Study On The Production Of Poly γ-glutamic Acid By Microorganism

Poly-γ-glutamic acid (y-PGA) is a kind of water-soluble and biodegradable anionic polypeptide that is made of D- and L-glutamic acid units linked by amide linkages between α -amino and γ-carboxylic acid groups. γ-PGA is of various applications in medicine, food and agriculture, etc.In this work, with y-PGA as the target molecule, a production process was studied which includes the screening and determination of start strain, the optimization of culture medium and cultivation conditions in shake-flask scale and jar fermentor scale, the establishment of fermentation kinetic model, and the development of separation and purification of γ-PGA from fermentation broth. A new co-cultivation system with Bacillus subtilis and Corynebacterium glutamicum was developed without the addition of L-glutamic acid, and the using of intermediate-product in glutamic acid production process as the precurser were also established in order to lower the production cost of γ-PGA. Furthermore, a pgsBCA gene was cloned and expressed in E. coli JM109. Finally, a recombined E.coli JM109/pTrc99a-pgsBCA strain with y-PGA producing ability was obtained.A strain with high y-PGA producing ability was screened from natural environment. The strain was named as Bacillus subtilis ZJU-7 according to the result of BLAST of 16s rDNA sequence as well as the physiological and biochemical properties. The strain was preserved in CGMCC with the accession number CGMCC No. 1250.The optimization of Bacillus subtilis ZJU-7 culture conditions and the scale-up of fermentation process were investigated. The optimal medium composition in shake-flask scale were determined as followings: sucrose 59.80 g/L;tryptone 53.54 g/L;L-glutamine 81.05g/L;NaCl 10 g/L;MgSO₄-7H₂O 1.0 g/L;CaCl₂ 1.0 g/L, and the cultivation should be performed at 37℃ and 200rpm as rotate speed. The final γ-PGA productivity of 58.3 g/L was achieved after 24-32 hour cultivation. The culture process was then scaled up in a 5 L fermentor and the y-PGA productivity reached 51.7g/L.A co-cultivation system coupling a glutamine producing strain, Corynebacterium glutamicum, with a y-PGA producer, Bacillus subtilis ZJU-7, was established and the factors affecting y-PGA production in the mixed-strain culture, such as age of seed, ratio of the starins, temperature and dissolved oxygen etc, on the productivity of the γ-PGA were evaluated. Without addition of L-glutamic acid, the y-PGA productivityof 32.8 g/L was achieved in the optimized conditions.Three kinds of the intermediate-product during glutamic acid producing process were used as the substitute of purified glutamic acid. The effects of sucrose, tryptone and glutamic acid concentration on y-PGA production were also examined. A y-PGA productivity of 45 g/L was achieved in the optimized conditions.ApgsBCA gene was cloned from Bacillus subtilis ZJU-7. An expression vector pTrc99a-p#sBCA was constructed and then transformed into E.coli JM109. A recombinant strain, E.coli JM109/pTrc99a-/>g.sBCA, was obtained, which was proven to be of the ability to synthesisy-PGA.