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Two new endo-1,4-beta-xylanases encoding genes EpXyn1 and EpXyn3 were isolated from mesophilic fungus Eupenicillium parvum 4–14. Based on analysis of catalytic domain and phylogenetic trees, the xylanases EpXYN1 (404 aa) and EpXYN3 (220 aa) belong to glycoside hydrolase (GH) family 10 and 11, respectively. Both EpXYN1 and EpXYN3 were successfully expressed in Pichia pastoris and the recombinant enzymes were characterized using beechwood xylan, birchwood xylan, or oat spelt xylan as substrates, respectively. The optimum temperatures and pH values were 75 °C and 5.5 for EpXYN1, and 55 °C and 5.0 for EpXYN3. EpXYN1 exhibited a high stability at high temperature (65 °C) or at pH values from 8 to 10. EpXYN3 kept over 80% enzymatic activity after treatment at pH values from 3 to 10. The specific activities of EpXYN1 and EpXYN3 were 384.42 and 214.20 U/mg using beechwood xylan as substrate, respectively. EpXYN1 showed lower Km values and higher specific activities toward different xylans compared to EpXYN3. Thin-layer chromatography analysis indicated that the hydrolysis profiles of xylans or xylo-oligosacharides were different by EpXYN1and EpXYN3. EpXYN3 had a higher efficiency than EpXYN1 in production of feruloylated oligosaccharides (FOs) from de-starched wheat bran. The maximum levels of FOs released by EpXYN1 and EpXYN3 were 11.1 and 14.4 μmol/g, respectively. In conclusion, the two xylanases are potential candidates for various industrial applications.

Feruloyl esterase (FAE) is a critical enzyme in bio-extraction of ferulic acid (FA) from plant cell wall. A new FAE (EpFAE1) encoding gene was isolated from Eupenicillium parvum and heterologously expressed in Pichia pastoris cells. Based on phylogenetic tree analysis, the protein EpFAE1 belongs to type A of the seventh FAE subfamily. Using methyl ferulate as substrate, the optimum temperature and pH for the catalytic activity of EpFAE1 were 50 °C and 5.5, respectively. The enzyme exhibited high stability at 50 °C, in a wide pH range (3.0–11.0), or in the presence of 2 M of NaCl. Together with the endo-xylanase EpXYN1, EpFAE1 released 72.32% and 4.00% of the alkali-extractable FA from de-starched wheat bran (DSWB) or de-starched corn bran (DSCB), respectively. Meanwhile, the substrates were pretreated with 1.75% (for DSWB) or 1.0% (for DSCB) of phosphoric acid (PA) at 90 °C for 12 h, followed by enzymatic hydrolysis of the soluble and insoluble fractions. The release efficiencies of FA were up to 84.64% for DSWB and 66.73% for DSCB. Combined dilute PA pretreatment with enzymatic hydrolysis is a low-cost and highly efficient method for the extraction of FA from cereal brans.