[Purification of L-sorbose/L-sorbosne dehydrogenase from Ketogulonigenium vulgare and construction and selection of genomic library].
L-sorbose/L-sorbosone dehydrogenase from Ketogulonigenium vulgare S2 can transform L-sorbose to 2-KLG, which is widely used in production of Vitamin C. In order to obtain the engineering strain producing L-sorbose/L-sorbosone dehydrogenase and simplify the fermentation technology, firstly, this enzyme was purified by the methods of ammonium sulfate precipitation, DEAE Sepharose Fast Flow and Q Sepharose High Performance. Then, the purified L-sorbose/L-sorbosone dehydrogenase was injected to rabbit to obtain antibody. Next, the genomic library of Ketogulonigenium vulgare S2 was constructed by inserting the restriction fragments of chromatosomal DNA digested with Sau3A I into cosmid pKC505 vector digested by Hpa I and Pst I, which were packed with lamda phage package protein and transferred into E. coli DH5alpha in vitro. Finally, the positive strain K719# was selected from more than 12,000 clones via Dot-ELISA. Through the test of SDS-PAGE and thin layer chromatography, the results showed that the engineering strain K719# had the same biological activity as Ketogulonigenium vulgare S2 after adding coenzyme PQQ.
Xie
Li
L
College of Life Science, Hebei Normal University, Shijiazhuang 050016, China.
Zhang
Duo
D
Dou
Yan-Feng
YF
Zhang
Li-Ping
LP
Zhao
Bao-Hua
BH
D002237:Carbohydrate Dehydrogenases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015698:Genomic Library; D020600:Gluconobacter oxydans / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013013:Sorbose / Q000378:metabolism; D013400:Sugar Acids / Q000378:metabolism; D014169:Transformation, Bacterial
D013400:Sugar Acids; D002237:Carbohydrate Dehydrogenases; C104557:sorbose dehydrogenase; C011491:provitamin C; D013013:Sorbose