Studies on the Production of Cyanophycin in Recombinant E. coli Carrying Cyanophycin Synthetase Gene and the Medium Optimization by Using Response Surface Method

• Main Authors: Huang, Huei-Ru, 黃蕙如
• Other Authors: Fang Tsuei-Yun
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/29tp3x

碩士 === 國立臺灣海洋大學 === 食品科學系 === 102 === Cyanophycin is a product from cyanobacteria, which is also called blue-green algae. Cyanophycin is composed by L-aspartic acid and L-arginine in impartial moles. The molecular weight of cyanophycin is around 25-100 kDa. It can be used to produce polyaspartic acids in industry, and can be widely applied in the biomedical and pharmaceutical manufacturing. Our laboratory had previously cloned the gene of cyanophycin synthetase into pRham N-His SUMO Kan Vector, expressed in E. coli-NEB-turbo to produce cyanophycin, and found that the optimal induction was carried out with 0.1% of rhamnose for 24 hours at 20℃ after the OD600 reaching 0.4. However, recombinant E. coli NEB-turbo carrying pRham-cphA vector grew poorly in TB medium, and has a low yield of cyanophycin. Thence, three vectors carrying cphA (pRham-cphA, pET-21b-cphA, and pET-21b-His-cphA) were respectively transferred into CleanColi BL21(DE3), CleanColi BL21(DE3)-CodonPlus, and E. Coli BL21(DE3)-CodonPlus-RIL cells, respectively. We found that these three vectors in CleanColi BL21(DE3) and CleanColi BL21(DE3)-CodonPlus all have resulted low yields of cyanophycin. Our laboratory also had previously transferred pET-21b-cphA and pET-21b-His-cphA, respectively, into E. coli BL21(DE3)-CodonPlus-RIL, and found that the yields of cyanophycin weren’t very high. When pRham N-His-cphA was transferred into E. coli BL21(DE3)-CodonPlus-RIL, a high yield of cyanophycin can be obtained by induction with 0.1% of rhamnose under 100 rpm, 20℃ for 24 hours. Tryprone, yeast extract and potassium phosphates were choosen to search for the optimal conditions, and then response surface methods was used for statistical analysis. We found that tryptone is an indispensable factor in cyanophycin production. Yeast extract has been upgraded to 1.37 times in optimal TB medium. Potassium phosphate plays a negative correlation with cyanophycin yield. Therefore, the optimal TB medium contains 2.4% tryptone, 3.28% yeast extract, 0.4% glycerol, 50 mM aspartic acid, 50 mM arginine, and induction with 0.2% of rhamnose under 100 rpm, 20℃ for 24 hours, which can lead to a higher yield of cyanophycin. Keywords：cyanophycin, medium optimiztion, response surface method.