Characterization and cloning of an (R)-specific trans-2,3-enoylacyl-CoA hydratase from Rhodospirillum rubrum and use of this enzyme for PHA production in Escherichia coli |
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Authors: | S E Reiser T A Mitsky K J Gruys |
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Institution: | (1) Monsanto Company, Ag Sector, 700 Chesterfield Parkway, St. Louis, MO 63198, USA e-mail: Kenneth.J.Gruys@monsanto.com Tel.: +1-636-7377345 Fax: +1-636-737-7015, US;(2) Monsanto Company, Nutrition Sector, 800 N. Lindbergh Blvd., St. Louis, MO 63167, USA, US |
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Abstract: | An (R)-trans-2,3-enoylacyl-CoA hydratase was purified to near-homogeneity from Rhodospirillum rubrum. Protein sequencing of enriched protein fractions allowed the construction of a degenerate oligonucleotide. The gene encoding
the (R)-specific hydratase activity was cloned following three rounds of colony hybridization using the oligonucleotide, and overexpression
of the gene in E. coli led to the purification of the enzyme to homogeneity. The purified enzyme used crotonyl-CoA, trans-2,3-pentenoyl-CoA, and trans-2,3-hexenoyl-CoA with approximately equal specificity as substrates in the hydration reaction. However, no activity was observed
using trans-2,3-octenoyl-CoA as a substrate, but this compound did partially inhibit crotonyl-CoA hydration. Based on the nucleotide
sequence, the protein has a monomeric molecular weight of 15.4 kDa and is a homotetramer in its native form as determined
by gel filtration chromatography and native PAGE. The hydratase was expressed together with the PHA synthase from Thiocapsa pfennigii in E. coli strain DH5α. Growth of these strains on oleic acid resulted in the production of the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate).
Received: 16 June 1999 / Received revision: 19 August 1999 / Accepted: 19 August 1999 |
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