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Ronny Martinez, Felix Jakob, Ran Tu, Petra Siegert, Karl-Heinz Maurer and Ulrich Schwaneberg Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution Biotechnology and Bioengineering 110

Article first published online: 1 NOV 2012 | DOI: 10.1002/bit.24766

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Directed evolution was used to simultaneously increase the activity and the thermal resistance of Bacillus gibsonii Alkaline Protease (BgAP). Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (Kcat) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted in variant MF1 with a 1.5-fold increased specific activity (15°C) and >100 times prolonged half-life at 60°C (224 min compared to 2 min of the WT BgAP). Activity-altering amino acid substitutions were from non-charged to non-charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which fostered ionic and hydrogen bonds interactions.

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