IntroductionL-Arabinose is a pentose sugar that can be obtained by the hydrolysis of various plant cell wall hemicellulosic polymers such as arabinoxylans and arabinans [1]. Due to the health-beneficial functions, L-arabinose and arabino-oligosaccharides (AOS) have been considered as promising candidates for low-calorie sweeteners and prebiotic materials [2][3][4]. For the enzymatic production of L-arabinose, α-L-arabinofuranosidase (ABF; E.C. 3.2.1.55) is the key player which catalyzes the exo-type hydrolysis of L-arabinose-containing polymers [5,6]. Especially, its synergistic actions with endo-α-(1,5)-L-arabinanase (ABN; E.C. 3.2.1.99) were suggested for the cost-effective L-arabinose production from sugar beet arabinan [7,8].The ABFs possess versatile hydrolyzing activities towards the terminal non-reducing α-(1,2)-, α-(1,3)-, and/or α-(1,5)-L-arabinofuranosidic linkages in various polymeric and oligomeric substrates [5,6]. Depending on their structural features, the common microbial ABFs can be mainly categorized to the members of glycoside hydrolase (GH) families 51 and 43. To date, various ABFs have been reported from mainly bacteria, as well as a few fungi and plants [5,6,9]. Based on the genome analyses, the probable gene clusters for the utilization of L-arabinose and arabinans were found from Bacillus [10, 11], Geobacillus [12], and Corynebacterium spp. [13]. Bacillus subtilis produces two intracellular exo-ABFs GH51 and two extracellular endo-ABNs GH43, which can synergistically degrade the arabinan polymers to L-arabinose [11]. On the contrary, only a limited number of eukaryotic ABFs were genetically and enzymatically characterized from the fungal microorganisms such as Penicillium [14], Aspergillus [15], Chrysosporium [16], and Aureobasidium spp. [17]. As the eukaryotic ABFs share relatively low Two genes encoding probable α-L-arabinofuranosidase (E.C. 3.2.1.55) isozymes (ABFs) with 92.3% amino acid sequence identity, ABF51A and ABF51B, were found from chromosomes 3 and 5 of Saccharomycopsis fibuligera KJJ81, an amylolytic yeast isolated from Korean wheat-based nuruk, respectively. Each open reading frame consists of 1,551 nucleotides and encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% amino acid sequence identity with eukaryotic ABFs from filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from Escherichia coli. SfABF51 A and SfABF51 B showed the highest activities on p-nitrophenyl arabinofuranoside at 40~45 o C and pH 7.0 in sodium phosphate buffer and at 50 o C and pH 6.0 in sodium acetate buffer, respectively. These exoacting enzymes belonging to the glycoside hydrolase (GH) family 51 could hydrolyze arabinoxylooligosaccharides (AXOS) and arabino-oligosaccharides (AOS) to produce only L-arabinose, whereas they could hardly degrade any polymeric substrates including arabinans and arabinoxylans. The detailed product analyses revealed that both SfABF51 isozymes can catalyze the ver...
Two α-L-arabinofuranosidases (BfdABF1 and BfdABF3) and a β-D-xylosidase (BfdXYL2) genes were cloned from Bifidobacterium dentium ATCC 27679, and functionally expressed in E. coli BL21(DE3). BfdABF1 showed the highest activity in 50 mM sodium acetate buffer at pH 5.0 and 25°C. This exo -enzyme could hydrolyze p -nitrophenyl arabinofuranoside, arabino-oligosaccharides (AOS), arabinoxylo-oligosaccharides (AXOS) such as 3 2 -α-L-arabinofuranosyl-xylobiose (A 3 X), and 2 3 -α-Larabinofuranosyl-xylotriose (A 2 XX), whereas hardly hydrolyzed polymeric substrates such as debranched arabinan and arabinoxylans. BfdABF1 is a typical exo -ABF with the higher specific activity on the oligomeric substrates than the polymers. It prefers to α-(1,2)-L-arabinofuranosidic linkages compared to α-(1,3)-linkages. Especially, BfdABF1 could slowly hydrolyze 2 3 ,3 3 -di-α-L-arabinofuranosyl-xylotriose (A 2+3 XX). Meanwhile, BfdABF3 showed the highest activity in sodium acetate at pH 6.0 and 50°C, and it has the exclusively high activities on AXOS such as A 3 X and A 2 XX. BfdABF3 mainly catalyzes the removal of L-arabinose side chains from various AXOS. BfdXYL2 exhibited the highest activity in sodium citrate at pH 5.0 and 55°C, and it specifically hydrolyzed p -nitrophenyl xylopyranoside and xylo-oligosaccharides (XOS). Also, BfdXYL2 could slowly hydrolyze AOS and AXOS such as A 3 X. Based on the detailed hydrolytic modes of action of three exo -hydrolases (BfdABF1, BfdABF3, and BfdXYL2) from Bf. dentium , their probable roles in the hemiceulloseutilization system of Bf. dentium are proposed in the present study. These intracellular exo -hydrolases can synergistically produce L-arabinose and D-xylose from various AOS, XOS, and AXOS.
PLA (3‐D‐phenyllactic acid) is an ideal antimicrobial and immune regulatory compound present in honey and fermented foods. Sporolactobacillus inulinus is regarded as a potent D‐PLA producer that reduces phenylpyruvate (PPA) with D‐lactate dehydrogenases. In this study, PLA was produced by whole‐cell bioconversion of S. inulinus ATCC 15538. Three genes encoding D‐lactate dehydrogenase (d‐ldh1, d‐ldh2, and d‐ldh3) were cloned and expressed in Escherichia coli BL21 (DE3), and their biochemical and structural properties were characterized. Consequently, a high concentration of pure D‐PLA (47 mM) was produced with a high conversion yield of 88%. Among the three enzymes, D‐LDH1 was responsible for the efficient conversion of PPA to PLA with kinetic parameters of Km (0.36 mM), kcat (481.10 s−1), and kcat/Km (1336.39 mM−1 s−1). In silico structural analysis and site‐directed mutagenesis revealed that the Ile307 in D‐LDH1 is a key residue for excellent PPA reduction with low steric hindrance at the substrate entrance. This study highlights that S. inulinus ATCC 15538 is an excellent PLA producer, equipped with a highly specific and efficient D‐LDH1 enzyme.
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