Kant Shashi, Vohra Anuja, Gupta Reena
Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171005, India.
Protein Expr Purif. 2013 Jan;87(1):11-6. doi: 10.1016/j.pep.2012.09.014. Epub 2012 Oct 13.
Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS-PAGE analysis, enzyme was found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0-5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3h. The addition of Mn(2+), K(+), Zn(2+), Ca(2+) and Al(3+) inhibited the enzyme activity; it increased in the presence of Mg(2+) and Cu(2+) ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K(m) and V(max) values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T(650) from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02.
多聚半乳糖醛酸酶是催化多聚半乳糖醛酸链水解裂解的果胶分解酶。在本研究中，对黑曲霉（MTCC 3323）的多聚半乳糖醛酸酶进行了纯化。用60%乙醇沉淀该酶，纯化倍数为1.68倍。通过Sephacryl S - 200凝胶过滤色谱法将该酶纯化至6.52倍。SDS - PAGE分析表明，该酶是由34 kDa和69 kDa亚基组成的异二聚体。通过非变性聚丙烯酰胺凝胶电泳（NATIVE - PAGE）检测该酶的均一性，发现其分子量为106 kDa。纯化后的酶在45℃、pH 4.8、反应时间15分钟的条件下，以多聚半乳糖醛酸为底物时表现出最大活性。该酶在pH 4.0 - 5.5范围内1小时内保持稳定。在4℃下，108小时后仍保留50%的活性，但在室温下，3小时后失去50%的活性。添加Mn(2+)、K(+)、Zn(2+)、Ca(2+)和Al(3+)会抑制酶活性；在Mg(2+)和Cu(2+)离子存在时酶活性增加。随着底物浓度从0.1%增加到0.5%，酶活性增强。该酶米氏常数（K(m)）和最大反应速度（V(max)）值分别为0.083 mg/ml和18.21 μmol/ml/min。该酶用于番石榴汁的提取和澄清。经酶处理的果肉的出汁率从6%提高到23%。添加纯化后的酶使650 nm处的透光率（%T(650)）从2.5提高到20.4，糖度（°Brix）从1.9提高到4.8。酶处理后果汁的pH从4.5降至3.02。
