Cornelius Bessler

Directed evolution of a bacterial α-amylase: Toward enhanced pH-performance and higher specific activity

α‐Amylases, in particular, microbial α‐amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for α‐amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the α‐amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild‐type BAA and the mutants BAA S201N and BAA N297D were subjected to error‐prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5‐fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4‐ and 1.5‐fold, respectively. Another mutant (BAA 29) possesses a wild‐type‐like pH profile but possesses a 40‐fold higher activity in periplasmic extracts and a 9‐fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial α‐amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial α‐amylases.

Engineering of choline oxidase from Arthrobacter nicotianae for potential use as biological bleach in detergents.

In order to engineer the choline oxidase from Arthrobacter nicotianae (An_CodA) for the potential application as biological bleach in detergents, the specific activity of the enzyme toward the synthetic substrate tris-(2-hydroxyethyl)-methylammonium methylsulfate (MTEA) was improved by methods of directed evolution and rational design. The best mutants (up to 520% wt-activity with MTEA) revealed mutations in the FAD- (A21V, G62D, I69V) and substrate-binding site (S348L, V349L, F351Y). In a separate screening of a library comprising of randomly mutagenised An_CodA, with the natural substrate choline, four mutations were identified, which were further combined in one clone. The constructed clone showed improved activity towards both substrates, MTEA and choline. Mapping these mutation sites onto the structural model of An_CodA revealed that Phe351 is positioned right in the active site of An_CodA and very likely interacts with the bound substrate. Ala21 is part of an α-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme.