Bioproduction, Synthesis Mechanism And Cryoprotective Studies Of Poly-γ-glutamic Acid

Poly-γ-glutamic acid(γ-PGA) is a naturally occurring poly-amino acid and a multifunctional polymer material. Generally, the molecular weight of γ-PGA ranges widely from 10 k Da- 1000 k Da. As a new macromolecular material, γ-PGA has several special physical, chemical and biological characteristics such as water-solubility, good thickening capacity, excellent absorbability, metal-binding capacity and biodegradability. This polymer is also edible, as it is non-toxic toward humans and the environment. These properties makeγ-PGA attractive for many potential applications in many fields such as food, agriculture,medicine, cosmetic and environmental protection. Microbial synthesis of γ-PGA is a complex metabolic process. Most of he present study focuses on the isolation of γ-PGA strains and the evaluation of γ-PGA production. There are few study and reliable conclusions on the biosynthesis mechanism of γ-PGA. In this study, a glutamic acid-independent γ-PGA producing stain was isolated and identified. The synthesis mechanism of glutamic acid monomers of γ-PGA was investigated, the synthetase genes pgs BCA were cloned and expressed in E.coli and the antifreeze activities of γ-PGA were researched.One glutamic acid-independent γ-PGA producing stain was isolated from the soil samples. It was classified as a B. methylotrophicus species and named B. methylotrophicus SK19.001 based on the 16 S r RNA sequences, as well as its physiological and biochemical characteristics. The 16 S r RNA sequence data was submitted to the Gen Bank databases under accession no. JQ723479. The chemical structure of the cultivation product was characterized by TLC, HPLC, FTIR, 1H NMR and 13 C NMR.The effects of various carbon and nitrogen sources on the γ-PGA productivity of SK19.001 were investigated. SK19.001 could use many kinds of carbon sources but had selectivity in nitrogen sources, only organic nitrogen source could be used by SK19.001.Sodium citrate with 15 g/L in medium could promote the production of γ-PGA by 76.5%. A high level of production of γ-PGA(33.84g/L) was obtained when SK19.001 was grown in a medium containing 30 g/L glycerol, 15 g/L sodium citrate and 50 g/L peptone for 36 h. The testing result of polysaccharides suggested that B. methylotrophicus SK19.001 did not produce exopolysaccharides. The γ-PGA product showed an ultra-high molecular weight of over 10,000 k Da, with 65%~70% portion of D-glutamic acid monomer.Tricarboxylic acid cycle intermediates and amino acids of glutamic acid family could participate in the γ-PGA synthesis via independent pathways and generated γ-PGA at similar efficiency except for the glutamine. The effect of citric acid, α-ketoglutaric acid, succinate and L-glutamine on the key enzymes around the 2-oxoglutarate branch(isocitrate dehydrogenase, α-ketoglutarate dehydrogenase complex and glutamate dehydrogenase) wereinvestigated. The citric acid(50 m M in medium) could raise the activity of isocitrate dehydrogenase and glutamate dehydrogenase for 8 times and 3 times, respectively. the activities of enzymes involved in L-glutamate synthesis in SK19.001 were assayed. The results suggested that the de novo synthesis of endogenous L-glutamic acid in B.methylotrophicus SK19.001 was carried out via the pathway of aminotransferase, glutamate synthase and glutaminase rather than the pathway of glutamate dehydrogenase. On further investigation it was found that glutamate racemase was responsible for the formation of D-glutamate for the synthesis of γ-PGA and the γ-PGA synthetase had stereoselectivity for glutamate substrate for the fact that the ratio of D- glutamate in cells was constantly changing,while the the ratio of D- glutamate in γ-PGA kept invariable during the process of cultivation(65%~70%).The synthetase genes pgs BCA were cloned by extraction of genomic DNA and PCR expansion, the sequenced genes were submitted to the Gen Bank databases under accession no.KP698949(pgs B), KP698950(pgs C) and KP698951(pgs A). Comparison study of the SK19.001 strain’s pgs BCA genes and Pgs BCA synthetase complex with those from otherγ-PGA-producing strains showed high similarity in glutamate-independent strains and glutamate-dependent strains, respectively. However, the pgs BCA genes and Pgs BCA synthetase complex from glutamate-independent strains had lower similarity with those from glutamate-dependent strains, which implied that pgs BCA genes were related to the glutamate dependence of γ-PGA producing strains. pgs B and pgs C from different strains had great similarity than pgs A. The p ET-28-pgs vector harboring pgs BCA genes were constructed and transformed into Escherichia coli BL21. E.coli BL21 could synthesize γ-PGA with a yield of0.65 g/L and 0.45 g/L when cultivated in the flask culture of LB plus 2% glucose and L-glutamate, respectively. The pgs BCA genes were proved be essential for the synthesis ofγ-PGA in B. methylotrophicus SK19.001.γ-PGA produced by B. methylotrophicus SK19.001 were added into frozen dough to research its antifreeze activity. The fermentation ability of the frozen dough added with0.2%~0.4% γ-PGA was commensurate with that of the frozen dough added with 12%trehalose, which implied that γ-PGA had high antifreeze activities at low concentrations. The antifreeze activity of γ-PGA decreased as its molecular weight decreased. γ-PGA showed slightly antifreeze activity when the molecular weight of γ-PGA decreased into 104 k D. The antifreeze activity was cation dependent decreasing in the order Ca2+﹥Mg2+﹥Na+﹥K+. The effect of γ-PGA on the survibility of yeast was studied. γ-PGA was found to raise the survibility of yeast significantly as compared to the cells with no γ-PGA protection. SEM images showed that the yeast was encapsulated within γ-PGA to prevent damage from icecrystal. DSC results indicated that γ-PGA decreased the freezable water of frozen dough.SEM images of frozen dough added with γ-PGA showed that its gluten network was better protected and large ice crystal formation was prevented.