| Pullulan, a kind of microbial polysaccharide consisting of maltotriose repeating units joined by α-1, 6 linkages, is generally produced by the polymorphic fungus Aureobasidium pullulans. Due to its unique linkage pattern, pullulan demonstrates distinctive physical properties, such as capability to form fibers, adhesiveness,impermeable to oxygen and biodegradation. As a result, pullulan has various application prospects in food, chemical, pharmaceutical industries, and environmental protection.In contrast to the former approach of fermentative pullulan production, pullulan biosynthesis was carried out by whole-cell bioconversion of glucose in this study.Aureobasidium pullulans CCTCC M 2012259 was used as the starting strain, and the feasibility of the strain to convert glucose to pullulan was explored. The seed medium for culturing the strain with high cell capability of pullulan bioconversion was optimized. The roles of surfactant on the enhanced pullulan biosynthesis, as well as the effect of metal ions on pullulan bioconversion were investigated. Furthermore, the mechanisms of improved pullulan production by surfactants and metal ions were also explored. The main contents and results of this study are summarized as follows:1. Response surface methodology was applied to optimize the seed medium of for improving the cells’ capability of pullulan bioconversion from glucose by A. pullulans CCTCC M 2012259. First, the nutrients in the basic seed medium were tested by single-factor experiments to investigate their effects on pullulan bioconversion. Based on these results, Plackett-Berman design was performed and three medium components,namely, glucose, yeast extract and Mg SO4·7H2O were identified as the most significant nutrients to affect the cells’ capability of pullulan bioconversion. A three-level Box-Behnken design was then employed to determine the optimal levels of the three components. The concentrations of the three nutrients were optimized using responsesurface analysis, and the optimal medium for culturing the seeds with high cells’ capability of pullulan bioconversion was achieved as follows: glucose 63.97 g/l, yeast extract 3.57 g/l,(NH4)2SO4 0.6 g/l, K2HPO4 5.0 g/l, Na Cl 1.0 g/l, Mg SO4·7H2O 0.18 g/l.The validation experiments showed that the cells’ pullulan bioconversion capability was32.73 mg/g/h with the optimal medium, which was improved by 23.1 % as compared to that using the basic medium. Moreover, a much quicker glucose consumption rate and a greater pullulan biosynthesis rate was obtained with the optimal medium, as compared to those obtained with the basic medium, which illustrated that the response surface analysis was feasible to optimize the seed medium. Furthermore, the gene expression and activities of the key enzymes involved in pullulan biosynthesis were evaluated.When the optimal medium was employed, the transcriptional levels of pgm1 and fks were up-regulated by 2.5- and 1.2-fold, respectively, and the activities of PGM and FKS were also increased by 17 and 19 %, respectively, when compared with those achieved using the basic medium. The above results explained the reason why the optimized seed medium improved the cells’ pullulan bioconversion capability. In the end, the cost of nutrients required for pullulan production by whole-cell bioconversion and fermentation was calculated, and the former method can save about 22.12% the cost as compared with the later approach for pullulan production.2. The effects of different types of surfactants on pullulan bioconversion were investigated, Tween and Span was found to have positive roles in improving pullulan production by bioconversion of glucose. Further studies on Tween 80 and Span 80 showed that both surfactants had the most significant effects on pullulan bioconversion.When the concentrations of Tween 80 and Span 80 were set at 5 g/l and 20 g/l, the yield of pullulan was increased by 36.10% and 38.39%, respectively, as compared with that of the control without surfactant addition. The complex addition of Tween 80 and Span80 was also studied, and the production of pullulan was further improved, but no cumulative effect of both surfactants was observed. In addition, the mechanism of improved pullulan biosynthesis by surfactants addition was investigated. Both Tween 80 and Span 80 had no significantly effect on cell survival rates, but increased the permeability of cell membrane. The activities of three key enzymes involved in pullulan biosynthesis were effectively regulated by the two surfactants, and the supply of the intracellular energy material ATP was also increased, which resulted in the significantly improved production of pullulan by the whole-cell bioconversion.3. The effects of metal ions on pullulan bioconversion were investigated and the copper ion(Cu2+) was found to significantly improve the production of pullulan by bioconversion. The additions of Cu2+ to the conversion broth and seed medium were tested, and the optimal concentrations of Cu2+ addition to both systems were obtained as0.2 mg/l, and the production of pullulan increased by 36.2% and 42.3%, respectively, as compared with the control without addition of Cu2+. Based on the above results,pullulan bioconversion by adding Cu2+ into both the conversion broth and seed medium was performed, pullulan production improved by 52% after the bioconversion of glucose for 48 h, compared with that of the control. The results indicated that Cu2+addition into the two systems had synergistic effect on pullulan production, but no cumulative effect could be found. Finally, the mechanism of Cu2+ addition improving pullulan production by bioconversion was studied. Neither negative effect of Cu2+ on cell survival rates nor the generation of melanin could be found at the moderate concentration of Cu2+. As a result, the addition of Cu2+ ion increased the activities of key enzymes involved in pullulan biosynthesis, improved the intracellular content of ATP, which in turn improved pullulan production by the whole-cell bioconversion. |
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