Abstract: A thermo-acidophilic bacterium, Alicyclobacillus mali FL18, was isolated from a hot spring of Pisciarelli, near Naples, Italy; following genome analysis, a novel putative β-xylosidase, AmβXyl, belonging to the glycosyl hydrolase (GH) family 3 was identified. A synthetic gene was produced, cloned in pET-30a(+), and expressed in Escherichia coli BL21 (DE3) RIL. The purified recombinant protein, which showed a dimeric structure, had optimal catalytic activity at 80 ◦C and pH 5.6, exhibiting 60% of its activity after 2 h at 50 ◦C and displaying high stability (more than 80%) at pH 5.0–8.0 after 16 h. AmβXyl is mainly active on both para-nitrophenyl-β-D-xylopyranoside (KM0.52 mM, kcat 1606 s−1, and kcat/KM 3088.46 mM−1·s−1) and para-nitrophenyl-α-L-arabinofuranoside(KM 10.56 mM, kcat 2395.8 s−1 , and kcat/KM 226.87 mM−1·s−1). Thin-layer chromatography showed its ability to convert xylooligomers (xylobiose and xylotriose) into xylose, confirming that AmβXyl isa true β-xylosidase. Furthermore, no inhibitory effect on enzymatic activity by metal ions, detergents,or EDTA was observed except for 5 mM Cu2+. AmβXyl showed an excellent tolerance to organic solvents; in particular, the enzyme increased its activity at high concentrations (30%) of organic solvents such as ethanol, methanol, and DMSO. Lastly, the enzyme showed not only a good tolerance to inhibition by xylose, arabinose, and glucose, but was activated by 0.75 M xylose and up to 1.5 Mby both arabinose and glucose. The high tolerance to organic solvents and monosaccharides togetherwith other characteristics reported above suggests that AmβXyl may have several applications inmany industrial fields

Alicyclobacillus mali FL18 as a Novel Source of Glycosyl Hydrolases: Characterization of a New Thermophilic β-Xylosidase Tolerant to Monosaccharides / Salzano, Flora; Aulitto, Martina; Fiorentino, Gabriella; Pedone, Emilia; Contursi, Patrizia; Limauro, Danila. - In: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. - ISSN 1422-0067. - 23:22(2022). [10.3390/ijms232214310]

Alicyclobacillus mali FL18 as a Novel Source of Glycosyl Hydrolases: Characterization of a New Thermophilic β-Xylosidase Tolerant to Monosaccharides

Flora Salzano;Martina Aulitto
Co-primo
;
Gabriella Fiorentino
Secondo
;
Patrizia Contursi
Penultimo
;
Danila Limauro
Ultimo
2022

Abstract

Abstract: A thermo-acidophilic bacterium, Alicyclobacillus mali FL18, was isolated from a hot spring of Pisciarelli, near Naples, Italy; following genome analysis, a novel putative β-xylosidase, AmβXyl, belonging to the glycosyl hydrolase (GH) family 3 was identified. A synthetic gene was produced, cloned in pET-30a(+), and expressed in Escherichia coli BL21 (DE3) RIL. The purified recombinant protein, which showed a dimeric structure, had optimal catalytic activity at 80 ◦C and pH 5.6, exhibiting 60% of its activity after 2 h at 50 ◦C and displaying high stability (more than 80%) at pH 5.0–8.0 after 16 h. AmβXyl is mainly active on both para-nitrophenyl-β-D-xylopyranoside (KM0.52 mM, kcat 1606 s−1, and kcat/KM 3088.46 mM−1·s−1) and para-nitrophenyl-α-L-arabinofuranoside(KM 10.56 mM, kcat 2395.8 s−1 , and kcat/KM 226.87 mM−1·s−1). Thin-layer chromatography showed its ability to convert xylooligomers (xylobiose and xylotriose) into xylose, confirming that AmβXyl isa true β-xylosidase. Furthermore, no inhibitory effect on enzymatic activity by metal ions, detergents,or EDTA was observed except for 5 mM Cu2+. AmβXyl showed an excellent tolerance to organic solvents; in particular, the enzyme increased its activity at high concentrations (30%) of organic solvents such as ethanol, methanol, and DMSO. Lastly, the enzyme showed not only a good tolerance to inhibition by xylose, arabinose, and glucose, but was activated by 0.75 M xylose and up to 1.5 Mby both arabinose and glucose. The high tolerance to organic solvents and monosaccharides togetherwith other characteristics reported above suggests that AmβXyl may have several applications inmany industrial fields
2022
Alicyclobacillus mali FL18 as a Novel Source of Glycosyl Hydrolases: Characterization of a New Thermophilic β-Xylosidase Tolerant to Monosaccharides / Salzano, Flora; Aulitto, Martina; Fiorentino, Gabriella; Pedone, Emilia; Contursi, Patrizia; Limauro, Danila. - In: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. - ISSN 1422-0067. - 23:22(2022). [10.3390/ijms232214310]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/901201
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