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Molecular cloning of a Clostridium B-xylosidase gene and enzyme purification from the recombinant Escherichia coli strain
Abstract
A gene encoding a B-xylosidase was cloned from a xylanolytic Clostridium via pBR322 into Escherichia coli. The gene was localized to a 3.1-kilobase EcoRI fragment. Either orientation of the fragment in pBR322 allowed for expression of B-xylosidase activity, indicating that the Clostridium promoter is utilized in E. coli. This activity was cell-associated and not inducible. B-Xylosidase was purified from cell extract of the recombinant strain E. coli DH5 (pIW1) by ammonium sulphate fractionation, hydrophobic interaction chromatography, ion exchange chromatography, and preparative isoelectric focusing. The molecular weight of B-xylosidase, as estimated by gel filtration, was 204,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified enzyme showed a single protein band with a molecular weight of 54,000. The isoelectric point of B-xylosidase was 4.8. B-Xylosidase activity was optimal at pH 6.5 and 55$\sp\circ$C. The enzyme was stable between pH 5.0 and 9.5 for 120 minutes at 20$\sp\circ$C and retained full activity at pH 6.0 after incubation at 50$\sp\circ$C for 30 minutes. Enzyme activity was totally inhibited by 1 mM Hg$\sp{2+}$, Cd$\sp{2+}$, Cu$\sp{2+}$, or Ni$\sp{2+}$. B-Xylosidase hydrolyzed p-nitrophenol-B-D-xylopyranoside (pNPX) and was slightly active on p-nitrophenol-B-D-galactopyranoside and p-nitrophenol-alpha-L-arabinopyranoside. With pNPX as substrate, the Km and Vmax values were 0.15 mM and 1.9 units/mg, respectively. Purified enzyme exhibited transferase activity with the substrate xylobiose. This activity was also detected with pNPX as substrate in the presence of methanol as a xylosyl acceptor.
Subject Area
Microbiology
Recommended Citation
Chambers, Kathleen Erin, "Molecular cloning of a Clostridium B-xylosidase gene and enzyme purification from the recombinant Escherichia coli strain" (1989). Doctoral Dissertations Available from Proquest. AAI9001490.
https://scholarworks.umass.edu/dissertations/AAI9001490