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

我的研究项目旨在研究酶在食品生产和加工中的利用，有三个研究主题。过去六年的研究：在豆类蛋白质改性方面，我们利用酶和发酵法对豆类蛋白质进行改性，以改善其功能和营养特性。我的第二个研究主题是利用昆虫和发酵的组合从农业副产品中回收营养物质。在我的第三个主题（Discovery Grant研究）中，我们基于X射线晶体学对乳酸乳球菌Xaa-Pro二肽酶（氨酰基脯氨酸二肽酶）进行了结构-功能关系（SFR）研究。我总共培训了35名HQP（包括。发表论文17篇，发表报告27篇，在Protein Data Bank中保存了4个晶体结构模型，并提交了另一份手稿。HQP找到了体面的工作，如大学教师和工业研究技术员/经理。拟议研究：该研究的总体目标是阐明我们发现的氨酰基脯氨酸二肽酶的独特功能的SFR，并将所得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，含有脯氨酸的肽是苦味的，这在食品发酵中至关重要。它们仅被Pro特异性肽酶（包括氨脯氨酸二肽酶）水解。我对脯氨酰二肽酶的研究表明，这种酶具有变构行为和底物抑制作用。我已经展示了一个环结构和活性位点之间的相互作用，形成一个假底物结合位点，影响亚基的相对定位，导致这些不寻常的行为。为了研究这些性质的SFR，我设定了四个短期目标，包括：1）使用定向进化（DE）策略获得突变体酶，2）解决所得突变体的X射线三维结构，3）使用基于三维模型的合理重新设计优化氨酰基脯氨酸二肽酶，和4）工程化L.使用所得突变体的乳酸菌。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。将筛选得到的突变体与快速选择方法（DE策略）组成的脯氨酸营养缺陷型表达，酶的细胞表面展示，和快速分光光度活性测定。所得到的突变体的动力学行为和3-D结构进行检查。所获得的信息将用于进行进一步的重新设计原理，以确认SFR。由于变构和底物抑制不是酶的共同特征，因此这项研究将为酶的SFR提供有意义的知识。此外，突变的氨酰基脯氨酸二肽酶将用于修饰L。用于发酵食品脱苦的乳酸。我计划培养1名博士。和2个硕士生在这项拟议的研究。目标1和2将主要由博士进行。学生，他将与MSC合作。学生在目标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.