Identification And Product Characterization Of Three Poly-γ-glutamic Acid Produing Strains And Fermentation Condition Optimization

Poly-y-glutamic acid(y-PGA) synthesized by microorganism is a kind of water-soluble extracellular homo-polyamide which is made of L-and D-glutamic acid units connected by amide linkages between α-amino and γ-carboxylic groups.γ-PGA is non-toxic to humans and the environment and biodegradable.γ-PGA and its derivatives have wide application in the fields such as agriculture, chemical industry and pharmaceutical industry.Up to now, most researches on γ-PGA are limited to lab level and γ-PGA has not been industrialized in mainland China. It is difficult to raise the yield of γ-PGA because of complex metabolism and complicated regulation mechanisms which also result in high production cost. So to screen γ-PGA-high-productive strains will be the key to γ-PGA industrialization.Three noval γ-PGA produing-strains(strain CC, DL and TW) were isolated by our lab, In order to to test the feasibility of the three strains uesd as industrial fermentation strains, taxonomy methods were used to identify them from morphological and molecular classification to get the preliminary understanding of their genetic background. Product characterization was also studied.Medium optimization both in liquid state fermentation and solid state fermentation was carried out in strain DL. Meanwhile, gene recombinant principle was used to construct a mutant TW strain whose γ-PGA degradative enzyme gene was knocked out to come up to the standard of industrial strain.The research content and summary results are as follows:1. Identification of three noval γ-PGA producing strains CC, DL and TWThree noval y-PGA producing strains(strain CC, DL and TW) isolated from our lab was identified as Bacillus subtilis based on morphological characters,16S rDNA sequences, RiboPrinter characterization, plasmid sequences and several related genes sequencing results. Comparing with the sequences of the degradation enzyme gene ywtD and y-PGA-synthesis-related gene capBCAE, the homologous rate of the three strains reached99.98%and100%respectively.2. Product Characterization of three noval y-PGA producing strains CC, DL and TWAccording to the fermentation experiments, the y-PGA yield of strain CC could reach25.81g/L with average molecular weight of820kDa after48h fermentation;the y-PGA yield of strain DL could reach31.51g/L with average molecular weight of700kDa after72h fermentation;the y-PGA yield of strain TW could reach25.80g/L with average molecular weight of900kDa after60h fermentation. Strain CC had the shortest fermentation period, strains DL got the highest y-PGA yield, strains TW possessed the largest molecular weight of y-PGA. The y-PGA of the three yield strain are D-L-glutamic acid mixed with different proportion. More L-glutamic acid exixted in y-PGA from strain CC,relatively.Comparively high flocculating rate possesd in the three strain and flocculating activity of γ-PGA from strain DL amounted to75.90%.3. Medium optimization of y-PGA production in liquid-state fermentation for strain DL with Plackett-Burman design and the steepest ascent experimentAccording to results of Plackett-Burman design, three major factors significantly affected the production of y-PGA:sodium glutamate, MgSO4·7H2O and NaCl.Then the fermentation conditions were optimized with the steepest ascent experiment.The optimum composition of medium was addition of maltose,yeast extract, sodium glutamate, NaCl, KH2PO4and MgSO4·7H2O with6.25%,1.25%,9%,3%,0.625%and0.15%, respectively.In the optimized fermentation medium, yeild of y-PGA produced by strain DL could reach81.56g/L which increased158.84%than the initial medium.4. Medium optimization of y-PGA production in solid-state fermentation for strain DLSodium glutamate, glucose and package materials significantly affected the production of y-PGA.The production of y-PGA could get up to14.86%as5%sodium glutamate added in the soybean meal substrate and is more than that of soybean substrate.5. Primary research of gene modification of Bacillus subtilis TWThis research aimed at knocking out the ywtD gene of Bacillus subtilis TW by using the homologous recombination principle.The PCR product consisted of a456bp-long homologous right arm of ywtD gene and a576bp-long homologous left arm of ywtD gene. Kanamycin resistant gene was inserted between the right arm and left arm as a selection marker through enzyme digestion and enzyme ligation. The recombined plasmid pUC19-kan-ywtD-R-L was construct successfully.lt laid the foundation for transforming the plasmid into strain TW.These findings suggested that three noval y-PGA producing strains(strain CC, and TW) isolated from our lab was identified as Bacillus subtilis.They are so different from one another at the aspect of production characterization that they can be used as industrial strains to meet different requirements, potentially..Bacillus subtilis DL has shown intriguing fermentative superiority in both liquid state fermentation and solid state fermentation.Therefore, it is of potential value in industry applications.