title: Characterization of UDP-glucose dehydrogenase from Pasteurella multocida CVCC 408 and its application in hyaluronic acid biosynthesis.
abstract: 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, 45.9 kDa) from Pasteurella multocida CVCC 408 was expressed in Escherichia coli BL21 (DE3). It was purified by two chromatographic columns with a specific activity of 6.58 IU/mg. The optimum pH and temperature were determined to be 10.0 and 37°C, respectively. The activity was stable across the pH range 6-10, and had a half-life of about 3 h at 45°C. The estimated apparent Km values for UDP-glucose and NAD(+) were 0.11 and 0.069 mM, 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 2.39 g/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 (0.15 IU/mg and 5.4 g/L) were higher than from pBPASB/BW25113 (0.013 IU/mg and 2.8 g/L) and pBPAEB/BW25113 (0.010 IU/mg and 2.22 g/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.
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: chux@im.ac.cn.
authors.forename: Xin
authors.initials: X
authors.lastname: Chu
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: hanjian148.student@sina.com.
authors.forename: Jian
authors.initials: J
authors.lastname: Han
authors.affiliation: School of Life Sciences, Taishan Medical University, Taian 271000, Shandong Province, China. Electronic address: gdonghui2007@163.com.
authors.forename: Donghui
authors.initials: D
authors.lastname: Guo
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: 425212527@qq.com.
authors.forename: Zhenzhou
authors.initials: Z
authors.lastname: Fu
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: liuwfv@163.com.
authors.forename: Weifeng
authors.initials: W
authors.lastname: Liu
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: taoyong@im.ac.cn.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tao
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D007700:Kinetics; D016979:Pasteurella multocida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006820:Hyaluronic Acid; D014533:Uridine Diphosphate Glucose Dehydrogenase
keywords: HA synthase; Hyaluronic acid; Pasteurella multocida; UDP-glucose dehydrogenase; Whole-cell catalysis