Abstract：
In order to better utilize chitinolytic enzymes to produce high-value N-acetyl-D-glucosamine (GlcNAc) from chitinous waste, there is an urgent need to explore bi-functional chitinases with exceptional properties of temperature, pH and metal tolerance. In this study, we cloned and characterized a novel bi-functional cold-adaptive chitinase called CaChi18A from a newly isolated strain, Chitinilyticum aquatile CSC-1, in Bama longevity village of Guangxi Province, China. The activity of CaChi18A at 50 °C was 4.07 U/mg. However, it exhibited significant catalytic activity even at 5 °C. Its truncated variant CaChi18A_ΔChBDs, containing only catalytic domain, demonstrated significant activity levels, exceeding 40 %, over a temperature range of 5-60 °C and a pH range of 3 to 10. It was noteworthy that it displayed tolerance towards most metal ions at a final concentration of 0.1 mM, including Fe3+ and Cu2+ ions, retaining 122.52 ± 0.17 % and 116.42 ± 1.52 % activity, respectively. Additionally, it exhibited favorable tolerance towards organic solvents with the exception of formic acid. Interestedly, CaChi18A and CaChi18A_ΔChBDs had a low Km value towards colloidal chitin (CC), 0.94 mg mL-1 and 2.13 mg mL-1, respectively. Both enzymes exhibited chitobiosidase and N-acetyl-D-glucosaminidase activities, producing GlcNAc as the primary product when hydrolyzing CC. The high activities across a broader temperature and pH range, strong environmental adaptability, and hydrolytic properties of CaChi18A_ΔChBDs suggested that it could be a promising candidate for GlcNAc production.
摘要：
为了更好地利用几丁质分解酶从几丁质废物中生产高价值的N-乙酰-D-氨基葡萄糖(GlcNAc),迫切需要探索具有特殊温度特性的双功能几丁质酶，pH和金属耐受性。在这项研究中,我们从一个新分离的菌株中克隆并鉴定了一种名为CaChi18A的新型双功能冷适应性几丁质酶，广西巴马长寿村的ChitinilyticumaquatileCSC-1，中国。CaChi18A在50°C下的活性为4.07U/mg。然而,即使在5°C下也表现出显著的催化活性。其截短变体CaChi18A_ΔChBDs，只含有催化结构域，表现出显著的活动水平，超过40%,在5-60°C的温度范围和3至10的pH范围内。值得注意的是，它在最终浓度为0.1mM时对大多数金属离子表现出耐受性，包括Fe3+和Cu2+离子，保留122.52±0.17%和116.42±1.52%的活性，分别。此外,除甲酸外，它对有机溶剂表现出良好的耐受性。有趣的是，CaChi18A和CaChi18A_ΔChBD对胶体甲壳素(CC)具有低Km值，分别为0.94mgmL-1和2.13mgmL-1。两种酶均表现出壳二糖苷酶和N-乙酰-D-氨基葡萄糖苷酶活性，水解CC时产生GlcNAc作为主要产物。在更宽的温度和pH范围内的高活性，环境适应性强,CaChi18A_ΔChBDs的水解特性表明它可能是GlcNAc生产的有希望的候选物。
