Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India.
Appl Biochem Biotechnol. 2011 May;164(1):77-88. doi: 10.1007/s12010-010-9116-8. Epub 2010 Oct 29.
A 56.56-kDa extracellular chitinase from Paenibacillus sp. D1 was purified to 52.3-fold by ion exchange chromatography using SP Sepharose. Maximum enzyme activity was recorded at pH 5.0 and 50 °C. MALDI-LC-MS/MS analysis identified the purified enzyme as chitinase with 60% similarity to chitinase Chi55 of Paenibacillus ehimensis. The activation energy (E (a)) for chitin hydrolysis and temperature quotient (Q (10)) at optimum temperature was found to be 19.14 kJ/mol and 1.25, respectively. Determination of kinetic constants k (m), V (max), k (cat), and k (cat)/k (m) and thermodynamic parameters ΔH*, ΔS*, ΔG*, ΔG*(E-S), and ΔG*(E-T) revealed high affinity of the enzyme for chitin. The enzyme exhibited higher stability in presence of commonly used protectant fungicides Captan, Carbendazim, and Mancozeb compared to control as reflected from the t (1/2) values suggesting its applicability in integrated pest management for control of soil-borne fungal phytopathogens. The order of stability of chitinase in presence of fungicides at 80 °C as revealed from t (1/2) values and thermodynamic parameters E (a(d)) (activation energy for irreversible deactivation), ΔH*, ΔG*, and ΔS* was: Captan > Carbendazim > Mancozeb > control. The present study is the first report on thermodynamic and kinetic characterization of chitinase from Paenibacillus sp. D1.
一株芽孢杆菌 D1 产生的 56.56kDa 细胞外几丁质酶，经 SP 琼脂糖凝胶离子交换层析纯化，比活提高 52.3 倍。最适酶活在 pH5.0 和 50°C 时达到最大。MALDI-LC-MS/MS 分析表明，纯化酶为几丁质酶，与芽孢杆菌 ehimensis 的几丁质酶 Chi55 有 60%的相似性。酶解几丁质的活化能（E(a)）和最适温度下的温度系数（Q(10)）分别为 19.14kJ/mol 和 1.25。动力学常数 k(m)、V(max)、k(cat)和 k(cat)/k(m)以及热力学参数ΔH*、ΔS*、ΔG*、ΔG*(E-S)和ΔG*(E-T)的测定表明，该酶对几丁质具有高亲和力。与对照相比，该酶在常用保护剂杀菌剂克菌丹、多菌灵和代森锰锌存在时表现出更高的稳定性，从 t(1/2)值反映出来，这表明它在综合虫害管理中控制土壤传播真菌病原体的应用潜力。根据 t(1/2)值和热力学参数 E(a(d))（不可逆失活的活化能）、ΔH*、ΔG和ΔS，在杀菌剂存在下几丁质酶的稳定性顺序为：克菌丹>多菌灵>代森锰锌>对照。本研究是首次报道芽孢杆菌 D1 几丁质酶的热力学和动力学特征。
