Effect of exchange of amino acid residues of the surface region of the PST-01 protease on its organic solvent-stability.
ABSTRACT The PST-01 protease from an organic solvent tolerant Pseudomonas aeruginosa has high stability and activity in the presence of various organic solvents. The structure gene of the PST-01 protease was amplified by the error-prone PCR method. The mutated proteases were incubated in the presence of acetonitrile. By measuring remaining activities, two kinds of mutated PST-01 proteases of which the stabilities were changed were selected. These mutations hardly changed the profile of the activity and stability at various pHs. Their activity and stability at higher temperatures were slightly lower than those of the wild-type PST-01 protease. The stabilities of the mutated enzymes in the presence of various organic solvents were greatly reduced. In both the mutated PST-01 proteases, amino acids located at the surface of the enzyme had been substituted.
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ABSTRACT: Enzymes ability to catalyze reactions in non-natural environments of organic solvents has 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 evolving 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 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 2-fold higher fatty acid methyl ester yield from soybean oil. Using in-silico modeling we suggest that the Q185L substitution facilitated a closed lid conformation which limited both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of new hydrogen bonds formation. These improved characteristics make this variant a potential biocatalyst for biodiesel production.Applied and Environmental Microbiology 12/2013; · 3.95 Impact Factor
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ABSTRACT: A novel lipase has been recently isolated from a local Pseudomonas sp. (GQ243724). In the present study, we have tried to increase the organic solvent stability of this lipase using site-directed mutagenesis. Eight variants N219L, N219I, N219P, N219A, N219R, N219D, S251L, and S251K were designed to change the surface hydrophobicity of this enzyme with respect to the wild-type. Among these variants, the stability of N219L and N219I significantly increased in the presence of all tested organic solvents, whereas two mutants (N219R and N219D) significantly exhibited decreased stabilities in all the organic solvent studied, suggesting that improvement of hydrophobic patches on the enzyme surface enhances the stability in organic media. Furthermore, replacing Ser(251) with hydrophobic residues on the enzyme surface dramatically diminished its stability in the tested condition. In spite of the distance of the mutated sites from the active site, the values of k cat and K m were affected. Finally, structural analysis of the wild-type and mutated variants was carried out in the presence and absence of some organic solvents using circular dichroism and fluorescence spectroscopy.Molecular Biotechnology 10/2013; · 2.26 Impact Factor
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ABSTRACT: A novel lipase lipB from Serratia marcescens ECU1010 is highly stable in the presence of organic solvents. By sequence and structure comparison with homologous lipase lipA, three amino acid residues were found to be different between them. To identify the residues which increase the organic solvent stability of lipB, residues that potentially provide this stability were mutated to the ones of lipA at equivalent positions. The replacement of Gly at position 33 by Asp obviously decreased its stability in organic solvents. Molecular modeling and structural analysis also suggested that the Gly33 residue is important for the organic solvent stability of lipB.Applied biochemistry and biotechnology 01/2014; · 1.94 Impact Factor