Quaglia_2019_PLoS.One_14_e0210100

Reference

Title : Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot - Quaglia_2019_PLoS.One_14_e0210100
Author(s) : Quaglia D , Alejaldre L , Ouadhi S , Rousseau O , Pelletier JN
Ref : PLoS ONE , 14 :e0210100 , 2019
Abstract :

Through the application of a region-focused saturation mutagenesis and randomization approach, protein engineering of the Cal-A enzyme was undertaken with the goal of conferring new triglyceride selectivity. Little is known about the mode of triglyceride binding to Cal-A. Engineering Cal-A thus requires a systemic approach. Targeted and randomized Cal-A libraries were created, recombined using the Golden Gate approach and screened to detect variants able to discriminate between long-chain (olive oil) and short-chain (tributyrin) triglyceride substrates using a high-throughput in vivo method to visualize hydrolytic activity. Discriminative variants were analyzed using an in-house script to identify predominant substitutions. This approach allowed identification of variants that exhibit strong discrimination for the hydrolysis of short-chain triglycerides and others that discriminate towards hydrolysis of long-chain triglycerides. A clear pattern emerged from the discriminative variants, identifying the 217-245 helix-loop-helix motif as being a hot-spot for triglyceride recognition. This was the consequence of introducing the entire mutational load in selected regions, without putting a strain on distal parts of the protein. Our results improve our understanding of the Cal-A lipase mode of action and selectivity. This holistic perspective to protein engineering, where parts of the gene are individually mutated and the impact evaluated in the context of the whole protein, can be applied to any protein scaffold.

PubMedSearch : Quaglia_2019_PLoS.One_14_e0210100
PubMedID: 30640952
Gene_locus related to this paper: canan-lipasA

Related information

Gene_locus canan-lipasA

Citations formats

Quaglia D, Alejaldre L, Ouadhi S, Rousseau O, Pelletier JN (2019)
Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot
PLoS ONE 14 :e0210100

Quaglia D, Alejaldre L, Ouadhi S, Rousseau O, Pelletier JN (2019)
PLoS ONE 14 :e0210100