| 论文题目: | Characterization of UDP-glucose dehydrogenase from Pasteurella multocida CVCC 408 and its application in hyaluronic acid biosynthesis |
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| 作者: | Chu Xin, Han Jian, Guo Donghui, Fu Zhenzhou, Liu Weifeng, and Tao Yong*. |
| 联系作者: | |
| 刊物名称: | Enzyme Microb Technol |
| 期: | |
| 卷: | 85 |
| 页: | 64-70 |
| 年份: | 2016 |
| 影响因子: | 2.932 |
| 论文下载: | http://www.sciencedirect.com/science/article/pii/S0141022915300946 |
| 摘要: | Hyaluronic acid (HA), a vital acid mucopolysaccharide, has immense applied value in foodstuffs, medicaments, and cosmetics among others. UDP-glucose dehydrogenase (UGDH, EC 1.1.1.22) is an essential enzyme for HA synthesis. In this study, a UGDH (PmuHasB, 45.9kDa) from Pasteurella multocida CVCC 408 was expressed in Escherichia coli BL21 (DE3). It was purified by two chromatographic columns with a specific activity of 6.58IU/mg. The optimum pH and temperature were determined to be 10.0 and 37 degrees C, respectively. The activity was stable across the pH range 6-10, and had a half-life of about 3h at 45 degrees C. The estimated apparent Km values for UDP-glucose and NAD(+) were 0.11 and 0.069mM, respectively. The results indicated that PmuHasB was an alkaline and mesophilic UGDH. PmuHasB and PmuHasA (HA synthase, HAS) were co-expressed in E. coli BW25113 to obtain a HA high-producing strain pBPAB/BW25113. It produced about 2.39g/L HA in shake flask by using the method of whole-cell catalysis. Investigation of the different UGDHs on HA synthesis revealed that intracellular UGDH activity and HA total yield of pBPAB/BW25113 (0.15IU/mg and 5.4g/L) were higher than from pBPASB/BW25113 (0.013IU/mg and 2.8g/L) and pBPAEB/BW25113 (0.010IU/mg and 2.22g/L). These results indicated that the activity and stability of UGDH plays a significant role in HA production, and should prove useful for further genetic engineering research with a view to construct other glucuronic acid polysaccharide synthesis pathways. |
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