Recombinant expression and characterization of high-specific activity α-l-arabinofuranosidase from Nomascus concolor fecal microbial metagenome

Abstract:

[Background] As a valuable hemicellulase, α-l-arabinofuranosidase (EC 3.2.1.55) can degrade xylan with other hemicellulases and is applied in food, medicine, and biomass energy conversion. [Objective] This study aimed to clone a novel α-l-arabinofuranosidase gene and analyze the heteroexpression, purification, and enzymatic properties of the protein. [Methods] The α-l-arabinofuranosidase gene was amplified from the fecal microbial metagenome of Nomascus concolor and heterologously expressed in Escherichia coli BL21(DE3), and then the enzymatic properties of the protein were examined. [Results] After α-l-arabinofuranosidase gene AbfNC2b_38 was amplified from the fecal microbial metagenome, a recombinant protein AbfNC2b_38 with a molecular weight of 57.04 kDa was expressed. At the optimum conditions of 55 °C and pH 6.0, the enzyme showed the K_m of (6.48±0.73) mmol/L, the V_max of (1 248.0±114.6) U/mg, and the highest specific activity of 300.81 U/mg compared with the α-l-arabinofuranosidases of other metagenomic sources. This enzyme was highly tolerant to ethanol and NaCl. It showed the relative activity of 68% after being treated with 30% ethanol for 1 h and relative activity of approximately 70% after being exposed to 25% NaCl for 1 h. Moreover, it had the highest synergistic rate of 1.21 when synergistically degrading beech xylan with xylanase. [Conclusion] A novel α-l-arabinofuranosidase gene AbfNC2b_38 was cloned from the fecal microbial metagenome of N. concolor, which was successfully heterologously expressed. AbfNC2b_38 was highly tolerant to both ethanol and NaCl, and can cooperate with xylanase to improve the degradation efficiency of xylan. Hence, this enzyme is of significant application potential in feed and food processing.