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18. Engineering Proteases for Industrial Applications

Author

Vojcic, Ljubica, primary, Jakob, Felix, additional, Martinez, Ronny, additional, Hellmuth, Hendrik, additional, O'Connell, Timothy, additional, Mühl, Helge, additional, Lorenz, Michael G., additional, and Schwaneberg, Ulrich, additional

Published
2016
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42. Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution.

Author

Martinez, Ronny, Jakob, Felix, Tu, Ran, Siegert, Petra, Maurer, Karl‐Heinz, and Schwaneberg, Ulrich

Abstract

Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. 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 finally in variant MF1 with a 1.5-fold increased specific activity (15°C) and over 100 times prolonged half-life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of 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 probably fostered ionic and hydrogen bonds interactions. Biotechnol. Bioeng. 2013; 110: 711-720. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Published
2013
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43. Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate

Author

Li, Zhenwei, Roccatano, Danilo, Lorenz, Michael, Martinez, Ronny, Schwaneberg, Ulrich, Li, Zhenwei, Roccatano, Danilo, Lorenz, Michael, Martinez, Ronny, and Schwaneberg, Ulrich

Abstract

A subtilisin E variant (M4) showing high activity and resistance towards guanidinium chloride (GdmCl) and sodium dodecylsulfate (SDS) was previously identified after three rounds of directed evolution [Li et al., ChemBioChem 2012, 13(5), 691–699.]. In this report, 10 additional positions, identified during directed subtilisin E evolution, were saturated on the previously reported SeSaM1-5 variant (S62/A153/G166/I205). Screening confirmed that chaotolerant variants included amino acid substitutions either in the active site, or the substrate binding pocket. Two variants, M5 (S62I/A153V/G166S/T224A/T240S) and M6 (S62I/A153V/G166S/I205V/N218S/T224A) were finally generated to maximize activity and stability in the presence of GdmCl or SDS. The inactivation concentration (IC50) of M6 using Suc-AAPF-pNA as substrate was significantly increased compared to M4 in the presence of GdmCl (IC50 (M4): 2.7 M; IC50 (M6): 4.6 M) and SDS (IC50 (M4): 1.5%; IC50 (M6): 4.0%). The half-life in 5 M GdmCl was also significantly improved for M6 compared to M4 (t 1/2 (M4): 2 min; t 1/2 (M6): 15 min). M5 retained resistance towards GdmCl or SDS as in M4. The activity of M5 towards a complex protein substrate (Azocasein) was increased by ∼1.5 fold compared to M4 and M6. Circular dichroism (CD) analysis for subtilisin E wild type (WT) and three variants (M4, M5 and M6) indicated that secondary structures of all variants including wild type at 1–2 M GdmCl (except M4) were not significantly perturbed, with unfolding occurring for WT and all three variants above 3 M GdmCl. In SDS, the secondary structures of WT and all three variants remained intact at concentrations of 0.5 to 2.0% (w/v) SDS. Results suggest that subtilisin E inactivation occurred most likely due to inhibitory effect, since a general unfolding of the enzyme was not observed through circular dichroism. Such inhibition could be avoided by limiting the access of GdmCl and SDS to the active site and/or to residues involved in substra

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