A catalase from a novel source Sechium edule (squash) has been purified to homogeneity using ammonium sulfate fractional precipitation, dialysis, and anion exchange chromatography on Diethylaminoethyl (DEAE) cellulose in sodium acetate buffer pH 6.0. The purity of the enzyme was analyzed by SDS‐PAGE. The molecular weight of the enzyme using the SDS‐PAGE method was found to be 55 kDa with specific activity 6.35 U/mg. The molecular weight of the enzyme was further confirmed by the INTACT mass spectrometric technique and found to be 52.5 kDa. The Reinheitzahl (Rz) value of the enzyme was 1.5. The MALDI‐TOF analysis of the purified enzyme showed that it contains 529 amino acid residues. The K_m, V_max, optimum pH, and optimum temperature of the free enzyme using H₂O₂ as the substrate were found to be 0.03 mM, 200 μmol/min, 7.6, and 24°C, respectively. The purified enzyme was immobilized on chitosan beads which was prepared by extracting the chitosan from Cornu aspersum (garden snail) having highest degree of deacetylation%. The kinetic characteristics, K_m, V_max, optimum pH, and optimum temperature of the immobilized catalase were found to be 0.065 mM, 250 μmol/min, 7.6, and 41°C, respectively. The immobilized catalase is more thermostable in comparison to free catalase.

中文翻译：

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新型来源Sechium毛囊过氧化氢酶的纯化，表征，固定化和动力学研究

使用硫酸铵分级沉淀，透析和阴离子交换色谱法，在乙酸钠缓冲液pH 6.0中的二乙氨基乙基（DEAE）纤维素上，使用硫酸铵分级沉淀，透析和阴离子交换色谱法，将来自新型来源Sechium edule（南瓜）的过氧化氢酶纯化至均质。通过SDS-PAGE分析酶的纯度。使用SDS-PAGE方法测得的酶分子量为55 kDa，比活性为6.35 U / mg。通过INTACT质谱技术进一步确认了该酶的分子量，为52.5kDa。该酶的Reinheitzahl（Rz）值为1.5。纯化的酶的MALDI-TOF分析表明，它含有529个氨基酸残基。的ķ_米，V _MAX，使用H ₂ O ₂作为底物的游离酶的最佳pH和最佳温度分别为0.03 mM，200μmol/ min，7.6和24°C。将纯化的酶固定在壳聚糖珠上，所述壳聚糖珠是通过从具有最高脱乙酰度％的Cornu aspersum（花园蜗牛）中提取壳聚糖而制得的。固定的过氧化氢酶的动力学特性，K _m，V _max，最适pH和最适温度分别为0.065 mM，250μmol/ min，7.6和41°C。固定的过氧化氢酶与游离的过氧化氢酶相比具有更高的热稳定性。