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  • Title: [Purification of L-sorbose/L-sorbosne dehydrogenase from Ketogulonigenium vulgare and construction and selection of genomic library].
    Author: Xie L, Zhang D, Dou YF, Zhang LP, Zhao BH.
    Journal: Sheng Wu Gong Cheng Xue Bao; 2007 Sep; 23(5):891-5. PubMed ID: 18051871.
    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.
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