[P001] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P01-019] Functional Characterization of Three Exo Acting α–L– Arabinofuranosidases from Levilactobacillus brevis and Their Synergistic Actions in Arabinan Degradation

○JunYeong Jung¹, MinJeong Son¹, Un Jo¹, YeEun Kang¹, JeongAh Yu¹, DaEun Jung¹, Tae-Jip Kim¹ (1. Chungbuk National University, Division of Animal, Horticultural and Food Sciences (Korea))

Keywords:

Levilactobacillus brevis KCTC 3498,α-L-Arabinofuranosidases (ABFs),Synergistic actions,Arabinan utilization

Levilactobacillus brevis KCTC 3498 is a Gram–positive, rod–shaped lactic acid bacterium isolated from the human gut, known to enhance immune function and suppress pathogenic microorganisms. Previous studies have shown that this strain can utilize a broad spectrum of carbohydrates metabolically, including pentoses, hexoses, and certain oligosaccharides derived from plant polysaccharides. In this study, genome analysis of L. brevis identified the gene clusters associated with arabinan utilization, within which three genes predicted to encode α–L–arabinofuranosidases (ABFs; EC 3.2.1.55) were found. These genes were designated LebABF51A1 (506 amino acids), LebABF51A2 (496 amino acids), and LebABF43C (326 amino acids). Amino acid sequence homology analysis revealed that LebABF51A1 shared 76.0% query coverage and 47.6% identity with ABF–A from Bifidobacterium longum, while LebABF51A2 and LebABF43C exhibited 61.1% and 71.4% of identity, respectively, to ABF–A and ABF–C from Schleiferilactobacillus shenzhenensis. ABFs are exo–acting glycoside hydrolases that remove α–L–arabinose residues from the non–reducing ends of arabinan and arabino–oligosaccharides (AOS). To validate the predicted functions of the three ABFs, the corresponding genes were cloned into the pET–21a expression vector and heterologously expressed in Escherichia coli BL21(DE3). Recombinant enzymes fused with a C–terminal 6ⅹHis–tag were purified using Ni–NTA affinity chromatography, and their hydrolysis products and enzymatic activities were assessed by TLC and quantitative assays. LebABF51A1 and LebABF51A2 hydrolyzed α–1,2–, α–1,3–, and α–1,5–linked L–arabinose bonds, exhibiting activity toward both linear and branched AOS and generating only L–arabinose as the final product. These enzymes also hydrolyzed polymeric substrates such as sugar beet arabinan (SA) and debranched arabinan (DA). In contrast, LebABF43C specifically targeted α–1,5–linked L–arabinose bonds, displaying activity toward linear substrates such as DA and LAOS, but not hydrolyzing branched substrates. These complementary substrate specificities indicate a potential synergistic role in arabinan degradation. LebABF51A1 and LebABF51A2 release L–arabinose from branched oligosaccharides and polymeric substrates, whereas LebABF43C specializes in the hydrolysis of linear arabinan. Through these distinct enzymatic actions, arabinan–derived oligosaccharides can be efficiently converted into L–arabinose, which subsequently enters the pentose phosphate pathway via enzymes encoded in the ara operon, contributing to cellular growth and energy production. This study elucidates the roles of intracellular ABFs encoded in the L. brevis genome in the hydrolysis and metabolism of hemicellulose–derived carbohydrates. These findings can expand microbiological understanding and highlight the potential application of these enzymes in the development of customized synbiotics based on probiotic–prebiotic interactions.