Department of Plant Molecular Biology and Biotechnology, ASPEE College of Horticulture and Forestry, Navsari Agricultural University, Navsari, 396450, India.
ASPEE SHAKILAM Biotechnology Institute, Navsari Agricultural University, Ghod Dod Road, Athwa Farm, Surat, 395 007, India.
Sci Rep. 2022 May 9;12(1):7564. doi: 10.1038/s41598-022-11022-0.
Pectate lyase is a hydrolytic enzyme used by diverse industries to clarify food. The enzyme occupies a 25% share of the total enzyme used in food industries, and their demand is increasing gradually. Most of the enzymes in the market belong to the fungal origin and take more time to produce with high viscosity in the fermentation medium, limiting its use. The bacteria belonging to the genus Bacillus have vast potential to produce diverse metabolites of industrial importance. The present experiment aimed to isolate pectate lyase-producing bacteria that can tolerate an alkaline environment at moderate temperatures. Bacillus subtilis PKC2, Bacillus licheniformis PKC4, Paenibacillus lactis PKC5, and Bacillus sonorensis ADCN produced pectate lyase. The Paenibacillus lactis PKC5 gave the highest protein at 48 h of incubation that was partially purified using 80% acetone and ammonium sulphate. Purification with 80% acetone resulted in a good enzyme yield with higher activity. SDS-PAGE revealed the presence of 44 kDa molecular weight of purified enzyme. The purified enzyme exhibits stability at diverse temperature and pH ranges, the maximum at 50 °C and 8.0 pH. The metal ions such as Mg, Zn, Fe, and Co significantly positively affect enzyme activity, while increasing the metal ion concentration to 5 mM showed detrimental effects on the enzyme activity. The organic solvents such as methanol and chloroform at 25% final concentration improved the enzyme activity. On the other hand, detergent showed inhibitory effects at 0.05% and 1% concentration. Pectate lyase from Paenibacillus lactis PKC5 had Km and Vmax values as 8.90 mg/ml and 4.578 μmol/ml/min. The Plackett-Burman and CCD designs were used to identify the significant process parameters, and optimum concentrations were found to be pectin (5 gm%) and ammonium sulphate (0.3 gm%). During incubation with pectate lyase, the clarity percentage of the grape juice, apple juice, and orange juice was 60.37%, 59.36%, and 49.91%, respectively.
果胶裂解酶是一种水解酶，被各种行业用于澄清食品。该酶占据食品工业中使用的总酶的 25%份额，其需求正在逐渐增加。市场上的大多数酶都来自真菌，生产时间较长，发酵介质的粘度较高，限制了其使用。属于芽孢杆菌属的细菌具有产生各种具有工业重要性的代谢物的巨大潜力。本实验旨在分离能够耐受中温和碱性环境的产果胶裂解酶细菌。枯草芽孢杆菌 PKC2、地衣芽孢杆菌 PKC4、解淀粉芽孢杆菌 PKC5 和苏云金芽孢杆菌 ADCN 产生果胶裂解酶。解淀粉芽孢杆菌 PKC5 在孵育 48 小时时产生最高量的蛋白质，并用 80%丙酮和硫酸铵部分纯化。用 80%丙酮纯化可获得较高活性的良好酶产量。SDS-PAGE 显示纯化酶的分子量为 44 kDa。纯化酶在不同的温度和 pH 范围内表现出稳定性，在 50°C 和 8.0 pH 下达到最大值。Mg、Zn、Fe 和 Co 等金属离子显著正向影响酶活性，而将金属离子浓度增加到 5 mM 会对酶活性产生不利影响。甲醇和氯仿等有机溶剂在最终浓度为 25%时可提高酶活性。另一方面，洗涤剂在 0.05%和 1%浓度下显示出抑制作用。解淀粉芽孢杆菌 PKC5 的果胶裂解酶的 Km 和 Vmax 值分别为 8.90 mg/ml 和 4.578 μmol/ml/min。Plackett-Burman 和 CCD 设计用于确定显著的工艺参数，发现最佳浓度为果胶（5 gm%）和硫酸铵（0.3 gm%）。在用果胶裂解酶孵育时，葡萄汁、苹果汁和橙汁的澄清度百分比分别为 60.37%、59.36%和 49.91%。
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