Background Xylanase, an important accessory enzyme that acts in synergy with cellulase, is widely used to degrade lignocellulosic biomass. Thermostable enzymes with good catalytic activity at lower temperatures have great potential for future applications in the feed and fuel industries, which have distinct demands; however, the potential of the enzymes is yet to be researched. Results In this study, a structure-based semi-rational design strategy was applied to enhance the low-temperature catalytic performance of Bispora sp. MEY-1 XYL10C_∆N wild-type (WT). Screening and comparisons were performed for the WT and mutant strains. Compared to the WT, the mutant M53S/F54L/N207G exhibited higher specific activity (2.9-fold; 2090 vs. 710 U/mg) and catalytic efficiency (2.8-fold; 1530 vs. 550 mL/s mg) at 40 °C, and also showed higher thermostability (the melting temperature and temperature of 50% activity loss after 30 min treatment increased by 7.7 °C and 3.5 °C, respectively). Compared with the cellulase-only treatment, combined treatment with M53S/F54L/N207G and cellulase increased the reducing sugar contents from corn stalk, wheat bran, and corn cob by 1.6-, 1.2-, and 1.4-folds, with 1.9, 1.2, and 1.6 as the highest degrees of synergy, respectively. Conclusions This study provides useful insights into the underlying mechanism and methods of xylanase modification for industrial utilization. We identified loop2 as a key functional area affecting the low-temperature catalytic efficiency of GH10 xylanase. The thermostable mutant M53S/F54L/N207G was selected for the highest low-temperature catalytic efficiency and reducing sugar yield in synergy with cellulase in the degradation of different types of lignocellulosic biomass. Graphic Abstract

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Background Xylanase can efficiently hydrolysis of hemicellulose, a barrier to the efficient saccharification of lignocellulose, into xylooligosaccharides and is widely used in the fields of feed, food and biomass utilization. High-temperature xylanase usually has low catalytic activity below 40°C, which limits its applications. Therefore, improving the properties of xylanases to enable synergistic degradation of lignocellulosic biomass with cellulase is of considerable significance in the field of bioconversion of lignocellulosic biomass. Results In this study, a structure-based rational design strategy was applied to enhance the low-temperature catalytic performance of XYL10C_∆N (WT). Screening and comparison were performed amoung the WT and mutants. In comparison with WT, MF53/53SL + N207G exhibited higher specific activity (2.9-fold; 2090 vs. 710 U/mg) and catalytic efficiency (2.8-fold; 1530 vs. 550 mL/s·mg) at 40°C, as well as higher thermostability (with Tm and T50 increased by 7.7°C and 3.5°C, respectively). Meanwhile, comparing with cellulase-only treatment, the combination of MF53/53SL + N207G and cellulase increased the reducing sugar by 1.6-, 1.2-, and 1.4-folds for degradating corn stalk, wheat bran, and corn cob, with the highest degree of synergy of 1.9, 1.2, and 1.6, respectively. Conclusions This study provided a successful strategy to improve the catalytic properties of enzymes and identified loop2 is a key functional area that affects the low-temperature catalytic efficiency of GH10 xylanase. Several xylanase candidates for applications in feed and bioenergy applications were obtained. Synergistic degradation experiments elucidated a possible mechanism of cellulase inhibition by xylan during the hydrolysis of different types of biomass.

Aspartic protease emerges as an optimistic hydrolytic agent to obtain several protein hydrolysates. An aspartic protease gene from Aspergillus fumigatus Af293 was successfully expressed in Pichia pastoris (GS115) and its hydrolytic potentials on silkworm (Bombyx mori) pupae protein were determined. It was optimum at pH 4.0 and 50 °C and stable over pH range 4.0-5.0 and temperatures 45-55 °C with a specific activity of 8408.9 ± 305.6 U/mg. SDS-PAGE analysis revealed the molecular weight of the recombinant protease to be 45 kDa. The half-life (t1/2) of the recombinant protease at 40, 50, 60, and 70 °C was 30, 25, 35, and 20 min, respectively. The protease showed enhanced activity in the presence of Cu2+, Pb2+ and SDS. Its substrate specificity studies were revealed in the order of cleaving ability to Bovine Serum Albumin (BSA) > Silkworm pupae powder (SPP) > Casein > Casein sodium salt (CSS). Upon hydrolysis of silkworm pupae protein, it showed enhanced and plausible hydrolytic potentials, increasing the degree of hydrolysis to 50 ± 6.1% at 6 h, increased solubility by 80%, and improved functional properties. The stable characteristics and hydrolytic performance of the recombinant aspartic protease qualify it for industrial application, especially within the food and related industries.