Engineering of thermostable endo-polygalacturonase to generate prebiotic-active pectic oligosaccharides

Abstract

Endo-polygalacturonases are valuable for decomposing pectin into functional oligosaccharides, which exhibit various bioactivities such as prebiotic, anticancer, and antibacterial effects. In this work, a novel endo-polygalacturonase from Penicillium arizonense (Kp-pePGB) was heterologously expressed in Komagataella phaffii. The recombinant enzyme showed optimal activity at 65 °C and pH 5.0, along with broad pH stability (pH 3.5–8.0). Nonetheless, its thermal instability limited its industrial utility. To address this, structure-based rational design was employed to engineer stabilizing mutations. The triple mutant E257C/N286C/D207N exhibited a remarkable 11.6-fold increase in residual activity after incubation at 55 °C for 60 min, without compromising catalytic performance. Molecular dynamics simulations suggested that the enhanced thermostability arose from newly introduced disulfide bonds and optimized electrostatic interactions. Importantly, the mutant efficiently generated pectic oligosaccharides, which significantly promoted the growth of beneficial probiotics including Pediococcus acidilactici, Lactobacillus paracasei, and Lactobacillus plantarum, demonstrating their prebiotic potential. This study not only provides a promising endo-polygalacturonase for the production of prebiotic oligosaccharides under thermally demanding conditions, but also offers an effective engineering strategy for improving the thermostability of other enzymes in the polygalacturonase family.

Keywords

Endo-polygalacturonase / Thermostability / Rational design / Pectic oligosaccharide / Prebiotic activity

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Funding

National Key Research and Development Program of China(2022YFD2101201)

Innovative Research Group Project of the National Natural Science Foundation of China(32072162)

RIGHTS & PERMISSIONS

Jiangnan University