Characterization and application of natural and recombinant butelase-1 to improve industrial enzymes by end-to-end circularization.

Abstract

Butelase-1, an asparaginyl endopeptidase or legumain, is the prototypical and fastest known Asn/Asp-specific peptide ligase. It is highly useful for engineering and macrocyclization of peptides and proteins. However, certain biochemical properties and applications of naturally occurring and recombinant butelase-1 remain unexplored. Here we report methods to increase the yield of natural and bacterial expressed recombinant butelase-1 and how they can be used to improve the stability and activity of two important industrial enzymes, lipase and phytase, by end-to-end circularization. First, the yield of natural butelase-1 was increased 3-fold to 15 mg kg−1 by determining its highest distribution which is found in young tissues, such as shoots. The yield of recombinantly-produced soluble butelase-1 was improved by promoting cytoplasmic disulfide folding, codon changes, and truncation of the N-terminal pro-domain. Natural and recombinant butelase-1 displayed similar ligase activity, physical stability, and salt tolerance. Furthermore, the processing and glycosylation sites of natural and recombinant butelase-1 were determined by proteomic analysis. Storage conditions for both forms of butelase-1, frozen or lyophilized, were also optimized. Cyclization of lipase and phytase mediated by either soluble or immobilized butelase-1 was highly efficient and simple, and resulted in increased thermal stability and enhanced enzymatic activity. Overall, improved production of butelase-1 can be exploited to improve the biocatalytic efficacy of lipase and phytase by end-to-end cyclization. In turn, ligase-improved enzymes could be a general and environmentally friendly strategy for producing more stable and efficient industrial enzymes.