Rade Letícia L, da Silva Melque N P, Vieira Plínio S, Milan Natalia, de Souza Claudia M, de Melo Ricardo R, Klein Bruno C, Bonomi Antonio, de Castro Heizir F, Murakami Mário T, Zanphorlin Leticia M
Brazilian Biorenewables National Laboratory, Brazilian Center for Research in Energy and Materials, Campinas, Brazil.
Department of Chemical Engineering, Engineering School of Lorena, University of São Paulo, Lorena, Brazil.
Front Bioeng Biotechnol. 2020 May 6;8:304. doi: 10.3389/fbioe.2020.00304. eCollection 2020.
Macaw palm is a highly oil-producing plant, which presents high contents of free fatty acids, being a promising feedstock for biofuel production. The current chemical routes are costly and complex, involving highly harsh industrial conditions. Enzymatic processing is a potential alternative; however, it is hampered by the scarce knowledge on biocatalysts adapted to this acidic feedstock. This work describes a novel lipase isolated from the thermophilic fungus (Lip), which tolerates extreme conditions such as the presence of methanol, high temperatures, and acidic medium. Among the tested feedstocks, the enzyme showed the highest preference for macaw palm oil, producing a hydrolyzate with a final free fatty acid content of 92%. Crystallographic studies revealed a closed conformation of the helical amphipathic that typically undergoes conformational changes in a mechanism of interfacial activation. Such conformation of the is stabilized by a salt bridge, not observed in other structurally characterized homologs, which is likely involved in the tolerance to organic solvents. Moreover, the lack of conservation of the aromatic cluster IxxWxxxxxF in the of Lip with the natural mutation of the phenylalanine by an alanine might be correlated with the preference of short acyl chains, although preserving catalytic activity on insoluble substrates. In addition, the presence of five acidic amino acids in the of Lip, a rare property reported in other lipases, may have contributed to its ability to tolerate and be effective in acidic environments. Therefore, our work describes a new fungal biocatalyst capable of efficiently hydrolyzing macaw oil, an attractive feedstock for the production of "drop-in" biofuels, with high desirable feature for industrial conditions such as thermal and methanol tolerance, and optimum acidic pH. Moreover, the crystallographic structure was elucidated, providing a structural basis for the enzyme substrate preference and tolerance to organic solvents.
金刚鹦鹉棕榈是一种高产油植物，其游离脂肪酸含量很高，是生物燃料生产的一种有前景的原料。目前的化学路线成本高且复杂，涉及高度苛刻的工业条件。酶促加工是一种潜在的替代方法；然而，由于对适应这种酸性原料的生物催化剂了解不足，其发展受到阻碍。这项工作描述了一种从嗜热真菌中分离出的新型脂肪酶（Lip），它能耐受极端条件，如甲醇的存在、高温和酸性介质。在测试的原料中，该酶对金刚鹦鹉棕榈油表现出最高的偏好，产生的水解产物最终游离脂肪酸含量为92%。晶体学研究揭示了螺旋两亲结构域的封闭构象，该结构域通常在界面激活机制中发生构象变化。这种结构域的构象通过一个盐桥得以稳定，在其他已进行结构表征的同源物中未观察到这种盐桥，它可能与对有机溶剂的耐受性有关。此外，Lip的结构域中芳香族簇IxxWxxxxxF缺乏保守性，苯丙氨酸自然突变为丙氨酸，这可能与对短酰基链的偏好有关，尽管它对不溶性底物仍保留催化活性。此外，Lip的结构域中存在五个酸性氨基酸，这是其他脂肪酶中罕见的特性，可能有助于其在酸性环境中耐受并发挥作用的能力。因此，我们的工作描述了一种新的真菌生物催化剂，它能够有效地水解金刚鹦鹉棕榈油，这是一种用于生产“即插即用”生物燃料的有吸引力的原料，具有热耐受性、甲醇耐受性和最佳酸性pH等工业条件所需的理想特性。此外，阐明了晶体学结构，为酶的底物偏好和对有机溶剂的耐受性提供了结构基础。
