Improving on nature's shortcomings: Evolving a lipase for increased lipolytic activity, expression and thermostability

Ana L. Alfaro-Chávez, Jian Wei Liu, Joanne L. Porter, Adrian Goldman, David L. Ollis*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    6 Citations (Scopus)

    Abstract

    An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties. Lipase Lip3 from Drosophila melanogaster had been previously optimised for expression in Escherichia coli. The expression levels were good, but Lip3 was mainly insoluble with poor activity. Directed evolution was used to identify variants with enhanced activity along with improved solubility. Five variants and the wild-type (wt) enzyme were purified and characterised. The yield of the wt enzyme was just 2.2 mg/L of culture, while a variant, produced under the same conditions, gave 351 mg. The improvement of activity of the best variant was 200 times higher than that of the wt when the crude lysates were analysed using pNP-C8, but with purified protein, the improvement observed was 1.5 times higher. This means that most of the increase of activity is due to increase in solubility and stability. All the purified variants showed increased thermal stability compared with the wt enzyme that had a T1/2 of 37°C, while the mutant with P291L of 42.2°C and the mutant R7-47D with five mutations had a value of 52.9°C, corresponding to an improvement of 16°C. The improved variants had between five and nine changes compared with the wt enzyme. There were four changes that were found in all 30 final round variants for which sequences were obtained; three of these changes were found in the substrate-binding domain.

    Original languageEnglish
    Pages (from-to)13-24
    Number of pages12
    JournalProtein Engineering, Design and Selection
    Volume32
    Issue number1
    DOIs
    Publication statusPublished - 10 Sept 2019

    Fingerprint

    Dive into the research topics of 'Improving on nature's shortcomings: Evolving a lipase for increased lipolytic activity, expression and thermostability'. Together they form a unique fingerprint.

    Cite this