Abstract
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.
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Acknowledgments
The authors thank Prof. Toshihisa Kotake, Saitama University, for kindly providing glucuronosyl β-1,6-GalOligos and endo-β-1,3-galactanase. We would like to thank Editage (http://www.editage.jp) for English language editing.
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This work was funded by Japan Society for the Promotion of Science KAKENHI (grant number 25450135). The funder had no role in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.
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Kondo, T., Nishimura, Y., Matsuyama, K. et al. Characterization of three GH35 β-galactosidases, enzymes able to shave galactosyl residues linked to rhamnogalacturonan in pectin, from Penicillium chrysogenum 31B. Appl Microbiol Biotechnol 104, 1135–1148 (2020). https://doi.org/10.1007/s00253-019-10299-y
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DOI: https://doi.org/10.1007/s00253-019-10299-y