title: Protein engineering and applications of Candida rugosa lipase isoforms.
abstract: Commercial preparations of Candida rugosa lipase (CRL) are mixtures of lipase isoforms used for the hydrolysis and synthesis of various esters. The presence of variable isoforms and the amount of lipolytic protein in the crude lipase preparations lead to a lack of reproducibility of biocatalytic reactions. Purification of crude CRL improve their substrate specificity, enantioselectivity, stability, and specific activities. The expression of the isoforms is governed by culture or fermentation conditions. Unfortunately, the nonsporogenic yeast C. rugosa does not utilize the universal codon CTG for leucine; therefore, most of the CTG codons were converted to universal serine triplets by site-directed mutagenesis to gain expression of functional lipase in heterologous hosts. Recombinant expressions by multiple-site mutagenesis or complete synthesis of the lipase gene are other possible ways of obtaining pure and different CRL isoforms, in addition to culture engineering. Protein engineering of purified CRL isoforms allows the tailoring of enzyme function. This involves computer modeling based on available 3-D structures of lipase isoforms. Lid swapping and DNA shuffling techniques can be used to improve the enantioselectivity, thermostability, and substrate specificity of CRL isoforms and increase their biotechnological applications. Lid swapping can result in chimera proteins with new functions. The sequence of the lid can affect the activity and specificity of recombinant CRL isoforms. Candida rugosa lipase is toxicologically safe for food applications. Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.
authors.affiliation: Department of Food Science and Technology, University of Georgia, Athens, Georgia 30602-7610, USA.
authors.forename: Casimir C
authors.initials: CC
authors.lastname: Akoh
authors.forename: Guan-Chiun
authors.initials: GC
authors.lastname: Lee
authors.forename: Jei-Fu
authors.initials: JF
authors.lastname: Shaw
mesh_terms: D002175:Candida / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods*; D020033:Protein Isoforms; D013379:Substrate Specificity
chemical_list: D020033:Protein Isoforms; D008049:Lipase