Dieselzymes: development of a stable and methanol tolerant lipase for biodiesel production by directed evolution

Abstract

Background: 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. Results: 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 halfinactivation 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. Conclusions: 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.

Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?

“…This would exclude cosolvent molecules from interacting with the surface and prevent the enzyme from denaturation and/or deactivation. In this respect, the observation made in this study is consistent with previous studies reporting the importance of introducing polar and charged amino acids [33,37,38] to increase resistance to water-miscible organic solvents. Similar trends were observed when the three-dimensional structures of 29 organic solvent-stable lipases were analyzed [26].…”

“…This ratio explains why beneficial substitutions were identified in previous directed evolution campaigns after the screening of only a few thousand clones [25,33]. However, our study clearly indicates that random mutagenesis methods fail to detect the vast majority (>88%) of the theoretically possible single substitution diversity that would be beneficial for resistance to the three selected organic cosolvents.…”

“…It is more likely that the majority of the introduced charges act in a water-attracting way to prevent cosolvent molecules from interacting with the enzyme. For example, the formation of new hydrogen bonds between residues on the enzyme's surface was observed in organic cosolvent-stable enzyme variants in previous studies, [33,35,40]. Such hydrogen bonds could be established between surface residues (e.g., after the substitution to polar or charged amino acids) as well as with water molecules.…”

Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus

“…Process engineering was also used to lower the methanol concentration in the reaction by stepwise addition (Bélafi-Bakó et al 2002;Nie et al 2006;Soumanou and Bornscheuer 2003) or by the use of cosolvent systems. In addition, in recent years, few attempts were made to enhance lipases stability in methanol by applying protein engineering methods (Dror et al 2014;Korman et al 2013;Park et al 2012). However, the protein engineering approach was hardly exploited due to the lack of structural information and profound understanding of methanol effect on the lipase structure and activity.…”

Identification and characterization of lipolytic bacteria from Thai fermented foods

“…Lipases are commonly used in the production of a variety of products, ranging from fruit juices, baked foods, vegetable fermentation and in flavour development (Hasan et al, 2005). Bacterial lipase is linked to their role as biocatalysts in many biochemical processes such as biodiesel synthesis (Joshi and Vinay, 2007;Korman et al, 2013). However, bacterial strains are varied in their enzymatic activity, which depend on the species and the cultivating conditions (Sharma et al, 2001;Mrozik et al, 2006).…”