Rational protein engineering and industrial application: structure prediction by homology and rational design of protein-variants with improved 'washing performance'--the alkaline protease from Bacillus alcalophilus.

The successful attempt is presented to engineer an enzyme with respect to its technical application by the use of computer-aided protein design techniques. Based on a modeled 3-D structure a number of mutants of a subtilisin-like protease was designed with the aim to increase its washing performance. The model of the highly alkaline subtilisin protease OPTICLEAN from Bacillus alcalophilus was developed by the process of 'modeling by homology' starting with the structure of subtilisin Carlsberg 1CSE.BRK from the Brookhaven protein databank. Amino acid changes and deletions were performed with the graphic protein design program BRAGI. Force field calculations and molecular dynamic simulations were made with AMBER 3.0. The comparison of the model and the later solved X-ray structure of OPTICLEAN shows a high similarity between the two structures. On the other hand, interesting deviations between the two structures were observed in some external loop regions. The comparison shows that the deviations are due to difficulties in the prediction of correct main chain torsion angles of additional prolines and the selection of correct loops in deletion or insertion regions.