嗜酸细菌,从Pisciarelli的温泉中分离出的alicyclobacillusmaliFL18,在那不勒斯附近,意大利;在基因组分析之后,一种新的推定β-木糖苷酶,AmβXyl,属于糖基水解酶(GH)家族3。产生了一个合成基因,克隆在pET-30a(+),并在大肠杆菌BL21(DE3)RIL中表达。纯化的重组蛋白,显示出二聚体结构,在80°C和pH5.6下具有最佳的催化活性,在50°C下2小时后表现出其活性的60%,并且在16小时后在pH5.0-8.0下表现出高稳定性(超过80%)。AmβXyl对这两种对硝基苯基-β-D-吡喃木糖苷(KM0.52mM,kcat1606s-1,和kcat/KM3088.46mM-1·s-1)和对硝基苯基-α-L-阿拉伯呋喃糖苷(KM10.56mM,kcat2395.8s-1和kcat/KM226.87mM-1·s-1)。薄层色谱显示其将木低聚物(木二糖和木三糖)转化为木糖的能力,确认AmβXyl是真正的β-木糖苷酶。此外,金属离子对酶活性没有抑制作用,洗涤剂,除5mMCu2+外,观察到EDTA。AmβXyl对有机溶剂表现出优异的耐受性;特别是,该酶在高浓度(30%)的有机溶剂如乙醇中增加了其活性,甲醇,DMSO。最后,该酶不仅表现出对木糖抑制的良好耐受性,阿拉伯糖,和葡萄糖,但被0.75M木糖激活,被阿拉伯糖和葡萄糖激活高达1.5M。对有机溶剂和单糖的高耐受性以及上面报道的其他特征表明AmβXyl可以在许多工业领域中具有多种应用。
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 (KM 0.52 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 is a 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 M by both arabinose and glucose. The high tolerance to organic solvents and monosaccharides together with other characteristics reported above suggests that AmβXyl may have several applications in many industrial fields.