Abstract
BackgroundIn the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust high-throughput protocols. The current work describes how to achieve this goal by engineering ligninolytic oxidoreductases (a high-redox potential laccase -HRPL- and a versatile peroxidase, -VP-) functionally expressed in Saccharomyces cerevisiae.ResultsTaking advantage of the eukaryotic machinery, complex mutant libraries were constructed by different in vivo recombination approaches and explored for improved stabilities and activities. A reliable high-throughput assay based on the analysis of T50 was employed for discovering thermostable oxidases from mutant libraries in yeast. Both VP and HRPL libraries contained variants with shifts in the T50 values. Stabilizing mutations were found at the surface of the protein establishing new interactions with the surrounding residues.ConclusionsThe existing tradeoff between activity and stability determined from many point mutations discovered by directed evolution and other protein engineering means can be circumvented combining different tools of in vitro evolution.
Highlights
In the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust highthroughput protocols
Among the enzymes forming the ligninolytic system of white-rot fungi, high redox potential laccases High Redox Potential Laccases (HRPL) (EC 1.10.3.2) and peroxidases, including versatile peroxidases (VP; EC 1.11.1.14) are outstanding biocatalysts finding potential applications in paper pulp bleaching and functionalization, bioremediation, organic synthesis, food and textile industries, nanobiodevice construction and more [21,22,23]
Library construction VP and HRPL variants come from laboratory evolution approaches to be functionally expressed in Saccharomyces cerevisiae ([29] and unpublished material)
Summary
In the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust highthroughput protocols. Few exceptions aside [7,8], the discovery of stabilizing mutations is not always straightforwardly accomplished without significant drops in turnover rates [9] Most of these mutations, which establish new interactions by salt bridges, hydrogen bonds, hydrophobic contacts or even disulfide bridges, are placed either at the protein surface or in internal. Among the enzymes forming the ligninolytic system of white-rot fungi (i.e. involved in lignin biodegradation), high redox potential laccases HRPL (EC 1.10.3.2) and peroxidases, including versatile peroxidases (VP; EC 1.11.1.14) are outstanding biocatalysts finding potential applications in paper pulp bleaching and functionalization, bioremediation, organic synthesis, food and textile industries, nanobiodevice construction and more [21,22,23]. The presence of different catalytic sites in a small and compact protein structure (around 300 amino acids) makes VP an ideal platform for laboratory evolution strategies [23,26,27]
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