Engineering of a novel fungal esterase by means of directed evolution and site-directed mutagenesis


Project assigned to: DANIEL KRACHER


Esterases occupy a prominent place among biocatalysts and play a major role in the modification and degradation of natural materials and industrial pollutants. Their manifold applications range from the production of chiral drugs to the de-colorization of paper or the improvement of flavors in the food industry. Esterases acting on specific types of esters are of particular interest in biotechnology and promising novel applications can be foreseen and achieved by enzyme engineering.

Aims and methods.

Previously, an esterase was identified and cloned from an ascomycetic fungus belonging to the order of Hypocreales. The enzymes unique ability to inactivate natural toxic compounds forms the basis of this project and eventually an implementation into industrial processes is aspired.

To optimize the enzyme for this purpose, commercially limiting factors such as low thermostability and unfavorable pH optimum have to be overcome. This goal shall be achieved by means of rational and semi-rational engineering on the basis of site-directed mutagenesis and directed evolution techniques (eg. error-prone PCR). Thereby, a library of mutants will be developed and screened by a high-throughput method.

A particular problem accompanying the investigation and engineering of esterases is the applicability and accuracy of the assay procedure. Due to the complex nature of their substrates, investigation of esterases usually relies on HPLC analysis and only to a minor extent on histochemical, electrochemical or photometric methods. Therefore, one aim of this project will be the development of a potent screening assay to detect enzymatic activities in micro-titer plates to provide the basis for directed evolution experiments based on a large number of clones. Another aim of this work will be the biochemical characterization of the wild-type enzyme and optimized enzyme variants. The substrate spectrum and specificity will be evaluated by spectroscopic methods and analysis of degradation products will be supported by HPLC/MS analysis. Structural studies based on X-ray crystallography shall provide insight in the detailed catalytic mechanism of the enzyme and possible key residues will be targeted by site-directed mutagenesis.

The optimized enzyme is expected to be tested in pilot-scale and if successful, foster commercial utilization of this novel esterase.

Cooperation partners: BIOMIN Holding GmbH, BIOMIN Research Center Tulln; Wulf-Dieter Moll Dept. of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna; Kristina Djinovic-Carugo; Irina Grishkovskaya

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