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Jundishapur Journal of Microbiology، جلد ۸، شماره ۱۲، صفحات ۰-۰
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| عنوان فارسی |
Optimization of Recombinant Expression of Synthetic Bacterial Phytase in Pichia pastoris Using Response Surface Methodology |
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| چکیده فارسی مقاله |
Results Escherichia coli phytase was expressed in P. pastoris under different cultivation conditions (post-induction temperature, methanol concentration, and post-induction pH). The optimized conditions by RSM using face centered central composite design were 1% (v/v) methanol, pH = 5.8, and 24.5°C. Under the optimized conditions, appA was successfully expressed in P. pastoris and the maximum phytase activity was 237.2 U/mL after 72 hours of expression. Conclusions By optimization of recombinant phytase expression in shake flask culture, we concluded that P. pastoris was a suitable host for high-level expression of phytase and it can possess high potential for industrial applications. Materials and Methods The appA gene with 410 amino acids was synthesized by P. pastoris codon preference and cloned in expression vector pPinkα-HC, under the control of AOX1 promoter, and it was transformed into P. pastoris GS115 by electroporation. Recombinant phytase was expressed in buffered methanol-complex medium (BMMY) and the expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic assay. To achieve the highest level of expression, methanol concentration, pH and temperature were optimized via RSM. Finally, the optimum pH and temperature for recombinant phytase activity was determined. Objectives In this study, the expression of synthetic appA gene in P. pastoris was greatly improved by adjusting the expression condition. Background Escherichia coli phytase is an acidic histidine phytase with great specific activity. Pichia pastoris is a powerful system for the heterologous expression of active and soluble proteins which can express recombinant proteins in high cell density fermenter without loss of product yield and efficiently secrete heterologous proteins into the media. Recombinant protein expression is influenced by expression conditions such as temperature, concentration of inducer, and pH. By optimization, the yield of expressed proteins can be increase. Response surface methodology (RSM) has been widely used for the optimization and studying of different parameters in biotechnological processes. |
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| کلیدواژه‌های فارسی مقاله |
6-Phytase،Genes،Synthetic،Pichia |
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| عنوان انگلیسی |
Optimization of Recombinant Expression of Synthetic Bacterial Phytase in Pichia pastoris Using Response Surface Methodology |
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| چکیده انگلیسی مقاله |
Results Escherichia coli phytase was expressed in P. pastoris under different cultivation conditions (post-induction temperature, methanol concentration, and post-induction pH). The optimized conditions by RSM using face centered central composite design were 1% (v/v) methanol, pH = 5.8, and 24.5°C. Under the optimized conditions, appA was successfully expressed in P. pastoris and the maximum phytase activity was 237.2 U/mL after 72 hours of expression. Conclusions By optimization of recombinant phytase expression in shake flask culture, we concluded that P. pastoris was a suitable host for high-level expression of phytase and it can possess high potential for industrial applications. Materials and Methods The appA gene with 410 amino acids was synthesized by P. pastoris codon preference and cloned in expression vector pPinkα-HC, under the control of AOX1 promoter, and it was transformed into P. pastoris GS115 by electroporation. Recombinant phytase was expressed in buffered methanol-complex medium (BMMY) and the expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic assay. To achieve the highest level of expression, methanol concentration, pH and temperature were optimized via RSM. Finally, the optimum pH and temperature for recombinant phytase activity was determined. Objectives In this study, the expression of synthetic appA gene in P. pastoris was greatly improved by adjusting the expression condition. Background Escherichia coli phytase is an acidic histidine phytase with great specific activity. Pichia pastoris is a powerful system for the heterologous expression of active and soluble proteins which can express recombinant proteins in high cell density fermenter without loss of product yield and efficiently secrete heterologous proteins into the media. Recombinant protein expression is influenced by expression conditions such as temperature, concentration of inducer, and pH. By optimization, the yield of expressed proteins can be increase. Response surface methodology (RSM) has been widely used for the optimization and studying of different parameters in biotechnological processes. |
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| کلیدواژه‌های انگلیسی مقاله |
6-Phytase,Genes,Synthetic,Pichia |
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| نویسندگان مقاله |
علی اکبرزاده | ali akbarzadeh
applied microbiology research center, baqiyatallah university of medical sciences, tehran, ir iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی بقیه الله (Baqiyatallah university of medical sciences)
احسان دهنوی | ehsan dehnavi
gene transfer pioneers research group, shahid beheshti university, tehran, ir iran
سازمان اصلی تایید شده: دانشگاه شهید بهشتی (Shahid beheshti university)
مجتبی آقایی پور | mojtaba aghaeepoor
gene transfer pioneers research group, shahid beheshti university, tehran, ir iran; semnan biotechnology research center, semnan university of medical sciences, semnan, ir iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی سمنان (Semnan university of medical sciences)
جعفر امانی | jafar amani
applied microbiology research center, baqiyatallah university of medical sciences, tehran, ir iran; applied microbiology research center, baqiyatallah university of medical sciences, vanak sq, molasadra st, p. o. box 193955487, tehran, ir iran. tel 98-2182482568, fax 98-2188068924
سازمان اصلی تایید شده: دانشگاه علوم پزشکی بقیه الله (Baqiyatallah university of medical sciences)
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| نشانی اینترنتی |
http://www.jjmicrobiol.com/index.php?page=article&article_id=27553 |
| فایل مقاله |
فایلی برای مقاله ذخیره نشده است |
| کد مقاله (doi) |
10.5812/jjm.27553 |
| زبان مقاله منتشر شده |
fa |
| موضوعات مقاله منتشر شده |
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| نوع مقاله منتشر شده |
research-article |
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