Recombinant Lipase Engineered with Amphipathic and Coiled-Coil Peptides

Abstract

Lipases have been utilized industrially to produce biodiesel, oleochemicals, and pharmaceuticals. Many efforts such as metagenomics, directed evolution and enzyme immobilization have been devoted to enhance the lipase activity.Here, we designed a recombinant lipase, NKC-M37-MAT, that was generated by incorporating an N-terminal amphipathic peptide (NKC) and a C-terminal coiled-coil peptide (MAT) into Photobacterium lipolyticum M37 lipase. The hydrophobic face of NKC improve the accessibility (K m ), and catalytic efficiency (K cat /K m ) of the soluble lipase toward hydrophobic substrate and tetrameric MAT further enhanced lipase catalytic activity (U/mg) through cooperative binding to its substrate such that the catalytic activity (U/mg) of NKC-M37-MAT was increased by a maximum of 54-fold compared with the wildtype, which decreased the biodiesel production time 5-fold from 30 h to 6 h. This novel approach shows promise as a platform technology to increase lipase catalytic efficiency for industrial-scale production of biodiesel and biochemicals synthesized from hydrophobic substrates.Triacylglycerol lipase (E.C. 3.1.1.3), 1 which catalyzes the hydrolysis of triglycerides to fatty acids and glycerols, plays an important role in industries that produce products such as detergents, food, leather, paper, cosmetics, and bioenergy. 1-3 Because of their broad substrate specificity and enantioselectivity, lipases have been utilized by the pharmaceutical industry to synthesize pure R-and S-optical isomers. 4 The energy industry is also using lipases to produce biodiesel from vegetable oil as well. 1,3,5,6 Biodiesel products, an emerging renewable energy source, are produced through trans-esterification in the presence of a catalyst which could be basic (generally, NaOH), acid (usually, HCl or H 2 SO 4 ), or enzymatic. 7-9 However, base-catalyzed trans-esterification is not feasible since alkaline can react with free fatty acid to form unwanted soap and water, which would affect the biodiesel quality and require extra investment for downstream separation and purification. In acid-catalyzed esterification, large excess amount of alcohol is required in order to reach high biodiesel yield, and if sulfuric acid is utilized as catalysts, it is difficult to remove them after reaction. Moreover, chemical-catalyzed trans-esterification produces toxic wastewater and consumes high amounts of energy to maintain high reaction temperature. 8 To overcome these problems, enzymatic methods using lipase are receiving special attention as an attractive alternative approach for biodiesel production. The great advantages of lipases compared with chemical catalysts are as follows: 1) relatively low reaction temperatures (approximately 40°C), 6 2) fewer process steps that generate reduced amounts of hazardous materials, 3) improved product separation that generates higher purity glycerol. 5,6 Since the enzymatic synthesis of biodiesel occurs at the lipid-water interface, increasing the accessibility of water-soluble lipase such as M37 to hydro...

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