Enzyme mutations for faster industrial processes
The EU-funded 'Xylanases as models for understanding enzymatic catalysis' (XYLANASES) project used common enzymes to study the role of remote residues in catalysis. Xylanases are glycosidases produced by fungi, bacteria and yeast, which break down hemicellulose, a major component of plant cell walls. Their commercial applications are most common in the paper industry. Project members aimed to understand how remote parts of an enzyme contribute to catalysis. Xylanases are among the best mechanistically and structurally characterised enzymes, making them ideal model systems for such research. The scientists investigated the participation of specific amino acids in catalysis by using a combination of mutagenesis, protein semisynthesis, enzyme kinetics, protein nuclear magnetic resonance (NMR) and X-ray crystallography. Systematically induced mutations resulted in changes in enzyme structure and activity. Remote amino acid mutations in the xylanase from Bacillus circulans resulted in substantially altered expression and purification properties. Protein semisynthesis was used to introduce unnatural amino acids. Kinetic studies of the mutants were followed by structural analysis using NMR. X-ray studies determined 3D structures of the selected mutants. It was thus possible to follow inactivation and reactivation of the mutants in real time. XYLANASES demonstrated for the first time in vivo site-specific unnatural amino acid mutagenesis on a carbohydrate active enzyme. The activity of the mutants was altered 8 to 55 times compared to wild-type enzyme. Mutations affecting enzyme inactivation and reactivation were distinct from one another. Structural studies using X-ray crystallography are expected to provide insight into the modulation of inactivation. The project established robust protein expression conditions yielding enough protein incorporating unnatural amino acids for X-ray crystallographic studies. Although xylanase from Bacillus circulans has been crystallised previously, no crystal proteins containing unnatural amino acids were produced. It is anticipated that crystallography data will help to clarify the kinetic effects of the incorporation of unnatural amino acids at key positions of xylanase.
Keywords
Enzyme, enzyme mutations, xylanases, enzymatic catalysis, Bacillus circulans
