RESUMO
Endo-1,4-ß-galactanase is an indispensable tool for preparing prebiotic ß-galacto-oligosaccharides (ß-GOS) from pectic galactan resources. In the present study, a novel endo-1,4-ß-galactanase (PoßGal53) 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 PoßGal53 exhibited a single band on SDS-PAGE with a molecular weight of 45.0 kDa. Using potato galactan as substrate, PoßGal53 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, PoßGal53 exhibited relatively conserved substrate specificity for (1 â 4)-ß-D-galactan with an activity of 6244 ± 282 U/mg. In this regard, the enzyme is in effect the most efficient endo-1,4-ß-galactanase identified to date. By using PoßGal53, ß-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 ß-GOS species ranged from 1,4-ß-D-galactobiose to 1,4-ß-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 ß-GOS.