Exogenous Expression Of Poly-γ-glutamic Acid And Research On Its Fermentation Process

Poly-γ-glutamic acid (γ-PGA) that is made of D- and L-glutamic acid units linked by amide linkages between α-amino and γ-carboxylic acid groups is a kind of water-soluble and biodegradable anionic polypeptide. Its molecular weight ranges from 100 to 1000kDa, and it is made of 500-5000 glutamic acid units. As a kind of biomaterial, γ-PGA is non-toxic to human and the environment, water soluble, biodegradable and even edible. It had been determined that pgsB, pgsC and pgsA are all indispensable for the synthesis of γ-PGA.In this dissertation, exogenous expression of γ-PGA was studied with E.coli and Corynebacterium glutamicum as host strains. Three genes pgsB, pgsC, pgsA were cloned from Bacillus subtilis ZJU-7, and two plasmids pTrc99 and pXMJ19 were used. All three genes pgsB, pgsC and pgsA were inserted to pTrc99 to construct recombinant plasmid pTrc99a-phdBCA, and recombinant plasmid pXMJ19-pgsBCA was constructed in the same way. E.coli JM109, E.coli BL21 and Corynebacterium glutamicum RES167 were chosen as host strains. Four recombinant strains (E.coli JM109/pTrc99a-pgsBCA, E.coli BL21/pXMJ19-pgsBCA, Corynebacterium glutamicum RES167/pTrc99a-pgsBCA and Corynebacterium glutamicum RES167/pXMJ19-pgsBCA) were constructed. The capability of producing γ-PGA by the four recombinant strains were investigated, and E.coli JM109/pTrc99a-pgsBCA, E.coli BL21/ pXMJ19-pgs5BCA and Corynebacterium glutamicum RES167/pTrc99a-pgsBCA were detected to have the capability of synthesizing γ-PGA. The fermentation condition of E.coli BL21/ pXMJ19-pgsBCA was optimized, and the suitable medium was founded. The optimal medium were determined as following: tryptone 10 g/L; yeast extract 5 g/L; sucrose 10 g/L; NaCl 10 g/L; L-glutamic acid 10 g/L; 1 mM MnSO4; chloramphenicol 34 μg/ml; 1 mM IPTG. The highest productivity of γ-PGA by E.coli BL21/pXMJ19-pgsBCA can reach 1.17g/L under the above condition.A strain with high γ-PGA synthesizing ability were screened from Bacillus subtilis ZJU-7 which was preserved in our laboratory, and its γ-PGA productivity canreach 48 g/L in shake-flask culture. The optimization of Bacillus subtilis ZJU-7 fermentation condition was studied in flask scale. Then the culture process was scaled up in a 15 L fermentor, and the γ-PGA productivity reached 23 g/L. To reduce the cost of medium for producing γ-PGA, several substitutes of tryptone were studied. With maize powder as nitrogen source, the cost of nitrogen source in medium was the lowest and reached 5 yuan/kg γ-PGA.