Three recombinant β-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze β-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-β-D-galactopyranoside and β-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the β-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked β-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.

中文翻译：

---

表征三个GH35β-半乳糖苷酶，该酶能够剃除果胶中鼠李糖半乳糖醛酸聚糖所连接的半乳​​糖基残基，该酶来自产黄青霉31B。

使用巴斯德毕赤酵母表达并鉴定了属于产黄青霉31B的糖苷水解酶（GH）家族35的三种重组β-半乳糖苷酶（BGAL； PcBGAL35A，PcBGAL35B和PcGALX35C）。PcBGAL35A显示了迄今为止尚未报道的独特底物特异性。根据酶学测试和1H核磁共振的结果，发现PcBGAL35A水解了果胶鼠李糖半乳糖醛酸-I（RG-I）和对硝基苯中与L-鼠李糖连接的β-1,4-半乳糖残基。 -β-D-半乳糖吡喃糖苷和β-D-半乳糖基寡糖。通过分子系统树和底物特异性分析，确定PcBGAL35B是常见的BGAL。发现PcGALX35C对β-1,4-半乳糖基低聚物和聚合物具有相似的催化能力。此外，PcGALX35C水解了果胶中聚合度为2或更高的RG-I连接的β-1,4-半乳糖基寡糖侧链。PcBGAL35A的氨基酸序列与大多数GH35 BGAL的酶相似性约为30％。PcBGAL35B的氨基酸序列与青霉属BGAL的氨基酸序列大约80％相同。PcGALX35C的氨基酸序列被分类为与PcBGAL35A相同的系统进化组。Pfam分析显示，三个BGAL具有五个结构域，包括催化结构域。我们的发现表明，PcBGAL35A和PcGALX35C是参与果胶中半乳糖基化RG-1降解的酶。本研究中表征的酶可用于需要果胶加工的产品以及果胶的结构分析。果胶中聚合度为2或更高的4-半乳糖基寡糖侧链。PcBGAL35A的氨基酸序列与大多数GH35 BGAL的酶相似性约为30％。PcBGAL35B的氨基酸序列与青霉属BGAL的氨基酸序列大约80％相同。PcGALX35C的氨基酸序列被分类为与PcBGAL35A相同的系统进化组。Pfam分析显示，三个BGAL具有五个结构域，包括催化结构域。我们的发现表明，PcBGAL35A和PcGALX35C是参与果胶中半乳糖基化RG-1降解的酶。本研究中表征的酶可用于需要果胶加工的产品以及果胶的结构分析。果胶中聚合度为2或更高的4-半乳糖基寡糖侧链。PcBGAL35A的氨基酸序列与大多数GH35 BGAL的酶相似性约为30％。PcBGAL35B的氨基酸序列与青霉属BGAL的氨基酸序列大约80％相同。PcGALX35C的氨基酸序列被分类为与PcBGAL35A相同的系统进化组。Pfam分析显示，三个BGAL具有五个结构域，包括催化结构域。我们的发现表明，PcBGAL35A和PcGALX35C是参与果胶中半乳糖基化RG-1降解的酶。本研究中表征的酶可用于需要果胶加工的产品以及果胶的结构分析。