Identification and characterization of ferulic acid esterase from Penicillium chrysogenum 31B: de-esterification of ferulic acid decorated with l-arabinofuranoses and d-galactopyranoses in sugar beet pectin

Abstract

We previously described the fungus Penicillium chrysogenum 31B, which has high performance to produce the ferulic acid esterase (FAE) for de-esterifying ferulic acids (FAs) from sugar beet pulp. However, the characteristics of this fungus have not yet been determined. Therefore, in this study, we evaluated the molecular characteristics and natural substrate specificity of the Pcfae1 gene from Penicillium chrysogenum and examined its synergistic effects on sugar beet pectin. The Pcfae1 gene was cloned and overexpressed in Pichia pastoris KM71H, and the recombinant enzyme, named PcFAE1, was characterized. The 505 amino acids of PcFAE1 possessed a GCSTG motif (Gly164 to Gly168), characteristic of the serine esterase family. By comparing the amino acid sequence of PcFAE1 with that of the FAE (AoFaeB) of Aspergillus oryzae, Ser166, Asp379, and His419 were identified as the catalytic triad. PcFAE1 was purified through two steps using anion-exchange column chromatography. Its molecular mass without the signal peptide was 75 kDa. Maximum PcFAE1 activity was achieved at pH 6.0-7.0 and 50 °C. The enzyme was stable up to 37 °C and at a pH range of 3-8. PcFAE1 activity was only inhibited by Hg²⁺, and the enzyme had activity toward methyl FA, methyl caffeic acid, and methyl p-coumaric acid, with specific activities of 6.97, 4.65, and 9.32 U/mg, respectively, but not on methyl sinapinic acid. These results indicated that PcFAE1 acted similar to FaeB type according the Crepin classification. PcFAE1 de-esterified O-[6-O-feruloyl-β-d-galactopyranosyl-(1→4)]-d-galactopyranose, O-[2-O-feruloyl-α-l-arabinofuranosyl-(1→5)]-l-arabinofuranose, and O-[5-O-feruloyl-α-l-arabinofuranosyl-(1→3)]-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose, indicating that the enzyme could de-esterify FAs decorated with both β-d-galactopyranosidic and α-l-arabinofuranosidic residues in pectin and xylan. PcFAE1 acted in synergy with endo-α-1,5-arabinanase and α-l-arabinofuranosidase, which releases FA linked to arabinan, to digest the sugar beet pectin. Moreover, when PcFAE1 was allowed to act on sugar beet pectin together with Driselase, approximately 90% of total FA in the substrate was released. Therefore, PcFAE1 may be an interesting candidate for hydrolysis of lignocellulosic materials and could have applications as a tool for production of FA from natural substrates.

Keywords: Ferulic acid; Ferulic acid esterase; Penicillium chrysogenum; Sugar beet pectin; Synergistic action.