Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Ernst-Moritz-Arndt University of Greifswald, Felix-Hausdorff Str. 4, 17487 Greifswald, Germany.
Appl Microbiol Biotechnol. 2012 Apr;94(1):141-50. doi: 10.1007/s00253-012-3903-9. Epub 2012 Feb 1.
The Ustilago maydis lipase UM03410 belongs to the mostly unexplored Candida antarctica lipase (CAL-A) subfamily. The two lipases with [corrected] the highest identity are a lipase from Sporisorium reilianum and the prototypic CAL-A. In contrast to the other CAL-A-type lipases, this hypothetical U. maydis lipase is annotated to possess a prolonged N-terminus of unknown function. Here, we show for the first time the recombinant expression of two versions of lipase UM03410: the full-length form (lipUMf) and an Nterminally truncated form (lipUMs). For comparison to the prototype, the expression of recombinant CAL-A in E. coli was investigated. Although both forms of lipase UM03410 could be expressed functionally in E. coli, the N-terminally truncated form (lipUMs) demonstrated significantly higher activities towards p-nitrophenyl esters. The functional expression of the N-terminally truncated lipase was further optimized by the appropriate choice of the E. coli strain, lowering the cultivation temperature to 20 °C and enrichment of the cultivation medium with glucose. Primary characteristics of the recombinant lipase are its pH optimum in the range of 6.5-7.0 and its temperature optimum at 55 °C. As is typical for lipases, lipUM03410 shows preference for long chain fatty acid esters with myristic acid ester (C14:0 ester) being the most preferred one.More importantly, lipUMs exhibits an inherent preference for C18:1Δ9 trans and C18:1Δ11 trans-fatty acid esters similar to CAL-A. Therefore, the short form of this U. maydis lipase is the only other currently known lipase with a distinct trans-fatty acid selectivity.
玉米黑粉菌脂肪酶 UM03410 属于研究甚少的南极假丝酵母脂肪酶 (CAL-A) 亚家族。与该酶具有最高同源性的两种脂肪酶是来自 Sporisorium reilianum 的脂肪酶和典型的 CAL-A。与其他 CAL-A 型脂肪酶不同，这种假设的玉米黑粉菌脂肪酶被注释为具有未知功能的延长 N 端。在这里，我们首次展示了两种形式的脂肪酶 UM03410 的重组表达：全长形式（lipUMf）和 N 端截断形式（lipUMs）。为了与原型进行比较，研究了重组 CAL-A 在大肠杆菌中的表达。尽管两种形式的脂肪酶 UM03410 都可以在大肠杆菌中功能性表达，但 N 端截断形式（lipUMs）对 p-硝基苯酯的活性显著更高。通过选择合适的大肠杆菌菌株、将培养温度降低至 20°C 和在培养介质中富集葡萄糖，进一步优化了 N 端截断脂肪酶的功能表达。重组脂肪酶的主要特性是其 pH 最适范围在 6.5-7.0 之间，最适温度在 55°C。与脂肪酶典型特征一样，lipUM03410 对长链脂肪酸酯具有偏好性，其中肉豆蔻酸酯（C14:0 酯）是最优选的。更重要的是，lipUMs 对 C18:1Δ9 反式和 C18:1Δ11 反式脂肪酸酯表现出固有偏好，类似于 CAL-A。因此，这种玉米黑粉菌脂肪酶的短形式是目前已知的唯一具有明显反式脂肪酸选择性的脂肪酶。
