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

我的研究项目旨在调查酶在食品生产和加工中的利用，有三个研究主题。近6年来的研究：在豆类蛋白改性主题中，我们利用酶和发酵技术对豆类蛋白进行处理，以改善其功能和营养特性。我的第二个研究主题是使用昆虫和发酵相结合的方法从农副产品中回收营养。在我的第三个主题(发现资助研究)中，我们基于X射线结晶学对乳酸乳球菌Xaa-Pro二肽酶(Prolidase)的结构-功能关系(SFR)进行了研究。我共培训了35名HQP(含目前已发表论文17篇，发表论文27篇，并将4个晶体结构模型存入蛋白质数据库，另有稿件提交。HQP找到了体面的工作，比如大学教员和行业的研究技术员/经理。建议研究：建议研究的主要目标是阐明我们发现的脯氨酸酶的独特功能的SFR，并将产生的突变酶用于发酵食品的脱苦过程。脯氨酸(Pro)是一种结构独特的氨基酸，含有Pro的多肽具有苦味，在食品发酵中起着至关重要的作用。它们只被Pro专一性的多肽酶，包括脯氨酸酶所降解。我对该酶的研究表明该酶具有变构行为和底物抑制作用。我已经展示了环结构和活性部位之间的相互作用，这形成了一个假底物结合部位，并影响亚基的相对定位，导致了这些不寻常的行为。为了研究这些性质的SFR，我设定了四个短期目标：1)使用定向进化(DE)策略获得突变酶，2)解决产生的突变株的X射线三维结构，3)使用基于三维模型的合理的重新设计来优化脯氨酸酶，以及4)利用得到的突变株来设计乳酸乳杆菌的脱苦能力。根据我们的结构、动力学和生物信息学研究，我选择了6个短区域，并将在这些区域进行随机突变。将用快速选择方法(DE策略)筛选得到的突变体，该方法包括：脯氨酸-营养缺陷型表达、细胞表面酶展示和快速分光光度活性测定。对产生的突变体进行了动力学行为和三维结构的检测。所获得的信息将被用来进行进一步的理论基础重新设计，以确认SFR。由于变构和底物抑制不是酶的共同特征，这项拟议的研究将为酶的SFR提供重要的知识。此外，突变的脯氨酸酶将被用来修饰乳酸乳杆菌，用于发酵食品的脱苦。我计划培养1名博士和2名硕士。这项拟议研究中的学生。目标1和目标2将主要由博士生进行，他将与硕士合作。在Obj的学生。3和4.

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.