فایل ورد کامل بهینه سازی آماری القای تولید فیتاز با آرابینوز در یک E. coli نوترکیب با استفاده از روش سطح پاسخ

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تعداد صفحات این فایل: ۱۴ صفحه

بخشی از مقاله انگلیسیعنوان انگلیسی:Statistical Optimization of the Induction of Phytase Production by Arabinose in a recombinant E. coli using Response Surface Methodology~~en~~

Abstract

The production of phytase in a recombinant E.coli using the pBAD expression system was optimized using response surface methodology with full-factorial faced centered central composite design. The ampicilin and arabinose concentration in the cultivation media and the incubation temperature were optimized in order to maximize phytase production using 23 central composite experimental design. With this design the number of actual experiment performed could be reduced while allowing eludidation of possible interactions among these factors. The most significant parameter was shown to be the linear and quadratic effect of the incubation temperature. Optimal conditions for phytase production were determined to be 100 µg/ml ampicilin, 0.2 % arabinose and an incubation temperature of 37C. The production of phytase in the recombinant E. coli was scaled up to 100 ml and 1000 ml.

۱ Introduction

Most cereals and legumes are rich in protein and fat but the presence of phytate discourages their use in food and feed. Approximately 70% of total phosphorus in cereal grains and legume seeds occurs in form of phytate. Phytate is poorly utilized by monogastric animals and acts as an anti-nutrient due to the chelation of various metals and the binding to protein. This diminishes the bioavailability of proteins and nutritionally important minerals. In animal diets, the supply of phosphorus, an essential mineral for animal growth and development, comes from either the feedstuffs or from inorganic phosphate added to the diets (Poulsen, 2000). These nutritional impediments result in the release of undigested phytate phosphorus in the feces and urine. Phosphorus in the environment accelerates eutrophication of fresh waters and is the main problem in surface water quality, resulting in restricted water use for fisheries, recreation, industry and domestic use (Krishna and Nokes, 2001). The phytate degrading enzymes or phytases convert phytate to partially phosphorylated myo-inositol phosphates and phosphate, making phosphorus available for bioabsorption (Irvin and Cosgrove, 1972). Supplementation of microbial phytase to animal diets alters the phytate complexes and also increases the bioavailability of proteins and essential minerals, providing growth performance equivalent or better than those with phosphate supplementation, and also reduces the amount of phosphorus in animal manure (Wodzinki and Ullah, 1996).

Escherichia coli is being used as the most important host organism for recombinant protein production. Expression of a novel bacterial phytase has been achieved in E. coli BL21 by arabinose induction under the control of the pBAD expression system (unpublished data). Optimization of the cultivation conditions for the productivity is becoming one of the major goals in biotechnology today. The productivity of any cultivation is affected by process parameters and media composition (Park, K.M. and Reardon, 1996). Preliminary experiments in our laboratory indicated that antibiotic concentration, arabinose percentage and incubation temperature of cultivation media play significant roles in recombinant phytase production. Therefore an investigation was performed to statistically optimize the cultivation media for the phytase production. Response surface methodology (RSM) is now being routinely used for optimization studies in several biotechnological and industrial processes (Beg et al., 2002; De Cornink et al., 2000; Puri et al., 2002). RSM is used to explain the interactions among the possible influencing parameters with limited numbers of experiment.

In this paper, we determine the optimum concentration of ampicilin supplemented in the media, concentration of arabinose for induction purpose, and incubation temperature during cultivation for maximum recombinant phytase production by using full factorial Face Centered Central Composite Design in RSM. The production of phytase was then scaled up to 100 ml and 1000 ml using the optimized cultivation conditions.

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