ArticleApplied and environmental microbiology2014
Protein engineering by random mutagenesis and structure-guided consensus of Geobacillus stearothermophilus Lipase T6 for enhanced stability in methanol.
Article in Applied and environmental microbiology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 42 papers.
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42 citing papers in PubMed, 134 citations in OpenAlex.
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- Protein Stability: Enhancement and Measurement.Methods in molecular biology (Clifton, N.J.) · 2023Article
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- Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase.ACS catalysis · 2022Article
- Thermostability engineering of industrial enzymes through structure modification.Applied microbiology and biotechnology · 2022Review
- Thermostable lipases and their dynamics of improved enzymatic properties.Applied microbiology and biotechnology · 2021Review
- Ethanol as additive enhance the performance of immobilized lipase LipA fromBiotechnology reports (Amsterdam, Netherlands) · 2021Article
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production.
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