Department of Chemistry and Biochemisty, UCLA-DOE Institute of Genomics and Proteomics, Molecular Biology Institute, University of California, Los Angeles, USA.
Biotechnol Biofuels. 2013 May 7;6(1):70. doi: 10.1186/1754-6834-6-70.
Biodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed transesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially.
We employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the half-inactivation time at 50°C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis.
Directed evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms the industrially used lipase from Burkholderia cepacia and provides a platform for still further evolution of desirable biodiesel production properties.
生物柴油是脂肪酸的甲酯，通常通过甘油三酯与甲醇的碱催化酯交换反应生产。一些脂肪酶是生物柴油合成的有效催化剂，与传统的碱或酸催化酯交换相比具有许多潜在优势。然而，天然脂肪酶通常会被高浓度的甲醇迅速失活，限制了它们的实际应用。变形假单胞菌脂肪酶是生物柴油合成的一种特别有前途的催化剂，因为它即使在大量水存在的情况下也能产生高产量的甲酯，并且在大肠杆菌中表达得非常好。然而，由于变形假单胞菌脂肪酶的稳定性和甲醇耐受性仅为中等，因此在该酶能够在工业上使用之前，需要对其进行改进。
我们采用定向进化技术，得到了一种具有 13 个突变的变形假单胞菌脂肪酶突变体，我们称之为 Dieselzyme 4。Dieselzyme 4 的热稳定性有了很大的提高，在 50°C 时半衰期延长了 30 倍。该进化酶的甲醇耐受性也有了显著提高，在 50%的甲醇水溶液中半衰期延长了 50 倍。固定化的 Dieselzyme 4 酶仍然具有合成生物柴油的能力，并且在多次生物柴油合成循环中比野生型或工业用的伯克霍尔德氏菌脂肪酶具有更长的使用寿命。Dieselzyme 4 的晶体结构显示，与野生型酶相比，Dieselzyme 4 中有更多的氢键和盐桥，这表明在用于生物柴油合成的油和甲醇混合物的低介电环境中，极性相互作用可能变得特别稳定。
定向进化产生了一种稳定的脂肪酶 Dieselzyme 4，可以固定化并重复用于生物柴油合成。Dieselzyme 4 优于工业用的伯克霍尔德氏菌脂肪酶，为进一步进化生物柴油生产所需的特性提供了一个平台。
