High throughput mutagenesis screening method

A method for obtaining a variant enzyme having one or more desired properties comprising:

• selecting amino acid sites utilizing a three-dimensional rendition of the enzyme;
• performing site-saturation mutagenesis at the selected mutation sites to create a library;
• screening the library for variants having one or more desired properties;
• grading the mutation sites of the variants for the one or more desired properties;
• selecting one or more variants having a desirable grade as a template;
• using the template and feedback to repeat site-saturation mutagenesis at mutation sites having desirable grades and to perform site-saturation mutagenesis on new libraries at new sites.

A process for the production of a cutinase variant with hydrolytic activity on polyester, the cutinase from Pseudomonas species, the process comprising:

• utilizing a three-dimensional model to select for mutation amino acid sites likely to demonstrate hydrolytic activity;
• performing site-saturation mutagenensis at the selected mutation sites on a library;
• screening the library for variants using assays to detect polyesterase activity and thermostability;
• grading the mutated sites as beneficial, neutral or detrimental for both polyesterase activity and thermostability;
• selecting a variant having at least one beneficial grade;
• creating new and repeat libraries using the selected variant and feedback from the grading.