A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis.

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: Km values of 19.6 µM, 64.1 µM, 72.5 µM, and 101.8 µM, respectively, and kcat/Km 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.

A halotolerant type A feruloyl esterase from Pleurotus eryngii.

An extracellular feruloyl esterase (PeFaeA) from the culture supernatant of Pleurotus eryngii was purified to homogeneity using cation exchange, hydrophobic interaction, and size exclusion chromatography. The length of the complete coding sequence of PeFaeA was determined to 1668 bp corresponding to a protein of 555 amino acids. The catalytic triad of Ser-Glu-His demonstrated the uniqueness of the enzyme compared to previously published FAEs. The purified PeFaeA was a monomer with an estimated molecular mass of 67 kDa. Maximum feruloyl esterase (FAE) activity was observed at pH 5.0 and 50 °C, respectively. Metal ions (5 mM), except Hg(2+), had no significant influence on the enzyme activity. Substrate specificity profiling characterized the enzyme as a type A FAE preferring bulky natural substrates, such as feruloylated saccharides, rather than small synthetic ones. Km and kcat of the purified enzyme for methyl ferulate were 0.15 mM and 0.85 s(-1). In the presence of 3 M NaCl activity of the enzyme increased by 28 %. PeFaeA alone released only little ferulic acid from destarched wheat bran (DSWB), whereas after addition of Trichoderma viride xylanase the concentration increased more than 20 fold.

Detection of feruloyl- and cinnamoyl esterases from basidiomycetes in the presence of interfering laccase.

Little is known on basidiomycete sources of feruloyl esterases (FAEs), although many wood-rotting representatives of these fungi typically grow on feruloyl-rich substrates. A major reason is that the almost ubiquitous presence of laccases interferes with the detection of FAE activity. Laccases polymerize the liberated ferulic acid (FA) in situ, thus detracting the product of enzymatic hydrolysis from its detection. A rapid HPLC-UV method was developed to detect the loss of FA, but also to quantify the hydrolysis of FA esters. The method allows at the same time to evaluate the substrate specificity of a FAE. Forty one basidiomycetes were tested for their FAE activities, and 25 out of the set were positive. The basidiomycetes hydrolyzing cinnamates with the highest conversion rates were Auricularia auricula-judae and Marasmius scorodonius. Moreover, a new FAE inducer, the nonionic detergent Tween 80, was found. This is the first comprehensive study on basidiomycete sources of FAEs.

Determination of halogenated natural products in passive samplers deployed along the Great Barrier Reef, Queensland/Australia.

Halogenated natural products (HNPs) have been increasingly reported to occur in marine wild life from all oceans. Several HNPs, such as 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (1) and 4,6-dibromo-2-(2',4'-dibromo)phenoxyanisole (2'-MeO-BDE 68 or BC-2), were detected at particularly high concentrations in dolphins from Queensland/Australia. About half of the coastline of Queensland (approximately 2500 km) is covered by the Great Barrier Reef, a rich ecosystem hosting a huge variety of species, many of which are known to produce natural compounds. In this study, semipermeable membrane devices (SPMDs) were deployed as passive samplers for about 30 days at 12 marine and 2 nonmarine sites (i.e., rivers) along the Great Barrier Reef as part of a routine monitoring program during November 2007 and May 2008. Q1 and 2'-MeO-BDE 68 were detected at the marine sites with frequencies of about 65% but not in any sample from the two rivers. Further HNPs (2,4,6-tribromophenol, TBP; 2,4,6-tribromoanisole, TBA; 2,2'-dimethoxy-3,3'5,5'-tetrabromobiphenyl, 2,2'-diMeO-BB 80 or BC-1; 3,5-dibromo-2-(2',4'-dibromo)phenoxyanisole, 6-MeO-BDE 47 or BC-3; and 3,5-dibromo-2-(3',5'-dibromo,2'-methoxy)phenoxyanisole, 2',6-diMeO-BDE 68 or BC-11) were detected as well with frequencies of 18-97% in the marine samples, but no polybrominated flame retardants were detected. The highest amount of a single HNP, 2.3 microg/SPMD, was determined for TBP, which had a frequency of detection of only 46%. The maximum (average) amount in the SPMDs from marine sites was 44 ng (12 ng) for (1 and 115 ng (20 ng) for 2'-MeO-BDE 68. A first order kinetic model was used to estimate concentrations of the HNPs in the water phase. Based on the depuration of performance reference compounds obtained at one of the sites, we assumed a sampling rate of 16 L/day. We used this sampling rate to estimate that the highest and average available concentrations of Q1 in the water during the deployment of the SPMD were 97 and 25 pg/L, respectively. The estimated maximum water concentrations of 2'-MeO-BDE 68, 2,2'-diMeO-BB 80, 6-MeO-BDE 47, and 2',6-diMeO-BDE 68 were on average 2-5.5 fold higher than that of Q1. The results confirm that the HNPs are produced throughout the Great Barrier Reef, which appears to be a significant source of these compounds.