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Enhancement of the stability of a prolipase from Rhizopus oryzae toward aldehydes by saturation mutagenesis.

ABSTRACT: A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.

SUBMITTER: Di Lorenzo M

PROVIDER: S-EPMC2168222 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Enhancement of the stability of a prolipase from Rhizopus oryzae toward aldehydes by saturation mutagenesis.

Di Lorenzo Mirella M Hidalgo Aurelio A Molina Rafael R Hermoso Juan A JA Pirozzi Domenico D Bornscheuer Uwe T UT

Applied and environmental microbiology 20070921 22

A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, ...[more]

PMID: 17890336

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Project description:Rhizopus oryzae lipase (ROL) is one of the most important enzymes used in the food, biofuel, and pharmaceutical industries. However, the highly demanding conditions of industrial processes can reduce its stability and activity. To seek a feasible method to improve both the catalytic activity and the thermostability of this lipase, first, the structure of ROL was divided into catalytic and noncatalytic regions by identifying critical amino acids in the crevice-like binding pocket. Second, a mutant screening library aimed at improvement of ROL catalytic performance by virtual saturation mutagenesis of residues in the catalytic region was constructed based on Rosetta's Cartesian_ddg protocol. A double mutant, E265V/S267W (with an E-to-V change at residue 265 and an S-to-W change at residue 267), with markedly improved catalytic activity toward diverse chain-length fatty acid esters was identified. Then, computational design of disulfide bonds was conducted for the noncatalytic amino acids of E265V/S267W, and two potential disulfide bonds, S61C-S115C and E190C-E238C, were identified as candidates. Experimental data validated that the variant E265V/S267W/S61C-S115C/E190C-E238C had superior stability, with an increase of 8.5°C in the melting temperature and a half-life of 31.7 min at 60°C, 4.2-fold longer than that of the wild-type enzyme. Moreover, the variant improved the lipase activity toward five 4-nitrophenyl esters by 1.5 to 3.8 times, exhibiting a potential to modify the catalytic efficiency. IMPORTANCE Rhizopus oryzae lipase (ROL) is very attractive in biotechnology and industry as a safe and environmentally friendly biocatalyst. Functional expression of ROL in Escherichia coli facilitates effective high-throughput screening for positive variants. This work highlights a method to improve both selectivity and thermostability based on a combination of virtual saturation mutagenesis in the substrate pocket and disulfide bond prediction in the noncatalytic region. Using the method, ROL thermostability and activity to diverse 4-nitrophenyl esters could be substantially improved. The strategy of rational introduction of multiple mutations in different functional domains of the enzyme is a great prospect in the modification of biocatalysts.

Dataset Information

Enhancement of the stability of a prolipase from Rhizopus oryzae toward aldehydes by saturation mutagenesis.

Publications

Enhancement of the stability of a prolipase from Rhizopus oryzae toward aldehydes by saturation mutagenesis.

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