Khaleghinejad Seyed Hossein, Motalleb Gholamreza, Karkhane Ali Asghar, Aminzadeh Saeed, Yakhchali Bagher
Department of Biology, Faculty of Sciences, University of Zabol, Zabol, Iran.
Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
J Genet Eng Biotechnol. 2016 Jun;14(1):83-89. doi: 10.1016/j.jgeb.2016.08.002. Epub 2016 Sep 3.
Lipases (, EC 3.1.1.3) are one of the highest value commercial enzymes as they have potential applications in biotechnology for detergents, food, pharmaceuticals, leather, textiles, cosmetics, and paper industries; and are currently receiving considerable attention because of their potential applications in biotechnology. Lipase 2 (BTL2) is one of the most important research targets, because of its potential industrial applications. In this study, the effect of substitution Phe17 with Ser in mutated BTL2 lipase, which conserved pentapeptide (Ala-His-Ser-Gln-Gly) was replaced with similar sequences (Gly-Glu-Ser-Ala-Gly) of lipase (CLR) at the nucleophilic elbow region. Docking results confirmed the mutated lipase to be better than the chimeric lipase. So, cloning was conducted, and the mutated and chimeric btl2 genes were expressed in , and then the enzymes were purified by anion exchange chromatography. The mutation increased lipase lipolytic activity against most of the applied substrates, with the exception of tributyrin when compared with chimeric lipase. Further, the mutated lipase exhibited higher activity than the chimeric lipase at all temperatures. Optimum pH of the mutated lipase was obtained at pH 9.5, which was more than the chimeric one. Enzyme activity of the mutated lipase in the presence of organic solvents, detergents, and metal ions was also improved than the chimeric lipase.
脂肪酶（EC 3.1.1.3）是最具商业价值的酶之一，因为它们在生物技术领域有潜在应用，可用于洗涤剂、食品、制药、皮革、纺织、化妆品和造纸工业；并且由于其在生物技术中的潜在应用，目前受到了广泛关注。脂肪酶2（BTL2）是最重要的研究对象之一，因其具有潜在的工业应用价值。在本研究中，对突变型BTL2脂肪酶中的苯丙氨酸17进行丝氨酸替换，在亲核肘部区域，保守的五肽（丙氨酸-组氨酸-丝氨酸-谷氨酰胺-甘氨酸）被脂肪酶（CLR）的类似序列（甘氨酸-谷氨酸-丝氨酸-丙氨酸-甘氨酸）所取代。对接结果证实突变型脂肪酶优于嵌合脂肪酶。因此，进行了克隆，将突变型和嵌合型btl2基因在[具体宿主]中表达，然后通过阴离子交换色谱法对酶进行纯化。与嵌合型脂肪酶相比，该突变提高了脂肪酶对大多数应用底物的脂解活性，但对三丁酸甘油酯除外。此外，在所有温度下，突变型脂肪酶的活性均高于嵌合型脂肪酶。突变型脂肪酶的最适pH值为9.5，高于嵌合型脂肪酶。在有机溶剂、洗涤剂和金属离子存在的情况下，突变型脂肪酶的酶活性也比嵌合型脂肪酶有所提高。
