Published November 1, 2022 | Version v1
Journal article Open

Design and Characterization of In-One Protease-Esterase PluriZyme

  • 1. Department of Applied Biocatalysis, ICP, CSIC, 28049 Madrid, Spain
  • 2. Department of Life Sciences, Barcelona Supercomputing Center (BSC), 08034 Barcelona, Spain
  • 3. Department of Life Sciences, Barcelona Supercomputing Center (BSC), 08034 Barcelona, Spain; Institution for Research and Advanced Studies (ICREA), 08010 Barcelona, Spain

Description

Proteases are abundant in prokaryotic genomes (~10 per genome), but their recovery encounters expression problems, as only 1% can be produced at high levels; this value differs from that of similarly abundant esterases (1–15 per genome), 50% of which can be expressed at good levels. Here, we design a catalytically efficient artificial protease that can be easily produced. The PluriZyme EH1AB1 with two active sites supporting the esterase activity was employed. A Leu24Cys mutation in EH1AB1, remodelled one of the esterase sites into a proteolytic one through the incorporation of a catalytic dyad (Cys24 and His214). The resulting artificial enzyme, EH1AB1C, efficiently hydrolysed (azo)casein at pH 6.5–8.0 and 60–70 °C. The presence of both esterase and protease activities in the same scaffold allowed the one-pot cascade synthesis (55.0 ± 0.6% conversion, 24 h) of L-histidine methyl ester from the dipeptide L-carnosine in the presence of methanol. This study demonstrates that active sites supporting proteolytic activity can be artificially introduced into an esterase scaffold to design easy-to-produce in-one protease-esterase PluriZymes for cascade reactions, namely, the synthesis of amino acid esters from dipeptides. It is also possible to design artificial proteases with good production yields, in contrast to natural proteases that are difficult to express

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Funding

OXIPRO – Transition towards environment-friendly consumer products by co-creation of an oxidoreductase foundry 101000607
European Commission
FuturEnzyme – Technologies of the Future for Low-Cost Enzymes for Environment-Friendly Products 101000327
European Commission