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Production and characterization of a plant α-hydroxynitrile lyase in Escherichia coli

✍ Scribed by J. Hughes; J. H. Lakey; M. A. Hughes

Publisher
John Wiley and Sons
Year
1997
Tongue
English
Weight
241 KB
Volume
53
Category
Article
ISSN
0006-3592

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✦ Synopsis

The coding sequence of the cyanogenic ␣-hydroxynitrile lyase gene of Manihot esculenta Crantz (cassava) was cloned in the plasmid vector pMal-c2 and expressed in Escherichia coli strain JM105. DNA sequencing showed that the recombinant plasmid contained the same sequence as the cDNA clone pHNL10. Peptide sequencing of the recombinant protein showed that the N-terminus was heterogeneous, with either four or six additional amino acid residues compared with the native protein. Circular dichroism spectra indicated similar secondary structure contents for both proteins. Enzyme assays showed that specific activity of native and recombinant proteins were 0.24 and 0.26 mmol CN -/mg/min, respectively; that both proteins had optimal activity at 40°C and pH 5.5; and that both proteins were inhibited by the serine protease inhibitor phenyl-methane sulfonyl flouride (PMSF). Isoelectric focusing of native and recombinant protein revealed multiple isoforms for both proteins; the recombinant protein had a more basic mean isoelectric point (pI) (5.1) than the native protein (4.5).

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