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.

中文翻译：

---

来自Penicillium chrysogenum 31B的阿魏酸酯酶的鉴定和表征：甜菜果胶中用l-阿拉伯呋喃糖和d-吡喃半乳糖修饰的阿魏酸的去酯化

我们之前描述了真菌 Penicillium chrysogenum 31B，它具有生产阿魏酸酯酶 (FAE) 的高性能，用于从甜菜浆中脱酯阿魏酸 (FA)。然而，这种真菌的特征尚未确定。因此，在本研究中，我们评估了来自 Penicillium chrysogenum 的 Pcfae1 基因的分子特征和天然底物特异性，并检查了其对甜菜果胶的协同作用。Pcfae1 基因被克隆并在毕赤酵母 KM71H 中过表达，并表征了名为 PcFAE1 的重组酶。PcFAE1 的 505 个氨基酸具有 GCSTG 基序（Gly164 到 Gly168），这是丝氨酸酯酶家族的特征。通过将 PcFAE1 的氨基酸序列与米曲霉的 FAE（AoFaeB）、Ser166、Asp379 的氨基酸序列进行比较，和 His419 被确定为催化三联体。使用阴离子交换柱色谱法通过两个步骤纯化 PcFAE1。其不含信号肽的分子量为 75 kDa。在 pH 6.0-7.0 和 50 °C 下实现了最大的 PcFAE1 活性。该酶在高达 37°C 和 3-8 的 pH 范围内是稳定的。PcFAE1 活性仅受 Hg2+ 抑制，该酶对甲基 FA、甲基咖啡酸和甲基对香豆酸有活性，比活性分别为 6.97、4.65 和 9.32 U/mg，但对甲基芥子酸无活性. 这些结果表明，根据 Crepin 分类，PcFAE1 的作用类似于 FaeB 型。PcFAE1 去酯化 O-[6-O-阿魏酰-β-d-吡喃半乳糖-(1→4)]-d-吡喃半乳糖，O-[2-O-阿魏酰-α-1-阿拉伯呋喃糖基-(1→5) ]-l-阿拉伯呋喃糖，和 O-[5-O-阿魏酰-α-1-阿拉伯呋喃糖基-(1→3)]-O-β-d-吡喃木糖基-(1→4)-d-吡喃木糖，表明该酶可以使 FA 脱酯在果胶和木聚糖中用 β-d-吡喃半乳糖苷和 α-l-阿拉伯呋喃糖苷残基装饰。PcFAE1 与内切-α-1,5-阿拉伯糖苷酶和 α-l-阿拉伯呋喃糖苷酶协同作用，后者释放与阿拉伯聚糖相连的 FA，以消化甜菜果胶。此外，当 PcFAE1 与 Driselase 一起作用于甜菜果胶时，底物中约 90% 的总 FA 被释放。因此，PcFAE1 可能是木质纤维素材料水解的有趣候选物，并且可以作为从天然底物生产 FA 的工具应用。表明该酶可以使果胶和木聚糖中用 β-d-吡喃半乳糖苷和 α-l-阿拉伯呋喃糖苷残基修饰的 FA 脱酯。PcFAE1 与内切-α-1,5-阿拉伯糖苷酶和 α-l-阿拉伯呋喃糖苷酶协同作用，后者释放与阿拉伯聚糖相连的 FA，以消化甜菜果胶。此外，当 PcFAE1 与 Driselase 一起作用于甜菜果胶时，底物中约 90% 的总 FA 被释放。因此，PcFAE1 可能是木质纤维素材料水解的有趣候选物，并且可以作为从天然底物生产 FA 的工具应用。表明该酶可以使果胶和木聚糖中用β-d-吡喃半乳糖苷和α-l-阿拉伯呋喃糖苷残基修饰的FA去酯化。PcFAE1 与内切-α-1,5-阿拉伯糖苷酶和 α-l-阿拉伯呋喃糖苷酶协同作用，后者释放与阿拉伯聚糖相连的 FA，以消化甜菜果胶。此外，当 PcFAE1 与 Driselase 一起作用于甜菜果胶时，底物中约 90% 的总 FA 被释放。因此，PcFAE1 可能是木质纤维素材料水解的一个有趣的候选物，并且可以用作从天然底物生产 FA 的工具。当 PcFAE1 与 Driselase 一起作用于甜菜果胶时，底物中大约 90% 的总 FA 被释放。因此，PcFAE1 可能是木质纤维素材料水解的有趣候选物，并且可以作为从天然底物生产 FA 的工具应用。当 PcFAE1 与 Driselase 一起作用于甜菜果胶时，底物中大约 90% 的总 FA 被释放。因此，PcFAE1 可能是木质纤维素材料水解的有趣候选物，并且可以作为从天然底物生产 FA 的工具应用。