Abstract A lipase gene was identified and isolated from a fosmid metagenomics library constructed from forest topsoil samples. Error prone PCR and saturation mutagenesis were used to generate seven lipase variants: S69 P/R194Q, S69 G, S69 P, S69E, S69 P/T99S/N190S/R194Q, R194S, and R194A; by testing five different fatty acids of p-nitrophenyl (p-NP) ester, positions S69 and R194 were found to influence the catalytic activity. Specifically, variant R194S was identified that had 5.0, 4.5, 2.1, 2.2, and 1.4-fold higher catalytic efficiency (vmax/Km) towards p-NP acetate, p-NP butyrate, p-NP octanoate, p-NP dodecanoate, and p-NP palmitate, respectively, compared to the metagenome-derived native lipase. Among the S69 variants, variants S69 P/R194Q and S69 P hydrolyzed p-NP acetate 1.8- and 1.5-fold faster than wild-type in terms of apparent vmax/Km values, respectively. The activity is lowered by introducing mutations S69E, R194A, and S69 P/T99S/N190S/R194Q. Here, two additional residues, T99 and N190, were also identified that may have important roles in catalysis. Present work confirms the advantages of combining directed evolution and saturation mutagenesis approaches of protein engineering and expands the knowledge on the architecture of the industrially important lipase enzyme and its relation to its catalytic activity.

中文翻译：

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宏基因组衍生脂肪酶的表面残留丝氨酸 69 和精氨酸 194 影响催化活性

摘要 从森林表土样品构建的fosmid宏基因组文库中鉴定并分离出脂肪酶基因。错误倾向 PCR 和饱和诱变用于产生七种脂肪酶变体：S69 P/R194Q、S69 G、S69 P、S69E、S69 P/T99S/N190S/R194Q、R194S 和 R194A；通过测试对硝基苯基 (p-NP) 酯的五种不同脂肪酸，发现 S69 和 R194 位影响催化活性。具体而言，发现变体 R194S 对 p-NP 乙酸盐、p-NP 丁酸盐、p-NP 辛酸、p-NP 十二酸和 p-NP 的催化效率 (vmax/Km) 高 5.0、4.5、2.1、2.2 和 1.4 倍。 p-NP 棕榈酸酯，分别与宏基因组衍生的天然脂肪酶相比。在 S69 变体中，变体 S69 P/R194Q 和 S69 P 水解了 p-NP 乙酸盐 1.8-和 1。就表观 vmax/Km 值而言，分别比野生型快 5 倍。通过引入突变 S69E、R194A 和 S69 P/T99S/N190S/R194Q 来降低活性。在这里，还鉴定了两个额外的残基，T99 和 N190，它们可能在催化中起重要作用。目前的工作证实了结合蛋白质工程的定向进化和饱和诱变方法的优势，并扩展了工业上重要的脂肪酶结构及其与其催化活性的关系的知识。