Protein structure-based enhancement of enzyme performance for food and bioproduct applications using X-ray crystallography, protein modification and metabolic engineering methods

我的DG研究计划的总体目标是采用结构-功能方法来提高酶作为食品和生物制品加工助剂的性能。在这种情况下，我们研究了脯氨酸特异性肽酶。脯氨酸的氮原子上有一条侧链，这使得脯氨酸在结构上与其他氨基酸不同，并能抵抗蛋白质水解。当食物发酵时，含有脯氨酸的多肽（Pro-peptides）的积累会引起不受欢迎的苦味。通过脯氨酸的释放可以降低前肽的苦味。已知和研究了至少9种脯氨酸特异性肽酶，其中Pro-peptides的最小单位被两种主要酶水解：Xaa-Pro二肽酶（X-Pase）和Pro-Xaa二肽酶（P-Xase）。已知X-Pases也能降解含有C-P（或S-P）键的有机磷化合物；用作杀虫剂和神经毒气)。在这项DG研究（2016-2021）中，选择这两种二肽酶作为模型系统，研究它们在食品和生物制品加工中的结构-功能关系。在这个计划中，我的目标是实现以下三个目标：1)利用x射线晶体学表征植物乳杆菌P-Xase的结构，这将是X-Pase的第一个结构模型；2)基于其晶体结构对P-Xase进行蛋白质工程；3)用突变的X-Pase和P-Xase对乳酸乳球菌进行代谢工程。博士生（HQP-1）将在第一年和第二年从事P-Xase的x射线晶体学研究。揭示的结构模型将被检查，以找到负责底物特异性和催化活性的P-Xase残基。这些残基的作用将被检查和修改使用定点诱变。突变酶将被x射线晶体学检查，以确认结构和功能之间的关系。在第三年，HQP-1将与第三年初招收的第二名博士生（HQP-2）合作开始这项蛋白质工程的研究，HQP-2将继续进行到第四年。从第3年开始，与蛋白质工程工作并行，HQP-2将通过克隆工程X-Pase（来自我之前的研究）和P-Xase（这项拟议的研究）来研究乳酸乳杆菌的代谢操纵。工程乳酸乳杆菌菌株将在第4年和第5年检查其减少苦味和OP的能力。该研究项目将以多学科的方式培养两名生物技术领域的博士生，并将为加拿大生物技术产业的发展做出贡献。在五年计划之后，脯氨酸特异性二肽酶的结构-功能关系信息将有助于生物制品的开发，如生物活性肽的生产。

The overarching goal of my DG research program is to take a structure-function approach to enhance the performance of enzymes as processing aids for food and bioproduct processing. We have worked on proline-specific peptidases in this context. Proline has a side chain attached to its nitrogen atom that makes it structurally distinct from the other amino acids and resistant to proteolysis. When foods are fermented, an accumulation of proline-containing peptides (Pro-peptides) occurs to elicit undesirable bitter tastes. Bitterness of Pro-peptides can be reduced through liberation of proline. At least 9 proline-specific peptidases are known and studied, with the smallest units of Pro-peptides being hydrolyzed by two principle enzymes: Xaa-Pro dipeptidase (X-Pase) and Pro-Xaa dipeptidase (P-Xase). X-Pases are also known to degrade organophosphorus (OP; compounds containing C-P (or S-P) bonds; used as pesticides and nerve gas agents). For this DG research (2016-2021), these two dipeptidases were chosen as model systems for investigating their structure-function relationships for applications in food and bioproduct processing. In this proposed plan, I aim to achieve the following three objectives: 1) To characterize the structure of Lactobacillus plantarum P-Xase using X-ray crystallography that will be the first structure model of X-Pases, 2) to protein engineer P-Xase based on its crystal structure, and 3) to metabolic engineer Lactococcus lactis with mutant X-Pase and P-Xase. A Ph.D. student (HQP-1) will work on the X-ray crystallography of P-Xase in year 1 and 2. The revealed structural models will be examined to find the residues responsible for substrate specificity and catalytic activity of P-Xase. Roles of these residues will be examined and modified using site-directed mutagenesis. The mutated enzymes will be examined by X-ray crystallography to confirm the relationship between structure and function. In year 3, HQP-1 and the second Ph.D. student (HQP-2), who will be recruited at the beginning of year 3, will collaboratively start this investigation of protein engineering, and HQP-2 will continue it into year 4. Starting in year 3, parallel to the protein engineering work, HQP-2 will work on the metabolic manipulation of L. lactis through cloning of engineered X-Pase (from my previous study) and P-Xase (this proposed study). Engineered L. lactis strains will be examined for their ability to reduce bitterness and OP in year 4 and 5. The research program will train two Ph.D. students in biotechnology fields in a multidisciplinary manner and will contribute to growing biotechnology industries in Canada. Beyond 5-year plan, the structure-function relationship information of proline-specific dipeptidases can contribute to the development of bioproducts, such as bioactive peptide production.