaustralis M9 medium; Na2HPO4. 2H2O 6, KH2PO4, NaCl 0.5, L- trichloroacetic acid. And centrifuged at 5,000rpm for 5min methionine 1, MgSO4. 7H2O 0.25, CaCl2 0.014, Glucose to remove precipitated protein and added to 3.7ml of 2 g/L was used as the fermentation medium. The bacterial distilled water where released ammonia was determined cultures incubated at 35±2°C. L-methioninase in the cell- using 0.2ml of Nessler reagent. L-methioninase was free filtrate was assayed after 72h of incubation. defined as liberates ammonia at 1μmol/ min under optimal Best incubation temperature and fermentation period were assay conditions. Specific activity for L-methioninase studied, cultures were incubated at different temperatures expressed as the enzyme in terms of units per milligram of (25-40±2°C). L-methioninase in cell-free filtrate CFF was protein [9]. assayed after 24, 48 and 72h. Different pH (4-8) were adjusted to determine the best pH value on L- RESULTS: methioninase production. Effect of incubation periods and different Determinethe carbon and nitrogen nutrition to temperatures on L-methioninase production optimize L-methioninase production The highest amount of the enzyme was accumulated when Optimizing L-methioninase production, the most suitable the culture incubated at 35±2°C0.05U/min/ml by carbon and nitrogen sources and concentrations were Pseudomonas extremaustralis after 48h of incubation, but studied. For the best carbon source, different carbon at 2g/ higher or lower of this degree, the production of the L was added to the fermentation medium. The optimal enzyme was decreased, results in Figures (1). concentration of carbon for L-methioninase production was determined, different concentrations (1-9g/ L) of the best carbon source were added. The optimal concentration of L-methionine was determined by using different concentrations (0.5– 1.5g\ L) of L-methionine. For the best nitrogen source, different amino acid and organic sources (1.25g\ L) were added to the fermentation medium. Mixed of L-methionine with different organic sources (tryptone, peptone and yeast extract) and different of 82 amino acids (L-form), (asparagine and glutamine) with L- methionine mixed with different nitrogen sources (1. 25g\ L) were used. Mixed of L-glutamine and peptone (0.75-1.75g\ L) were added to the fermentation medium, in the presence and absence of glycerol (3ml). Fig. 1: Effect of incubation periods and temperatures The influence of different inorganic nitrogen sources was on L-methioninase production studied. Inorganic sources as KNO₃, NaNO₃ and (NH₄)2SO₄ were substituted individually in place of L- Different carbon sources effect on L-methioninase methionine. production The optimal concentration of the inorganic nitrogen source Different carbon sources (Glucose, Mannitol, Sucrose, for L-methioninase production was determined. Different Xylose, Maltose, Lactose, Galactose, Fructose, and concentrations (1–1.45g/ L) of the best inorganic nitrogen Glycerol) Effect on L-methioninase production by the source were tested. The cultures were prepared in selected isolate of bacteria were studied. duplicates and were incubated at the propriety From Data, Figure (2) obviously, the glycerol was the best temperature, the CFF culture was assayed by Nessler carbon source for the highest amount of L-methioninase assay for L-methioninase after different incubation periods by Pseudomonasextremaustralis 0.06U/min/ml followed in hours. The culture without L-methionine (substrate) by xylose 0.04U/min/ml and mannitol, galactose and was set as control. fructose 0.03U/min/ml, compared to control 0.009U/min/ ml. L-methioninase assay by Nesslerization method L-methioninase activity was determined by the Nesslerization method [11] with modifications. The standard reaction was 1 ml of 1% L-methionine in 0.5M phosphate buffer (pH 7.0), 0.1ml of pyridoxal phosphate, and 1ml of crude enzyme and incubated at 30°C for 1 h. and stopped activity by adding 0.5ml of 1.5mol/L International Journal of Pharmaceutical and Phytopharmacological Research (eIJPPR) | December 2019| Volume 9| Issue 6| Page 81-88 AAl-Zahrani, SHM,Optimization of Production of the Anticancer Agent L-methioninasebyPseudomonasextremaustralis Fig. 2: Different carbon sources effects of on L- Fig. 4:The effect of pH on L-methioninase production methioninase production Effectof carbon source concentrations on L- Effect of different L-methionine concentrations on the methioninase production production of L-methioninase The maximum amount of L-methioninase Resultsin Figure (5) show that the highest L-methioninase production0.08U/min/ml was optained when 3ml of was 0.09U/min/ml was produced by Pseudomonas glycerol was addedto the fermentation medium Figure (3). extremaustralis when 1.25g/ L of L-methionine was added to the medium. The enzyme production was reduced by increasing of L-methionine concentration. 83 Fig. 3: Effect ofcarbon source concentrations on L- methioninase production Effect of pH on L-methioninase production Fig. 5: Effect of L-methionine concentrations onL- Result in Figure (4) revealed the maximum productivity of methioninase production L-methioninase by Pseudomonas extremaustralis by decreasing the pH value of the medium to pH 6, it was Effect of Magnesium Sulfate concentrations on L- 0.09U/ min/ ml. These amounts decreased at lower or methioninase production above pH 6 of the fermentation medium. Result in

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