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Cui, Haiyang; Jaeger, Karl‐Erich; Davari, Mehdi D.; Schwaneberg, Ulrich
Chemistry : a European journal, February 5, 2021, Letnik: 27, Številka: 8Journal Article
The CompassR (computer‐assisted recombination) rule enables, among beneficial substitutions, the identification of those that can be recombined in directed evolution. Herein, a recombination strategy is systematically investigated to minimize experimental efforts and maximize possible improvements. In total, 15 beneficial substitutions from Bacillus subtilis lipase A (BSLA), which improves resistance to the organic cosolvent 1,4‐dioxane (DOX), were studied to compare two recombination strategies, the two‐gene recombination process (2GenReP) and the in silico guided recombination process (InSiReP), employing CompassR. Remarkably, both strategies yielded a highly DOX‐resistant variant, M4 (I12R/Y49R/E65H/N98R/K122E/L124K), with up to 14.6‐fold improvement after screening of about 270 clones. M4 has a remarkably enhanced resistance in 60 % (v/v) acetone (6.0‐fold), 30 % (v/v) ethanol (2.1‐fold), and 60 % (v/v) methanol (2.4‐fold) compared with wild‐type BSLA. Molecular dynamics simulations revealed that attracting water molecules by charged surface substitutions is the main driver for increasing the DOX resistance of BSLA M4. Both strategies and obtained molecular knowledge can likely be used to improve the properties of other enzymes with a similar α/β‐hydrolase fold. Smarter variation: Two recombination strategies, the two‐gene recombination process (2GenReP) and the in silico guided recombination process (InSiReP), employing the CompassR (computer‐assisted recombination) rule are used as guides to obtain highly improved variants from huge protein sequence space.
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JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
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