Targeted Selection of Amino Acid Residues to Create Variant Libraries of Glaciozyma antarctica Proline Iminopeptidase

摘要

Proline iminopeptidase is an exopeptidase which catalyses the cleavage of prolines from the N-termini of peptides. In industries, this enzyme can be used as a biocatalyst especially in food processing such as in debittering of proteolysates and flavour development of cheeses. Thus for the purpose of applications, an enzyme engineering approach was undertaken to enhance the characteristics of Glaciozyma antarctica proline iminopeptidase through development of enzyme variants that possess optimum enzyme activities in lower temperatures. Six amino acid residues of G. antarctica proline iminopeptidase Glu189, Thr143, Ala195, Lue131, Lue144 and Ala277 were selected based on its low B-factor value determined via the B-FITTER programme and alignment guided approach. Sets of mutagenising primers were designed to introduce mutations at the chosen sites by randomisation using NNK degenerate primers. CASTER tool analyses determined that a minimum of 94 colonies should be screened in these focused libraries for a 95% coverage of the possible variants. This study provides valuable information in developing enhanced thermolability of proline iminopeptidase using directed evolution.