Department of Bioinformatics and Biosciences, Capital University of Science and Technology, Islamabad, Pakistan.
College of Life Sciences and Biotechnology, The State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, China.
Arch Microbiol. 2020 Aug;202(6):1497-1506. doi: 10.1007/s00203-020-01869-0. Epub 2020 Mar 26.
Lipases with high tolerance to temperature play a significant role in industry from food manufacturing to waste management systems. Thus, there is a need to investigate these enzymes from different geographical areas to look out for a more thermo-stable one. Characterization of lipases through experimental approaches is time consuming process and sometimes the results are ambiguous due to errors. However, integration of computational technologies is quite useful for prediction of optimized conditions. Such technologies can be applied as synthetic biology, which has many major applications in engineered biological approaches for accurate prediction of effects of different physical and chemical parameters on the system. In this study, cloning and expression of a lipase gene from Bacillus amyloliquefaciens, isolated from a novel geographical region of Pakistan, in Escherichia coli DH5α cells followed by sequencing was carried out. To isolate thermostable lipase producing strains, all the samples were kept at 50 °C. Genomic DNA was isolated and signal peptide (1-32 residues) sequence was chopped (ΔSPLipase). The ΔSPLipase was amplified and expressed in Linearized p15TV-L vector. The purified lipase appeared as single band of approximately 26 kDa. Suitable conditions of factors required for maximum lipase activity such as temperature, pH, substrate, organic solvent, detergents and metal ions were predicted through synthetic biology approach and further confirmed in wet lab. The predicted suitable factors for enzyme were almost similar to those determined experimentally. The optimum enzyme activity was recorded at pH 8 and 50 °C temperature. Interestingly, the activity of enzyme was found on a number of solvents, metal ions, detergents, and surfactants. The predicted optimum values and their experimental confirmations highlights the importance of integrated synthetic biology approaches in wet lab experiments. The characterized lipase of B. amyloliquefaciens at molecular level from Pakistani strains displayed good activity on a range of factors that implies this strain to be used for application in industrial level production.
具有较高温度耐受性的脂肪酶在食品制造到废物管理系统等工业领域发挥着重要作用。因此，需要从不同的地理区域研究这些酶，以寻找更耐热的脂肪酶。通过实验方法对脂肪酶进行表征是一个耗时的过程，有时由于误差，结果还存在歧义。然而，计算技术的集成对于预测优化条件非常有用。这些技术可应用于合成生物学，它在工程生物学方法中有许多重要应用，可准确预测不同物理和化学参数对系统的影响。在这项研究中，从巴基斯坦一个新的地理区域分离出的解淀粉芽孢杆菌的脂肪酶基因进行了克隆和在大肠杆菌 DH5α细胞中的表达，然后进行了测序。为了分离产热稳定脂肪酶的菌株，所有样品都保存在 50°C。提取基因组 DNA 并切除信号肽（1-32 个残基）序列（ΔSPLipase）。扩增 ΔSPLipase 并在线性化 p15TV-L 载体中表达。纯化的脂肪酶出现约 26 kDa 的单一条带。通过合成生物学方法预测了最大脂肪酶活性所需的因素（如温度、pH 值、底物、有机溶剂、洗涤剂和金属离子）的适宜条件，并在湿实验室进一步验证。预测的酶适宜因素与实验确定的因素非常相似。在 pH 8 和 50°C 的温度下记录到最佳酶活性。有趣的是，该酶在许多溶剂、金属离子、洗涤剂和表面活性剂上都具有活性。预测的最佳值及其实验验证突出了综合合成生物学方法在湿实验室实验中的重要性。从巴基斯坦菌株的分子水平对 B. amyloliquefaciens 进行了表征的脂肪酶在一系列因素上表现出良好的活性，这意味着该菌株可用于工业水平的生产应用。
