Abstract
Fibrobacter succinogenes is a major cellulolytic anaerobic bacterium in the gut of herbivores and possesses genes with a wide range of hemicellulolytic activities. Endo-l,4-β-xylanase (EC.3.2.1.8) is a key member of the xylanolytic enzyme system and is widely used in animal feed, food, papermaking and medicine. In this research, the nucleotide-optimized endo-l,4-β-xylanase gene Xynm derived from the Fisuc_2442 gene of F. succinogenes was expressed in Pichia pastoris GS115. Carbon sources were optimized to facilitate recombinant xylanase Xynm production in high-cell-density fermentation. Characterization of the enzymatic properties of Xynm showed that it was overexpressed in P. pastoris by high-cell-density fermentation. Xynm had high specific activities toward various xylose polymers but low activity toward glucose-based polysaccharides. By methanol or a mixture of sorbitol/methanol (2 : 20 (wt/vol)) induction, the maximum specific activity of 6382 and 13821 U/mg protein was achieved with wheat flour arabinoxylan (high viscosity) as the substrate, and the maximum biomass was 111 and 161 g/L, respectively. Xynm exhibited optimal catalytic activity at pH 5.5 and 37°C and maintained over 80% of the initial activity after incubation at pH from 4.5 to 6.5 or from 10 to 50°C for 1 h. The enzyme activity was increased by Ba2+ and inhibited by Fe2+, Zn2+, and Ag+. Compared with corn, Xynm hydrolyzed wheat and released more reducing sugars. In summary, Xynm has high enzyme activity and moderate reaction conditions and shows promising application prospects for animal husbandry to improve the digestion of plant feedstuffs.
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This work was funded by the Department of Xinjiang Science and Technology, China (no. ZYYD2023B09).
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T.T. Fu and L. Wang are co-first authors with equal contributions.
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Fu, T.T., Wang, L., Li, W.J. et al. Overexpression and Characterization of Endo-1,4-β-xylanase from Fibrobacter succinogenes in Pichia pastoris. Appl Biochem Microbiol 59, 900–908 (2023). https://doi.org/10.1134/S0003683823060030
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DOI: https://doi.org/10.1134/S0003683823060030