Chemistry of Natural and Microbial Products Dept., National Research Centre, Dokki, Giza, Egypt.
Chemistry of Natural and Microbial Products Dept., National Research Centre, Dokki, Giza, Egypt; Biology Department, Faculty of Education, Hafr Al Batin University, Saudi Arabia.
Int J Biol Macromol. 2018 Jun;112:179-187. doi: 10.1016/j.ijbiomac.2018.01.156. Epub 2018 Feb 26.
Our study full filled in two main goals preparation of constitutive exochitinase with low cost, utilizing non-chitin containing agricultural wastes, and improving the thermodynamics of purified Trichoderma longibrachiatum KT693225 exochitinase by covalent coupling to sodium periodate activated agar. Central composite design (CCD) was used to improve the chemical modification of Trichoderma longibrachiatum KT693225 exochitinase. Optimum temperature for conjugated exochitinase 60 °C was higher than native form 40 °C. Covalent coupling to oxidized agar caused 4.32, 2.75 and 2.44-fold increase in half-life values at 50, 55 and 60 °C, respectively. Also, conjugated exochitinase showed higher D-values (decimal reduction time) 1790.49 compared to 733.08 min for native form at 60 °C. Moreover, conjugated form had lower deactivation constant rate (k) 0.39 × 10 minat 60 °C than native form 1.7 × 10 min. Native exochitinase exhibited higher activation energy (E) 3.39 Kcal·mol and lower energy for denaturation (E) 6.88 Kcal·mol compared to 3.21 and 13.05 Kcal·mol, respectively for conjugated form. The values of thermodynamic parameters for inactivation of native and conjugated exochitinase indicated that conjugation significantly decreased entropy (ΔS°) and increased enthalpy (ΔH°) and free energy (ΔG°) of deactivation. Conjugated exochitinase exhibited higher antifungal effect against Alternaria alternata, Fusarium oxysporium and Aspergillus niger than native form.
利用不含几丁质的农业废弃物，以低成本制备结构型几丁外切酶；通过共价偶联到高碘酸钠活化琼脂来提高纯化的长枝木霉 KT693225 几丁外切酶的热力学稳定性。采用中心复合设计（CCD）来优化长枝木霉 KT693225 几丁外切酶的化学修饰。共轭几丁外切酶的最适温度为 60°C，高于天然形式的 40°C。与氧化琼脂的共价偶联分别使 50、55 和 60°C 时的半衰期值增加了 4.32、2.75 和 2.44 倍。此外，共轭几丁外切酶在 60°C 时的 D 值（十进制减少时间）为 1790.49，比天然形式的 733.08 min 更高。此外，共轭形式在 60°C 时的失活常数速率（k）为 0.39×10 min，比天然形式的 1.7×10 min 低。与共轭形式相比，天然几丁外切酶的活化能（E）为 3.39 Kcal·mol，变性能（E）为 6.88 Kcal·mol，分别更高，而天然形式的变性熵（ΔS°）和焓（ΔH°）和自由能（ΔG°）分别为 13.05 Kcal·mol 和 3.21 Kcal·mol，分别更低。天然和共轭几丁外切酶失活动力学参数的值表明，共轭作用显著降低了失活的熵（ΔS°），增加了焓（ΔH°）和自由能（ΔG°）。与天然形式相比，共轭几丁外切酶对Alternaria alternata、Fusarium oxysporium 和 Aspergillus niger 具有更高的抗真菌作用。
