Directed Evolution of Lactococcus lactis Xaa-Pro dipeptidase based on the rationales given through X-ray crystallographic studies

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My research program aims to investigate the utilization of enzymes in food production and processing, and has three research themes. Research in last 6 years: In legume protein modification theme, we treated legume proteins using enzymes and fermentation to improve their functional and nutritional properties. My second research theme investigated recovering nutrients from agro-byproducts using combinations of insects and fermentation. In my third theme (Discovery Grant research), we conducted structure-function relationship (SFR) study of Lactococcus lactis Xaa-Pro dipeptidase (prolidase) based on X-ray crystallography. I have trained a total of 35 HQP (incl. 10 current), and have published total 17 articles and 27 presentations, and deposited four crystal structure models in Protein Data Bank, with another manuscript submitted. HQP found decent jobs, such as university faculty and research technician/manager in industries. Proposed research: The overarching goals for the proposed study are to clarify SFR of the unique functionality of prolidase we found and to use resulting mutant enzymes in debittering process of fermented foods. Proline (Pro) is a structurally-unique amino acid, and peptides containing Pro are bitter which is critical in food fermentation. They are hydrolyzed only by Pro-specific peptidases, including prolidase. My study of prolidase showed this enzyme has allosteric behaviour and substrate inhibition. I have shown the interactions between a loop structure and the active site, which form a pseudo-substrate binding site and influence the relative positioning of subunits, leading these uncommon behaviours. To investigate the SFR of these properties, I have set four short-term objectives including: 1) to obtain mutant enzymes using the Directed Evolution (DE) strategy, 2) to solve X-ray 3-D structures of resulting mutants, 3) to optimize prolidase using rationale redesign based on 3-D models, and 4) to engineer the debittering capacity of L. lactis using the resulting mutants. I choose 6 short-regions based on our structural, kinetic and bioinformatic studies, and random mutations will be conducted in these regions. The resulting mutants will be screened with rapid selection methods (DE strategy) consisting of proline-auxotroph expression, cell-surface display of enzyme, and rapid spectrophotometric activity determination. The resulting mutants are examined for kinetic behaviour and 3-D structures. The obtained information will be used to conduct further rationale redesign to confirm the SFR. Since allostery and substrate inhibition are not common characters of enzymes, this proposed study will give significant knowledge in SFR of enzymes. Further, mutant prolidases will be used to modify L. lactis for debittering of fermented foods. I plan to train 1 Ph.D. and 2 M.Sc. students in this proposed study. The Obj 1 and 2 will be conducted mainly by the Ph.D. student, and he will cooperate with the MSc. students in the Obj. 3 and 4.