| Summary: | 碩士 === 國立臺灣科技大學 === 化學工程系 === 99 === Cyanophycin granule polypeptide (CGP) is a non-ribosomal protein found in most groups of cyanobacteria. It was discovered in 1887 by Borzi during microscopic studies of cyanobacteria. Most cyanobacteria harbor a functional cyanophycin synthetase gene (cphA) and synthesize CGP. The branched polymer consists of an equimolar amount of arginine and aspartic acid.
In this study, we used Synechocystis sp. PCC 6803 cphA in E. coli BL21-CodonPlus(DE3)-RIL to produce CGP. The pET expression system was employed to produce CGP. After induction by 0.01 mM IPTG and different amounts of supplemented amino acids to the culture medium, the yields of purified CGP were compared. The molecular weights were analyzed by SDS-PAGE and MALDI-TOF MS. The amino acid compositions were assayed by HPLC. The physical properties of CGP were further analyzed by XRD, TGA, and FTIR.
The results showed that the medium supplemented with either glutamate or aspartic acid had no effect on the amino acid compositions of CGP. The bacteria grown in the presence of arginine produced a higher amount of insoluble CGP contents with a reduced yield of soluble CGP contents. The molecular weights of both insoluble (17.4-34.8 kDa) and soluble (8.3-20.3 kDa) CGP analyzed by SDS-PAGE were lower than the CGP from the non-supplemented culture condition. The purified soluble CGP from the supplemented arginine culture showed 36.46±0.49 mole% arginine, higher than the purified soluble CGP (22.38±0.63 mole% arginine) from the unsupplemented culture condition. The molecular weight of arginine-rich soluble CGP analyzed by MALDI-TOF MS is 18.84 kDa, lower than the typical soluble CGP (20.19 kDa). In addition, the medium supplemented with arginine might decrease the crystallinity of soluble CGP. The medium supplemented with lysine showed a higher soluble CGP content with a decreased insoluble CGP content, and had no effect on the amino acid compositions of CGP. The TGA and FTIR analysis showed that the CGP exhibited similar properties irrespective of the supplemented amino acids.
In conclusion, the bacteria grown in the presence of supplemented arginine may affect the molecular weight, yield, and amino acid compositions of CGP. The supplementation of the medium with lysine could increase the soluble CGP yield.
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