Bo Lu, Liang Xian, Jing Zhu, Yunyi Wei, Chengwei Yang, and Zhong Cheng. J. Microbiol. Biotechnol. 2022;32:464-72. https://doi.org/10.4014/jmb.2112.12023

Chengwei Yang, Ting Zhang, Jing Zhu, Yunyi Wei, Furong Zhu, and Zhong Cheng. J. Microbiol. Biotechnol. 2023;33:533-42. https://doi.org/10.4014/jmb.2211.11003

Agricultural residues like sugarcane bagasse and rice straw are rich in cellulose and xylan. Their efficient conversion into (oligo)saccharides and value-added products requires microbial cellulases and xylanases, but low enzyme yields and high production costs hinder industrial application. This study isolated Penicillium oxalicum UNN1, a high-xylanase-producing strain with an initial activity of 51.63 U/mL. Submerged fermentation conditions were optimized using different carbon/nitrogen sources to enhance enzyme production. The optimized xylanase activity reached 191.22 U/mL (sugarcane bagasse xylan as sole carbon source) and 142.32 U/mL (combined with Avicel), with filter paper cellulase activity of 0.76 U/mL. The crude enzymes exhibited optimal activity at pH 5.0 and 50 °C. Cellulase retained over 75% activity after 7 h at pH 4.0–6.0 (4 °C) or 40 °C (pH 5.0), while xylanase activity remained nearly unchanged, even after over 21 days of storage at 4 °C (pH 5.0). However, the half-life of xylanase was less than 1 h at 50 °C, though it exceeded 72 h at 40 °C (pH 5.5). 3–5 mM Ca²⁺ and Cu²⁺ strongly inhibited both enzymes. Crude enzyme addition (about 7 U cellulase and 1,400 U xylanase) effectively enhanced reducing sugar production from agricultural residues. Single-factor and response surface optimization yielded optimal hydrolysis conditions: 480 U/g sugarcane bagasse xylan of xylanase, hydrolysate pH of 5.5, hydrolysis temperature of 40 °C, achieving a maximum reducing sugar yield of 0.355 g/g dry biomass. This work demonstrates the potential of P. oxalicum UNN1 enzymes for efficient and stable saccharification of agricultural residues, offering a viable approach for their valorization and environmental management. Graphical Abstract

The development of thermostable and pH-robust endo-polygalacturonases (endo-PGases) is crucial for industrial applications such as food processing. This study aimed to engineer the thermostability of an acidic, thermophilic endo-PGase (PoxaEnPG28B) by rigidifying its flexible regions. We employed an integrated computational strategy combining molecular dynamics (MD) simulations at elevated temperatures with in silico analyses of unfolding free-energy changes to identify and design stabilizing mutations. This approach successfully yielded the mutant D249K, which exhibited a 5 °C higher optimal temperature (70 °C) and a 68.8% longer half-life at 55 °C, and it retained over 76.8% activity at 75 °C. Notably, D249K maintained the wild-type’s optimal pH (5.0) and broad pH stability (3.0–8.0). Although it is not the absolute top performer in every single metric, D249K achieves the best overall balance between thermostability and pH robustness among all reported thermophilic endo-PGases. MD simulations revealed that its enhanced stability sems from reduced global and local flexibility and a more compact structure. In juice extraction applications, D249K increased yields by up to 98.5%, significantly surpassing the wild-type. This study demonstrates the efficacy of MD-guided flexible region engineering for the GH28 family and presents D249K as a highly promising industrial biocatalyst.