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S-selective hydroxynitrile lyase from a plant Baliospermum montanum: Molecular characterization of recombinant enzyme

Paper ID Volume ID Publish Year Pages File Format Full-Text
23825 43476 2011 11 PDF Available
Title
S-selective hydroxynitrile lyase from a plant Baliospermum montanum: Molecular characterization of recombinant enzyme
Abstract

A novel S-hydroxynitrile lyase (HNL) was purified from leaves of a plant, Baliospermum montanum, by ammonium sulfate fractionation and column chromatographies. Full-length cDNA and genomic DNA were cloned and sequenced. The latter contained two introns and one ORF encoding a 263-residue protein (subunit: 29.5 kDa). The hnl gene was expressed in Escherichia coli and the enzyme was characterized including detailed kinetic studies of 20 substrates for (S)-cyanohydrin synthesis. The enzyme exhibited the highest specific activity (178 U/mg), kcat (98/s) and kcat/Km ratio for piperonal. kcat/Km ratio for aromatic aldehydes was much larger than those of aliphatic aldehydes and ketones. It was strongly inhibited by AgNO3, PMSF, phenol and methyl ethyl ketone, showed an optimum at pH 5, while having activity at range of 4–6.5. It exhibited stability at wide pH range 2.4–11, the highest activity at 20 °C, being active at 0–65 °C. The enzyme showed variations in residues involved in substrate pocket and substrate entrance channel compared to other S-selective HNLs, based on a model was built. C-terminal short truncations provided more enzyme production. Gel filtration revealed a 60–65 kDa molecular mass for this non-FAD enzyme and its C-terminally truncated forms using three buffer compositions, indicating dimeric structures.

Keywords
S-BmHNL, S-selective hydroxynitrile lyase from plant Baliospermum montanum; E. coli, Escherichia coli; MeHNL, hydroxynitrile lyase from Manihot esculenta; HbHNL, hydroxynitrile lyase from Hevea brasiliensis; cDNA, complementary DNA; CTAB, cetyl trimethyl
First Page Preview
S-selective hydroxynitrile lyase from a plant Baliospermum montanum: Molecular characterization of recombinant enzyme
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 153, Issues 3–4, 20 May 2011, Pages 100–110
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering