title: Gene cloning and expression of a glucoside 3-dehydrogenase from Sphingobacterium faecium ZJF-D6, and used it to produce N-p-nitrophenyl-3-ketovalidamine.
abstract: In this study, we report the cloning and expression of a functional glucoside 3-dehydrogenase (G3DH) gene from Sphingobacterium faecium ZJF-D6. This gene is 1686 bp in length and encodes a peptide of 562 amino acids. The G3DH gene was successfully expressed in E. coli, and the recombinant enzyme could oxidize glucosides, galactosides and analogues at C-3 position. The sequence and multiple alignment analysis showed that the enzyme has highest identity with G3DHs from Paraglaciecola polaris LMG 21857, Aliiglaciecola lipolytica E3 and Halomonas sp. alpha-15. The recombinant G3DH was purified on Ni-NTA column and exhibited the highest activity at pH 7.6 and 30 °C. It was sensitive to acid and alkali, and showed well thermostability. The SfG3DH could oxidize a wild range of sugars. When recombinant E. coli BL21 cells were used as catalyst, a high rate of conversion to N-p-nitrophenyl-3-ketovalidamine was achieved, and no p-nitroaniline was detected. This process offers a promising approach to fulfill substrate of 3-ketovalidoxylamine A C-N lyase production.
authors.affiliation: College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou, 310015, China. grace_zjf@126.com.|Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China. grace_zjf@126.com.
authors.forename: Jian-Fen
authors.initials: JF
authors.lastname: Zhang
authors.affiliation: College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou, 310015, China.
authors.forename: Wei-Qing
authors.initials: WQ
authors.lastname: Chen
authors.affiliation: College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou, 310015, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005697:Galactosides / Q000378:metabolism; D005948:Glucose Dehydrogenases / Q000235:genetics* / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009596:Nitrophenols / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D045249:Sphingobacterium / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D005697:Galactosides; D005960:Glucosides; C519801:N-p-nitrophenyl-3-ketovalidamine; D009596:Nitrophenols; D011994:Recombinant Proteins; D005948:Glucose Dehydrogenases; C051543:glucoside 3-dehydrogenase
keywords: 3-Ketovalidoxylamine A C–N lyase; Gene cloning; Glucoside 3-dehydrogenase; N-p-nitrophenyl-3-ketovalidamine; Valienamine