In:
Applied and Environmental Microbiology, American Society for Microbiology, Vol. 81, No. 5 ( 2015-03), p. 1679-1688
Abstract:
An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p -coumarate, methyl caffeate, and methyl ferulate were as follows: K m values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and k cat / K m values of 25.83 mM −1 s −1 , 7.63 mM −1 s −1 , 3.83 mM −1 s −1 and 3.75 mM −1 s −1 , respectively. UmChlE released ferulic, p -coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.
Type of Medium:
Online Resource
ISSN:
0099-2240
,
1098-5336
DOI:
10.1128/AEM.02911-14
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2015
detail.hit.zdb_id:
223011-2
detail.hit.zdb_id:
1478346-0
SSG:
12
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