• Journal of Microbiology and Biotechnology
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• 제28권4호
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• pp.571-578
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• 2018

Biofuel production using lignocellulosic biomass is gaining attention because it can be substituted for fossil fuels without competing with edible resources. However, because Saccharomyces cerevisiae does not have a ${\text\tiny{D}}$-xylose metabolic pathway, oxidoreductase or isomerase pathways must be introduced to utilize ${\text\tiny{D}}$-xylose from lignocellulosic biomass in S. cerevisiae. To elucidate the biochemical properties of xylose isomerase (XI) from Piromyces sp. E2 (PsXI), we determine its crystal structure in complex with substrate mimic glycerol. An amino-acid sequence comparison with other reported XIs and relative activity measurements using five kinds of divalent metal ions confirmed that PsXI belongs to class II XIs. Moreover kinetic analysis of PsXI was also performed using $Mn^{2+}$, the preferred divalent metal ion for PsXI. In addition, the substrate-binding mode of PsXI could be predicted with the substrate mimic glycerol bound to the active site. These studies may provide structural information to enhance ${\text\tiny{D}}$-xylose utilization for biofuel production.

• Journal of Microbiology and Biotechnology
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• 제32권2호
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• pp.187-194
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• 2022

Two α-ʟ-arabinofuranosidases (BfdABF1 and BfdABF3) and a β-ᴅ-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℃. This exo-enzyme could hydrolyze p-nitrophenyl arabinofuranoside, arabino-oligosaccharides (AOS), arabinoxylo-oligosaccharides (AXOS) such as 3²-α-ʟ-arabinofuranosyl-xylobiose (A³X), and 2³-α-ʟ-arabinofuranosyl-xylotriose (A²XX), 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)-ʟ-arabinofuranosidic linkages compared to α-(1,3)-linkages. Especially, BfdABF1 could slowly hydrolyze 2³,3³-di-α-ʟ-arabinofuranosyl-xylotriose (A²⁺³XX). Meanwhile, BfdABF3 showed the highest activity in sodium acetate at pH 6.0 and 50℃, and it has the exclusively high activities on AXOS such as A³X and A²XX. BfdABF3 mainly catalyzes the removal of ʟ-arabinose side chains from various AXOS. BfdXYL2 exhibited the highest activity in sodium citrate at pH 5.0 and 55℃, and it specifically hydrolyzed p-nitrophenyl xylopyranoside and xylo-oligosaccharides (XOS). Also, BfdXYL2 could slowly hydrolyze AOS and AXOS such as A³X. Based on the detailed hydrolytic modes of action of three exo-hydrolases (BfdABF1, BfdABF3, and BfdXYL2) from Bf. dentium, their probable roles in the hemiceullose-utilization system of Bf. dentium are proposed in the present study. These intracellular exo-hydrolases can synergistically produce ʟ-arabinose and ᴅ-xylose from various AOS, XOS, and AXOS.