TY - JOUR
T1 - Preparation of β-galacto-oligosaccharides using a novel endo-1,4-β-galactanase from Penicillium oxalicum
AU - Yan, Xuecui
AU - Wang, Yibing
AU - Zhang, Yaxin
AU - Wang, Xiang
AU - Liu, Yunxia
AU - Cui, Jing
AU - Mayo, Kevin H.
AU - Zhou, Yifa
AU - Cui, Liangnan
N1 - Publisher Copyright:
© 2023 Elsevier B.V.
PY - 2024/1
Y1 - 2024/1
N2 - Endo-1,4-β-galactanase is an indispensable tool for preparing prebiotic β-galacto-oligosaccharides (β-GOS) from pectic galactan resources. In the present study, a novel endo-1,4-β-galactanase (PoβGal53) belonging to glycoside hydrolase family 53 from Penicillium oxalicum sp. 68 was cloned and expressed in Pichia pastoris GS115. Upon purification by affinity chromatography, recombinant PoβGal53 exhibited a single band on SDS-PAGE with a molecular weight of 45.0 kDa. Using potato galactan as substrate, PoβGal53 showed optimal reaction conditions of pH 4.0, 40 °C, and was thermostable, retaining >80 % activity after incubating below 45 °C for 12 h. Significantly, PoβGal53 exhibited relatively conserved substrate specificity for (1 → 4)-β-D-galactan with an activity of 6244 ± 282 U/mg. In this regard, the enzyme is in effect the most efficient endo-1,4-β-galactanase identified to date. By using PoβGal53, β-GOS monomers were prepared from potato galactan and separated using medium pressure liquid chromatography. HPAEC-PAD, MALDI-TOF-MS and ESI-MS/MS analyses demonstrated that these β-GOS species ranged from 1,4-β-D-galactobiose to 1,4-β-D-galactooctaose (DP 2–8) with high purity. This work provides not only a highly active tool for enzymatic degradation of pectic galactan, but an efficient protocol for preparing β-GOS.
AB - Endo-1,4-β-galactanase is an indispensable tool for preparing prebiotic β-galacto-oligosaccharides (β-GOS) from pectic galactan resources. In the present study, a novel endo-1,4-β-galactanase (PoβGal53) belonging to glycoside hydrolase family 53 from Penicillium oxalicum sp. 68 was cloned and expressed in Pichia pastoris GS115. Upon purification by affinity chromatography, recombinant PoβGal53 exhibited a single band on SDS-PAGE with a molecular weight of 45.0 kDa. Using potato galactan as substrate, PoβGal53 showed optimal reaction conditions of pH 4.0, 40 °C, and was thermostable, retaining >80 % activity after incubating below 45 °C for 12 h. Significantly, PoβGal53 exhibited relatively conserved substrate specificity for (1 → 4)-β-D-galactan with an activity of 6244 ± 282 U/mg. In this regard, the enzyme is in effect the most efficient endo-1,4-β-galactanase identified to date. By using PoβGal53, β-GOS monomers were prepared from potato galactan and separated using medium pressure liquid chromatography. HPAEC-PAD, MALDI-TOF-MS and ESI-MS/MS analyses demonstrated that these β-GOS species ranged from 1,4-β-D-galactobiose to 1,4-β-D-galactooctaose (DP 2–8) with high purity. This work provides not only a highly active tool for enzymatic degradation of pectic galactan, but an efficient protocol for preparing β-GOS.
KW - Endo-1,4-β-galactanase
KW - Enzymatic preparation
KW - Galacto-oligosaccharides
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U2 - 10.1016/j.ijbiomac.2023.127966
DO - 10.1016/j.ijbiomac.2023.127966
M3 - Article
C2 - 37944726
AN - SCOPUS:85176363229
SN - 0141-8130
VL - 254
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
M1 - 127966
ER -