Purification of L-sorbose/L-sorbosne dehydrogenase from Ketogulonigenium vulgare and construction and selection of genomic library.
- Author:
Li XIE
1
;
Duo ZHANG
;
Yan-Feng DOU
;
Li-Ping ZHANG
;
Bao-Hua ZHAO
Author Information
1. College of Life Science, Hebei Normal University, Shijiazhuang 050016, China.
- Publication Type:Journal Article
- MeSH:
Carbohydrate Dehydrogenases;
genetics;
isolation & purification;
metabolism;
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Genomic Library;
Gluconobacter oxydans;
enzymology;
genetics;
growth & development;
Sorbose;
metabolism;
Sugar Acids;
metabolism;
Transformation, Bacterial
- From:
Chinese Journal of Biotechnology
2007;23(5):891-895
- CountryChina
- Language:Chinese
-
Abstract:
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.
