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

¹ Henkel KGaA, Henkelstrasse 67, 40191 Düsseldorf, Germany
² Institute of Technical Biochemistry, Allmandring 31, 70569 Stuttgart, Germany

Reprint requests to: Rolf D. Schmid, Institute of Technical Biochemistry, Allmandring 31, 70569 Stuttgart, Germany; e-mail: Rolf.D.Schmid{at}rus.uni-stuttgart.de; fax: 49-711-685-3196.

-Amylases, in particular, microbial -amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for -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 -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 -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 -amylases.

Keywords: Directed evolution; -amylase; pH activity profile; specific activity; high throughput assay

Abbreviations: BAA, -amylase from Bacillus amyloliquefaciens • BLA, -amylase from Bacillus licheniformis • BstA, -amylase from Bacillus stearothermophilus • LAMY, -amylase from Bacillus sp. KSM-1376 • S707, -amylase from Bacillus strain #707 • TS-23, -amylase from Bacillus sp. TS-23 • BRP, Bacteriocin release protein

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