Lipase properties

Recent advances in recombinant DNA technology, high-throughput technologies, genomics and proteomics, have fuelled the development of new biocatalysts and biocatalytic processes. In particular, site directed mutagenesis, directed evolution or metagenome approach are very valuable tools to enhance lipase properties. 

Palomo JM, Munoz G, Fernandez-Lorente G et al. (2003) Modification of Mucor miehei lipase properties via directed immobilization on different heterofunctional epoxy resins. Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid. J Mol Catal B Enzym 21 201-210 Palomo JM, Ortiz C, Fernandez-Lorente G et al. (2005) Lipase-lipase interaction as a new tool to immobilize and modulate the lipase properties. Enzyme Microb Technol 36 447-454 Park EY, Sato M, Kojima S (2006) Fatty acid methyl ester production using Upase-immobilizing silica particles with different particle sizes and different specific surface areas. Enzyme Microb Technol 39(4) 889-896... 

Directed evolution is a powerful tool used to improve lipase properties that does not depend on a comprehensive understanding of the relationship between enzyme structure and function. It rather depends on simple, yet powerfnl, random mutation and selection. The targeted genes are exposed to iterative cycles of random mutagenesis, expressed in an appropriate host and subsequently screened (Johannes and Zhao, 2006). Bacillus lipase was engineered by directed evolntion, where a lip gene was cloned and expressed in E. coli. The mutagenesis was executed by error-prone polymerase chain reaction (PCR). The mutation enhanced the specific activity of the lipase by twofold (Khurana et al., 2011). 

Palomo, J., G. Munoz, G. Femandez-Lorente, C. Mateo, M. Fuentes, J. M. Guisan, and R. Fernandez-Lafuente. 2003. Modulation of Mucor Miehei Lipase Properties Via Directed Immobilization on Different Hetero-Functional Epoxy Resins Hydrolytic Resolution of (R,S)-2-Butyroyl-2-Phenylacetic Acid. Journal of Molecular Catalysis B Enzymatic 21 (4-6) 201-210. 

Ability to improve lipase properties (e.g. by genetic engineering and/or directed evolution and/or rational protein design and/or mutagenesis) Immobilized lipases can be used many times... 

Thanks to their special properties and potential advantages, ionic liquids may be interesting solvents for biocatalytic reactions to solve some of the problems discussed above. After initial trials more than 15 years ago, in which ethylammonium nitrate was used in salt/water mixtures [29], results from the use of ionic liquids as pure solvent, as co-solvent, or for biphasic systems have recently been reported. The reaction systems are summarized in Tables 8.3-1 and 8.3-2, below. Table 8.3-1 compiles all biocatalytic systems except lipases, which are shown separately in 8.3-2. Some of the entries are discussed in more detail below. 

Cocoa butter has the desired "melt in the mouth property and is of high commercial value in comparison with palm oil. On the other hand, palm oil is more abundant than cocoa butter. The question is, can we convert palm oil to a product which has the desired properties of cocoa butter The answer is yes, by using lipases. 

We recently prepared various types of differently fiuorinated alkyl sulfate ILs and discovered that the hydrophobicity was dependent on the content ratio of the fluorine on the alkyl sulfate anion and 2,2,3,3,4,4,5,5-octafiuoropentyl sulfate salts showed hydrophobic properties. Melting point and viscosity were also dependent on the fluorine contents of the anionic part, while conductivity was determined by the cationic part and not influenced by the fluorine contents. Efficient lipase-catalyzed transesterificafion was demonstrated using hydrophobic 1-butyl-3-methylimidazolium 2,2,3,3,4,4,5,5-octafiuoropentyl sulfate ([bmim][C5E8]) as solvent (Eig. 6). ... 

Furthermore, in the system with coupled lipase and lipoxygenase, the production rate of HP is governed by the first enzymatic reaction and mass transfer. When TL,- is small (0 to 1 mM equiv. 3 mM LA), the kinetic curve has a sigmoid shape due to surface active properties of LA and HP [25]. Hydrolysis of TL and the increase of LA favor the transfer of LA. Such a transfer allows the lipoxygenase reaction to progress. Since lipox-ygenation consumes LA and produces HP, catalysis and transfer demonstrates a reciprocal influence. 

In vitro synthesis of polyesters using isolated enzymes as catalyst via non-biosynthetic pathways is reviewed. In most cases, lipase was used as catalyst and various monomer combinations, typically oxyacids or their esters, dicarboxylic acids or their derivatives/glycols, and lactones, afforded the polyesters. The enzymatic polymerization often proceeded under mild reaction conditions in comparison with chemical processes. By utilizing characteristic properties of lipases, regio- and enantioselective polymerizations proceeded to give functional polymers, most of which are difficult to synthesize by conventional methodologies. 

Layered phosphate/phosphonate and phosphonate materials, obtained by substitution of the phosphate moiety by phosphonate groups, display interesting tunable hydrophilic/organophilic properties for adsorption processes. When Candida rugosa lipase (CRL) is simply equilibrated with zirconium phosphate and phosphonate [135,136], immobilization was demonstrated to take place at the surface of the microcrystals. However, because lipase exhibits a strong hydrophobic character, its uptake by zirconium phosphate and phosphonate was much more related to the hydrophobic/hydrophilic character of the supports than to the surface area properties. A higher uptake is observed for zirconium-phenylphosphonate (78 %)... 

The concept of zeolite action was tested in a particular reaction where the enzyme is exposed from the beginning to an acidic environment the esterification of geraniol with acetic acid catalyzed by Candida antarctica lipase B immobilized on zeolite NaA [219]. Lipases have been used for the hydrolysis of triglycerides and due to their ambivalent hydrophobic/hydrophilic properties they are effective biocatalysts for the hydrolysis of hydrophobic substrates [220]. When water-soluble lipases are used in organic media they have to be immobilized on solid supports in order to exhibit significant catalytic activity. 

At present, margarine producers are moving to use fractionation and interesterification to produce the required properties. A new technology uses lipase enzymes to rearrange fatty acids in a controlled way. 

One of the most intriguing and best explored specificities of hydrolases is their product enantiospecificity, a property that is not restricted to the biotransformation of xenobiotics since it is displayed by lipases acting on their physiological substrates. Indeed, prochiral triglycerides have been found to be hydrolyzed with marked product enantioselectivity by various lipases [51] [52], Such specificity can hardly be fortuitous and must have a physiological significance, which remains to be understood. 

Other examples of efficient enzymatic resolutions by reaction at a remote position from stereocentres have been reported, such as the lipase-catalysed resolution of a synthetic intermediate of escitalopram." This property of enzymes has also been effectively used to resolve sterically hindered compounds by the introduction of a tether so that the enzyme-catalysed reaction can be performed at an artificially created, but less hindered, remote location. An example is the resolution of tertiary alcohols by the introduction of a glyoxylate ester. 

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