(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Eukaryota: NE > Opisthokonta: NE > Fungi: NE > Dikarya: NE > Ascomycota: NE > saccharomyceta: NE > Pezizomycotina: NE > leotiomyceta: NE > sordariomyceta: NE > Sordariomycetes: NE > Sordariomycetidae: NE > Sordariales: NE > Chaetomiaceae: NE > Thielavia: NE > Thielavia terrestris: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA EEAEAEAEPKASLQQVTNFGTNPTSIQMFIYVPAKLAANPPIIVALHPCG GSAQQWYSGTKLPSYADSNGFIIIYPQTTRYSNCWDVNNAGSLTHGGSGD ALGIVSMVNYTLSKYNGDKTKVYAMGASSGAMMTNVLAGSYPDVFEAGAS YSGVAFACFAGSTSPPTPFGANQTCAQGLQHTPEEWAAFVHNAYPGYSGK RPRMQVIHGLADTLVRPQCGYEQLKQWSAVLGLTNTRNVTGSSVPEGAQY TEIVYGDGSQLVGYFGQGVGHFAPPNEPVMLKFFGLL
Feruloyl esterases (FAEs, EC 3.1.1.73) catalyze the hydrolytic cleavage of ester bonds between feruloyl and arabinosyl moieties in arabinoxylans. Recently, we discovered that two bacterial FAEs could catalyze release of diferulic acid moieties (diFAs) from highly substituted, cross-linked corn bran arabinoxylan. Here, we show that several fungal FAEs, notably AnFae1 (Aspergillus niger), AoFae1 (A. oryzae), and MgFae1 (Magnaporthe oryzae (also known as M. grisae)) also catalyze liberation of diFAs from complex arabinoxylan. By comparing the enzyme kinetics of diFA release to feruloyl esterase activity of the enzymes on methyl- and arabinosyl-ferulate substrates we demonstrate that the diFA release activity cannot be predicted from the activity of the enzymes on these synthetic substrates. A detailed structure-function analysis, based on AlphaFold2 modeled enzyme structures and docking with the relevant di-feruloyl ligands, reveal how distinct differences in the active site topology and surroundings may explain the diFA releasing action of the enzymes. Interestingly, the analysis also unveils that the carbohydrate binding module of the MgFae1 may play a key role in the diFA releasing ability of this enzyme. The findings contribute further understanding of the function of FAEs in the deconstruction of complex arabinoxylans and provide new opportunities for enzyme assisted upgrading of complex bran arabinoxylans.
Title: Cloning, Characterization, and Functional Expression of a Thermostable Type B Feruloyl Esterase from Thermophilic Thielavia Terrestris Meng Z, Yang QZ, Wang JZ, Hou YH Ref: Appl Biochem Biotechnol, 189:1304, 2019 : PubMed
Feruloyl esterases (FAEs) have great potential applications in paper and breeding industry. A new thermo-stable feruloyl esterase gene, TtfaeB was identified from the thermophilic fungus Thielavia terrestris h408. Deduced protein sequence shares the identity of 67% with FAEB from Neurospora crassa. The expression vector pPIC9K-TtfaeB was successfully constructed and electro-transformed into GS115 strain of Pichia pastoris. One transformant with high feruloyl esterase yield was obtained through plate screening and named TtFAEB1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TtFAEB1 showed a distinct protein band appearing at the position of about 35-kDa, indicating that TtfaeB gene has been successfully expressed in P. pastoris. The recombinant TtFAEB was purified by affinity chromatography and the specific activity of purified TtFAEB was 6.06 +/- 0.72 U/mg. The optimal temperature and pH for purified recombinant TtFAEB was 60 degrees C and 7.0, respectively. TtFAEB was thermostable, retaining 96.89 and 84.16% of the maximum activity after being treated for 1 h at 50 degrees C and 60 degrees C, respectively. Additionally, the enzyme was stable in the pH range 4.5-8.0. The homology model of TtFAEB showed that it consists of a single domain adopting a typical alpha/beta-hydrolase fold and contains a catalytic triad formed by Ser117, Asp201, and His260. TtFAEB in association with xylanase from Trichoderma reesei could release 77.1% of FA from destarched wheat bran. The present results indicated that the recombinant TtFAEB with excellent enzymatic properties is a promising candidate for potential applications in biomass deconstruction and biorefinery.
