NUCLEOTIDE SEQUENCE OF MEMBRANE-BOUND FORM 2-KETO-D-GLUCONATE DEHYDROGENASE AND BIOTRANSFORMATION OF 2-KETO-D-GLUCONATE INTO 2,5-DIKETO-D-GLUCONATE BY THE ENZYME EXPRESSED IN E.COLI

摘要

PURPOSE: Nucleotide sequence of membrane bound 2-keto-D-gluconate dehydrogenase gene from Erwinia herbicola is determined and used for bio transformation of 2-keto-D-gluconate into 2,5-diketo-D-gluconate. 2,5-diketo-D-gluconate is an intermediate to produce vitamin C and pharmaceutics CONSTITUTION: Nucleotide sequence of membrane bound 2-keto-D-glaciate dehydrogenate is determined and bio transformation of 2-keto-D-gluconate into 2,5-diketo-D-gluconate is achieved by the recombinant enzyme introduced into other bacteria lacking of cytoplasm metabolic pathway of it. Membrane-bound 2-keto-D-gluconate dehydrogenase gene from Erwinia herbicola ATCC 08111 is isolated from cosmid library and sub cloned into PSTV28 for sequencing. The 2-keto-D-gluconate dehydrogenase gene is cloned into pKI111 and transformed into E. coli. The recombinant E.coli is cultured in LB media containing 2-keto-D-gluconate for 24hrs. Bio transformation takes place at per plasma of E.coli and conversion reaction is very fast.